
==== Front
Nat Commun
Nat Commun
Nature Communications
2041-1723
Nature Publishing Group UK London

39300175
52568
10.1038/s41467-024-52568-z
Review Article
Discovering the hidden function in fungal genomes
http://orcid.org/0000-0002-9971-0031
Gervais Nicholas C.
http://orcid.org/0000-0002-7119-8865
Shapiro Rebecca S. shapiror@uoguelph.ca

https://ror.org/01r7awg59 grid.34429.38 0000 0004 1936 8198 Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON Canada
19 9 2024
19 9 2024
2024
15 821925 4 2024
11 9 2024
© The Author(s) 2024
2024
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New molecular technologies have helped unveil previously unexplored facets of the genome beyond the canonical proteome, including microproteins and short ORFs, products of alternative splicing, regulatory non-coding RNAs, as well as transposable elements, cis-regulatory DNA, and other highly repetitive regions of DNA. In this Review, we highlight what is known about this ‘hidden genome’ within the fungal kingdom. Using well-established model systems as a contextual framework, we describe key elements of this hidden genome in diverse fungal species, and explore how these factors perform critical functions in regulating fungal metabolism, stress tolerance, and pathogenesis. Finally, we discuss new technologies that may be adapted to further characterize the hidden genome in fungi.

New molecular technologies have helped unveil facets of the genome beyond the canonical proteome, such as microproteins and short ORFs, products of alternative splicing, regulatory non-coding RNAs, transposable elements, and cis-regulatory DNA. In this Review, Gervais & Shapiro highlight what is known about this ‘hidden genome’ in fungi.

Subject terms

Fungal genetics
Genomics
Genome
Non-coding RNAs
Fungal genomics
https://doi.org/10.13039/501100000038 Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (Conseil de Recherches en Sciences Naturelles et en Génie du Canada) issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

The canonical human proteome represents a universally recognized and comprehensive set of proteins encoded by the human genome, and its establishment has been critical to understanding fundamental cellular processes1. However, traditional annotations of this canonical proteome often overlook the polycistronic nature of genes and the factors encoded by non-canonical open reading frames (ORFs) that exist within and outside of the primarily recognized protein-coding genome2,3. Indeed, the combination of many non-ORF genomic features, including microproteins encoded within and adjacent to larger canonical protein sequences, non-coding RNA and DNA, and products of alternative splicing, all contribute to the emerging concept of the ‘hidden genome’ (Fig. 1). For example, in human cell lines, many unique proteins with differing functions can be encoded within a single gene sequence2,4. This growing appreciation of the nested and entangled nature of ORFs in the translatome has demonstrated that our traditional understanding of the functional potential within cells has been historically underestimated5. Our understanding of cellular function is further complicated by the classification of features such as long non-coding RNAs, which can function as RNAs, though, other times, actually do encode proteins critical to cell function6,7. Non-coding DNA has become of particular interest since genome-wide association studies suggest that over 90% of human disease-associated DNA variants are found in the non-coding genome8. Certainly, defining the canonical human proteome appears to have been just the beginning of characterizing the many intricate genetic products that contribute to cellular fitness in the human cell.Fig. 1 Functional products of DNA expression, including components of the hidden genome.

Sources of function from DNA include: production of mRNA and the resulting canonical proteins that are the focus of most genomic research; alternatively spliced RNA and protein products, including introns that are capable of becoming fixed in a cell and regulating gene expression; ubiquitous transcription of diverse non-coding RNAs that play important roles via their interaction with DNA, RNA, and proteins; the expression of pseudogenized DNA and dubious ORFs into functional proteins; the existence of non-coding regulatory elements (NCREs) that regulate gene expression; transposable elements that may move within the genome or between cells within or outside of the originating species, along with other repetitive DNA that may facilitate structural rearrangements in the genome. Figure 1, created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

This phenomenon of the hidden genome extends across the tree of life and includes fungal species, where non-ORF genes that contribute to fitness have traditionally been neglected and are only recently beginning to be identified and characterized9,10. Indeed, the genomes of many fungal species important to human health and disease, agriculture, and biotechnology, remain incompletely characterized11. There is substantial emerging evidence for a critical role of the hidden genome, including microproteins and alternatively spliced proteins, microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), as well as other regions of non-coding and repetitive DNA, in numerous key facets of fungal biology (Table 1). Therefore, the ability to fully characterize these cryptic components of the fungal genome will be a critical step towards a comprehensive understanding of the genomics and biology of the fungal kingdom. The fact that genomic tools generally take longer to adapt and utilize in fungi has meant that many components of the hidden genome have gone relatively underexplored compared to other model species, though advances in other model organisms do offer a glimpse into how new tools may be employed in fungi. As the vast diversity of the fungal kingdom plays significant roles in all aspects of human life, it is imperative to fully dissect the functional potential of these fungal genomes.Table 1 Selected ‘hidden genome’ elements and their corresponding studies that are referenced by this Review, by species

Hidden genome element	Species	Referenced studies	
Microproteins/sORFs	A. fumigatus, A. nidulans, A. niger, A. oryzae, Ag. bisporus, C. albicans, C. dubliniensis, Cr. gattii, Cr. neoformans, D. hansenii, E. cuniculi, E. intestinalis, Er. cymbalariae, Er. gossypii,, G. zeae, K. africana, Kl. lactis, L. thermotolerans, M. grisea, My. thermophila, N. glabrata, Na. castellii, Na. dairenensis, P. pastoris, P. stipitis, S. cerevisiae, Sc. pombe, T. delbrueckii, Te. phaffii, Th. terrestris, Y. lipolytica, Z. rouxii	16–21	
Alternatively spliced proteinsd	A. fumigatus, A. nidulans, C. albicans, C. parapsilosis, Cr. neoformans, H. capsulatum, Li. corymbifera, M. oryzae, N. glabrata, Ne. crassa, S. cerevisiae, Sch. commune, Sh. bambusicola	29,31–35,38–40,42–47	
Long non-coding RNAs	A. flavus, A. sydowii, C. albicans, C. auris, C. parapsilosis, C. tropicalis, Co. militaris, Cr. neoformans, F. graminearum, Me. robertsii, N. glabrata, Ne. crassa, No. ceranae, S. cerevisiae, Sc. pombe, P. pastoris, U. maydis	58,60–81	
Micro/Micro-like RNAs, small non-coding RNAs	A. fumigatus, B. bassiana, Bo. cinerea, C. albicans, Cr. neoformans, F. graminearum, F. oxysporum, F. verticillioides, Me. anisopliae, Mu. circinelloides, Ne. crassa, Ne. tetrasperma, Neo. fischeri, Pe. marneffei, Pu. graminis, Py. tritici-repentis, Scl. sclerotiorum, So. macrospora, Sp. reilianum, Tr. reesei, Tri. rubrum, U. maydis, V. dahliae, Va. mali	58,82–96,97a	
Circular RNAs	As. apis, Ga. lucidum, No. ceranae, M. oryzae, S. cerevisiae, Sc. pombe, Tri. rubrum	98–103	
Transposable elements	A. fumigatus, Am. brunnescens, Am. inopinata, Am. muscaria, Am. polypyramis, Am. thiersii, Bo. cinerea, C. albicans, Cr. deneoformans, Cr. neoformans, F. graminearum, F. oxysporum, La. bicolor, M. oryzae, Mi. osmundae, N. glabrata, Ne. crassa, Ph. carnosa, Ph. chrysosporium, Pl. ostreatus, Ps. antarctica, Ps. hubeiensis, Pu. graminis, Pu. striiformis, S. cerevisiae, Sc. pombe, Se. lacrymans, Vo. volvacea, Zy. tritici	127,128,129b,130–136	
Starshipse	A. felis, A. flavus, A. fumigatus, A. niger, A. oryzae, A. sojae, Al. alternata, Al. gaisen, Al. solani, Al. tenuissima, Ar. ostoyae, Bot. dothidea, Bi. sorokiniana, Cl. psammophila, F. culmorum, F. fujikuroi, F. oxysporum, Fo. nubica, Fo. pedrosoi, Le. lupina, Mo. importuna, Ma. phaseolina, O. oligospora, Pa. lecythidis, Pa. variotii, Par. nodorum, Pe. fuscoglaucum, Pe. roqueforti, Po. anserina, Po. comata, Po. pauciseta, Py. tritici-repentis, Tr. asperellum, Ta. marneffei	128,137–143,144c	
Abbreviatious used for genera: A., Aspergillus; Ag., Agaricus; Al., Alternaria; Am., Amanita; Ar., Armillaria; As., Ascosphaera; B., Beauveria; Bi., Bipolaris; Bo., Botrytis; Bot., Botryosphaeria; C., Candida; Cl., Cladophialophora; Co., Cordyceps; Cr., Cryptococcus; D., Debaryomyces; E., Encephalitozoon; Er., Eremothecium; F., Fusarium; Fo., Fonsecaea; H., Histoplasma; G., Gibberella; Ga., Ganoderma; K., Kazachstania; Kl., Kluyveromyces; L., Lachancea; La., Laccaria; Le., Letharia; Li., Lichtheimia; M., Magnaporthe; Ma., Macrophomina; Me., Metarhizium; Mi., Mixia; Mo., Morchella; Mu., Mucor; My., Myceliophthora; N., Nakaseomyces; Na., Naumovozyma; Ne., Neurospora; Neo., Neosartorya; No., Nosema; O., Orbilia; P., Pichia; Pa., Paecilomyces; Par., Parastagonospora; Pe., Penicillium; Ph., Phanerochaete; Pl., Pleurotus; Po., Podospora; Ps., Pseudozyma; Pu., Puccinia; Py., Pyrenophora; S., Saccharomyces; Sc., Schizosaccharomyces; Sch., Schizophyllum; Scl., Sclerotinia; Se., Serpula; Sh., Shiraia; So., Sordaria; Sp., Sporisorium; T., Torulaspora; Ta., Talaromyces; Te., Tetrapisispora; Th., Thielavia; Tr., Trichoderma; Tri., Trichophyton; U., Ustilago; V., Verticillium; Va., Valsa; Vo., Volvariella; Y., Yarrowia; Z., Zygosaccharomyces; Zy., Zymoseptoria.

aThe comprehensive set of species in ref. 97 is not listed due to the contentious presence of bona fide miRNA/milRNA/sRNAs (or lack thereof) in some species.

bNot all species could be listed here as ref. 129 included 625 publicly available genomic assemblies, including 27 Aspergillus isolates, 30 Candida isolates, 19 Cryptococcus isolates, 26 Fusarium isolates, 15 Penicillium isolates, 25 Saccharomyces isolates, 20 Trichophyton isolates, and more.

cComputational annotations of Starships from ref. 144 have been left out due to space constraints, encompassing 2899 genomes from 1649 fungal species.

dAlternative splicing has already been described in a number of fungal taxa that would exceed the space available in this table (see refs. 28,30,41). This list only includes those species with well characterized instances of alternatively spliced proteins that are specifically discussed in this Review.

eThe existence of bona fide Starships in some listed species remains putative. This list of species that contain characterized Starships is not necessarily comprehensive, even amongst the literature cited in this Review.

Here, we describe the emergent findings that have been made into the hidden genome of diverse fungal species. We describe key components of the genome outside the canonical proteome, using both fungal and other species as relevant context, describe the important role of these factors in mediating important facets of fungal biology, and discuss new technologies that may be adapted to study the hidden genome in fungal species.

Non-canonical ORFs

Microproteins

Microproteins are proteins made up of a polypeptide chain shorter than 100 amino acids in length, encoded by short open reading frames (sORFs)12. These proteins were originally omitted from most functional analysis research due to the expectation that they would only rarely impact fitness, and for practical reasons due to their massive abundance in the genome12. However, the use of newer, more sensitive technologies has allowed for the confident detection of thousands of microproteins translated from sORFs in human cells, many of which have been characterized as playing important roles, including their involvement in stress response pathways13. Foundational studies in characterizing microprotein function in human cell lines have revealed the potentially profound impact of the microproteome on the development of disease14. In bacteria, many microproteins have been implicated in drug resistance, as well as in toxin-antitoxin systems and oxidative stress15.

Microproteins encoded by sORFs have also been recognized as having putative regulatory functions in fungi for decades, though, by their nature, it is difficult to distinguish between sORFs that are transcribed and translated and those randomly occurring incidental small ORFs that are not16. Regardless, bioinformatic approaches for annotating genomic sORFs have improved substantially over the past few years, and have been used to predict thousands of sORFs in 31 different fungal genomes17. One study in Saccharomyces cerevisiae leveraged ribosome profiling datasets to determine that translation of non-canonical ORFs may occur within the DNA sequences of at least 15% of canonical ORF-encoding genes18. Hypothetical annotations such as these can serve as the foundation for experimental approaches while attempting to successfully identify microproteins. One combinatorial method using ribosome profiling and proteomics, for example, was used in the fission yeast Schizosaccharomyces pombe to verify the existence of peptides corresponding to hypothesized sORFs19. However, in this case, only 9/373 of the presumed sORFs had a detectable peptide, implying that new technologies will be required for the reliable detection of microproteins in fungi19. Despite the technological limitations faced by fungal researchers, several studies have showcased the function of microproteins across different growth conditions. In one investigation, researchers were able to identify the microprotein Nrs1 from a genome-wide overexpression screen in S. cerevisiae, whose upregulation rescued an otherwise inviable double gene-deletion mutant20. Nrs1 itself allows cells to overcome nitrogen-starved conditions and plays an important role in the regulatory circuitry involved in yeast budding20. Several other singular instances of microproteins serving as key players in regulatory pathways have been described, mostly from research on S. cerevisiae16. In another study in S. cerevisiae, researchers were able to identify 225 microproteins and plot how they are differentially- and sometimes, exclusively- expressed in response to UV stress, heat shock, and nutrient limitations, suggesting critical functions in different cellular adaptation contexts21.

There is therefore a strong implication that improved tools to investigate the fungal microproteome would result in numerous insights into stress response pathways and diverse aspects of fungal biology. While ribosome profiling and mass spectrometry-based proteomics may be adapted in other fungal species, these strategies could also be augmented by combining CRISPR screening and single-cell RNA sequencing to allow for the function of microproteins in fungi to be determined at scale, including the genome-wide effects of their perturbance on gene expression22. Similarly, improved models for the in silico prediction of microproteins that are not limited to one coding sequence per transcript, and that can predict sORFs and ORFs translated from non-AUG start codons, could now be applied to fungal species23. In addition, the optimization of new RNA-targeting CRISPR-based tools for fungi may allow for the discriminate targeting of sORF mRNA and characterization of microprotein function24.

Alternatively spliced proteins

Similar to microprotein formation, the capacity to include and exclude different sets of exons from a single gene through alternative splicing is also known to massively increase mRNA and protein diversity in eukaryotes25. Alternative splicing events have been demonstrated to play an important role in many facets of cell biology, including the establishment of drug resistance in human cancer cells and the pathogenesis of microbial parasites26,27. While alternative splicing is generally regarded as being less relevant in bacteria due to introns being either absent or very rare in prokaryotic species, splicing events are distinct in eukaryotes, where the number of genes that contain introns in fungi, for example, ranges massively from 4% to 99% across different species28. Despite this, alternative splicing was long seen as inconsequential to the cell in fungi, perceived as resulting in transcripts with either redundant or inoperative functionality29.

Recently, however, numerous mechanistic effects of alternative splicing have been established in fungi with impacts on growth, stress adaptation, infection, and immune recognition30. In the plant-parasitic fungus Shiraia bambusicola and the filamentous fungi Neurospora crassa and Aspergillus nidulans, alternative splicing has been observed to increase proteomic complexity and downstream functionality, particularly in response to different environmental stressors31–33. In the mushroom-forming Schizophyllum commune, alternative splicing was found to increase the total number of transcripts in the cell by 20%, and the majority of spliced transcripts were predicted to have alternative functions based on the fact that 70% of them had either lost or gained a functional domain compared to the non-spliced isoform29. In many yeast species, the rate of alternative splicing can be altered in response to stress, and further research into the molecular basis for the augmentation of genomic complexity from alternative splicing has demonstrated the capacity for dual localization of proteins encoded by the same gene to serve distinct functions in metabolism9,34,35. One study created a deletion set of all known introns in S. cerevisiae and identified the important roles introns have during competition and nutrient starvation36. Interestingly, in S. cerevisiae, once spliced out, introns themselves can become stably fixed in a cell and can act on different metabolic pathways, thus acting as regulatory ncRNAs37. In addition, expression via alternative transcriptional start sites and antisense transcription can be triggered in response to environmental cues in many fungi, including Metarhizium robertsii, A. nidulans, and Cryptococcus species, often resulting in changes in protein localization and downstream regulatory effects on gene expression38–40.

Alternative splicing may also be broadly associated with fungal pathogenicity, as it appears to be more prevalent in fungal pathogens, especially human fungal pathogens, than non-pathogenic taxa41. In the rice blast pathogen Magnaporthe oryzae, for example, deletion of the MoGrp1 protein involved in different splicing processes leads to a dramatic decrease in the virulence of the pathogen42. It has further been suggested that alternative splicing is potentially linked to drug susceptibility phenotypes in pathogenic fungi, and could even act as a target for the development of new therapeutics43. Indeed, alternative splicing seems to be differentially regulated specifically in response to certain antifungal drugs in N. crassa44. Another example involves the human pathogen Candida albicans with the oxidative stress-generating drug menadione45. In this study, researchers identified a case of differential drug resistance following deletion of the superoxide dismutase gene SOD3, where only overexpression of the spliced isoform of SOD3 rescued the mutant’s increased susceptibility to menadione45. As expected, menadione’s antifungal effect is partly based on its inhibition of the cell’s ability to perform alternative splicing45. While alternative splicing of introns clearly has underexplored and important roles in metabolism, antifungal drug resistance, and pathogenicity, the splicing of inteins — intervening sequences that are spliced out at the protein level — is also emerging as a process important to fungal biology and antifungal drug susceptibility. In the human pathogenic yeast Cryptococcus neoformans, different antifungal agents have been found to prevent the Prp8 intein from performing its essential role in cell viability and virulence via intein splicing, which has opened up new avenues for potential therapeutics against pathogenic fungi46,47.

Coinciding with the advent of long-read RNA sequencing technologies, new computational tools that allow for the sensitive detection of alternatively spliced transcripts have emerged in the past few years48. As these platforms continue to be improved upon, they may be adapted for the detection of mRNA isoforms in fungal species at a genome-wide level48. Further, adapting CRISPR-based platforms that allow for targeted deletion of single exons49, as well as multiplexed repression and activation of exons50, may allow fungal researchers to investigate the functional differences of spliced mRNA isoforms.

Non-coding RNAs

Non-coding RNAs (ncRNAs) include any RNA molecule in the cell that is generally not translated into a protein. While some classes of ncRNAs have long been acknowledged for playing crucial roles in the cell, including ribosomal RNA (rRNA) and transfer RNA (tRNA), the majority of ncRNA molecules were historically overlooked as being inert by-products of transcription51. However, more recent large-scale sequencing efforts have found that the majority of the genome can be transcribed, mostly into ncRNAs, and many divergent families of ncRNAs have been recognized for their unique and critical functions51. Among the many different classes of ncRNAs, regulatory RNAs include lncRNAs, miRNAs, and circRNAs. LncRNAs are ncRNAs longer than 200 nucleotides (nt), miRNAs have a length of around 19-25nt, and circRNAs differ in being non-linear and having a closed-loop structure. Each of these three types have different mechanisms of action, though in many cases exert epigenetic, RNA processing, and translational regulatory actions in the cell52–54. In human cells, all three of these regulatory ncRNA classes have been well studied in contributing to disease55,56. Despite their relatively simpler genomes, bacterial pathogens also utilize regulatory ncRNAs in diverse ways, some of which have been proposed as potential drug targets57. Regulatory ncRNAs, primarily lncRNAs, miRNAs, and circRNAs have all been identified in a myriad of disparate fungal taxa, though the extent to which these ncRNAs have been functionally characterized differ.

Long non-coding RNAs

Perhaps at the forefront of these inquiries are lncRNAs. In many fungi, lncRNA-encoding DNA can exist within and be transcribed from intergenic, intronic, sense, and antisense regions (Fig. 2)58. lncRNAs can be especially difficult to identify and characterize, as, unlike protein-coding genes, they seem to lack sequence conservation in mammalian cell lines, for example59. Despite this, lncRNAs have been described in many different fungal cellular processes including gene silencing and regulation, nutrient metabolism, histone modification, drug resistance, and virulence58,60. In fungi, lncRNAs have been most comprehensively studied in model yeasts. In S. cerevisiae, differential lncRNA abundance in distinct cell subpopulations has been suggested to play a regulatory role in cell and colony development61. Efforts have been made to characterize ncRNAs on a large scale in S. cerevisiae, where barcoded ncRNA deletion libraries have been screened to identify several intergenic ncRNAs essential to cell survival62. Another relatively large-scale approach taken in S. cerevisiae to explore lncRNA biology involved knocking out non-essential ORFs in combination with lncRNAs to generate double deletion mutant cells, allowing the characterization of lncRNAs via genetic interaction analysis63. This resulted in the identification of one lncRNA that acts in trans to regulate levels of distant telomeric single-stranded DNA, which is a necessary component of telomeric replication63. The researchers were, however, also able to implicate many other lncRNAs operating in a wide range of biological processes in the cell63. Indeed, there are several other well-defined cases of lncRNAs influencing the cell in yeast, including the specific regulatory roles that lncRNAs have on cell wall-related gene expression64. In the model fission yeast S. pombe, thousands of lncRNAs have been detected and have had their expressions monitored in response to different perturbations65. Amongst the lncRNAs in S. pombe, transcription of the well-characterized nc-tgp1 was found to play an important role in sensitivity to the drug thiabendazole as well as to hydroxyurea and caffeine by simply increasing nucleosome density, thereby preventing transcription of the neighboring tgp1 ORF (Fig. 2)66. lncRNA biology is also quickly expanding into other fungal taxa, including the yeast Pichia pastoris and the filamentous fungi N. crassa, where a vast amount of lncRNAs have been identified and assigned putative functions67,68.Fig. 2 The nature of lncRNA transcription and examples of intergenic lncRNAs in fungi.

LncRNAs exist in several different orientations relative to canonical ORFs within fungal genomes, including in between exons (intronic), in between genes (intergenic), as well as within genes in either the same (sense) or opposite (antisense) direction as the larger ORF. Many lncRNAs have been characterized in diverse fungal species, including: the lncRNA DINOR in Candida auris that affects fungal morphology and drug resistance; the putatively cis-acting lncRNA sequence RZE1 in Cryptococcus neoformans that recapitulates virulence phenotypes of the neighboring gene ZNF2 when deleted and appears to affect the proportion of mRNA of ZNF2 that is localized to the nucleus; and the lncRNA nc-tgp1 that alters sensitivity to environmental stressors in Schizosaccharomyces pombe by increasing nucleosome occupancy when actively transcribed, excluding the transcription factor Pho7 from binding and activating expression of the downstream tgp1 gene. Figure 2, created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

The field of fungal lncRNAs has also had a recent surge of interest in the context of pathogenic fungi. In many pathogenic Candida species, new bioinformatic and transcriptomic approaches continue to unveil more and more lncRNA transcripts in the cell69–72. Further, certain lncRNAs have been validated to exhibit significance in biological processes. One of the most striking examples involved a showcasing of the lncRNA named DINOR in the rapidly emerging multidrug-resistant human pathogen Candida auris, and its critical role in governing fungal pathogenicity, filamentous growth, and antifungal drug resistance (Fig. 2)73. DINOR was discovered via screening a genome-wide transposon mutagenesis library, therefore also inadvertently presenting a strong argument for constructing and screening more comprehensive mutant libraries that are not limited to canonical proteins73. On a larger scale, the differential expression of hundreds of lncRNAs cataloged across several important pathogenic Candida spp. during infection has also been characterized74.

The relevance of lncRNAs to virulence and metabolism is becoming clear in other crucial human pathogens as well, as a random insertional mutagenesis screen in C. neoformans uncovered RZE1, an lncRNA essential to pathogenicity via its predominately cis-acting regulatory function of the yeast-to-hypha transition (Fig. 2)75. In the pathogen Aspergillus sydowii, lncRNAs were found to play a role in tolerance to high NaCl stress, and in the related pathogen Aspergillus flavus, expression profiling of hundreds of lncRNAs suggested they are expressed and differentially localized in response to important environmental triggers, such as changes in temperature, osmotic stress, and CO276,77. In other fungal pathogens, including those that primarily infect and parasitize plants and insects, there has also been a rapid increase in interest in lncRNA function, where reports on the lncRNA landscape in Fusarium graminearum, Nosema ceranae, M. robertsii, and Cordyceps militaris, have all produced valuable preliminary insight into their functions in the cell78–81. Our understanding of lncRNAs in fungi and the diverse aspects of fungal biology they impact is continuing to expand.

MicroRNAs and micro-like RNAs

Research into other types of regulatory ncRNAs, namely miRNAs and circRNAs, has also shown promise for our improved understanding of fungal biology. RNA interference (RNAi) is a conserved mechanism in eukaryotes that typically involves small non-coding RNAs (sRNAs) ~ 19–24 nucleotides in length82. sRNAs work in concert with effector proteins to form an RNA-induced silencing complex (RISC) that can together silence mRNA translation via complementary binding to target RNA by the sRNA82. One category of sRNA in these systems is miRNA, which differs from other types due to their relatively indiscriminate binding patterns82. miRNAs also have many regulatory roles in the cell, rather than being simply a genome defense mechanism against invading viruses and transposons, as is the case for other sRNAs82. miRNAs were first discovered in Caenorhabditis elegans in 1993 and were initially considered to be absent in fungi until they were discovered in N. crassa in 201083,84. Since then, miRNAs and miRNA-like RNAs (milRNAs), which do not meet certain criteria established in other eukaryotic taxa to be included as miRNAs, have been identified in a considerable number of fungi, including Penicillium marneffei, Aspergillus fumigatus, Trichoderma reesei, Metarhizium anisopliae, Trichophyton rubrum, Sclerotinia sclerotiorum, C. albicans, and more83,85–90.

Much of the research into miRNAs and milRNAs in fungal species has involved employing sequencing approaches to validate their presence, and then analyzing their differential expression patterns in response to different growth conditions. Strategies such as these have allowed researchers to putatively classify miRNA and milRNA activity in important fungal cellular processes including thermal dimorphism, defense against mycoviral infection, cellulase production, mycelial growth and conidiogenesis, and sclerotial development, and also to predict their target RNAs in some cases83,85–89. Research in C. neoformans identified and profiled miRNAs, and found that miRNA sequences align to genomically-encoded transposons and pseudogenes, suggesting a role of miRNAs in regulating transposable element activity and the cryptic expression of pseudogenes91. Another phenomenon involves the spontaneous acquisition of drug resistance in the human fungal pathogen Mucor circinelloides via its RNAi-mediated gene silencing during growth in the presence of the antifungal drug tacrolimus92. Other studies have adopted a wider approach, such as in one case which involved employing computational tools to predict milRNAs and their RNA targets across 13 different plant fungal pathogen species93. This work identified several milRNA targets within the genome of their respective host plants, confirming a role for fungal milRNAs in suppressing plant host defense genes during infection93. Other examples of sRNA/milRNA-mediated silencing of host immunity by the plant pathogens Valsa mali and Botrytis cinerea have also been demonstrated and mechanistically characterized94,95. Despite these findings, it remains difficult to identify the activities of milRNAs in many cases, and it has been suggested that they may have other roles in genetic regulation besides mRNA cleaving, including translational repression or even DNA methylation96. Potential functions for sRNAs/miRNAs can also be overlooked, such as in one case where functional sRNA discovery in C. albicans was long impeded due to the widely used reference strain being uniquely deficient in a functional RNAi pathway, while the majority of other C. albicans isolates employ RNAi and sRNAs in the repression of telomere-associated genes90. As mi/milRNA annotations and their proposed functions in fungi continue to be assessed and improved upon97, the diverse roles of miRNAs and milRNAs across the fungal kingdom will become increasingly understood.

Circular RNAs

Circular RNAs (circRNAs) are the product of backsplicing, where the acceptor site of an upstream exon is towards its 5’ end, and the donor site of a downstream exon is towards its 3’ end, such that the exonic RNA folds in on itself and circularizes54. CircRNAs encode diverse cellular products, though they also seem to execute specific actions in the cell without being translated, including the inhibition of both miRNAs and the translation and activities of proteins54. It is also apparent that the ratio of circRNAs to linear RNA molecules is a tightly regulated process that has implications for disease and aging in humans54,98. CircRNAs of this kind were identified in fungi in 2014 in S. cerevisiae and S. pombe, but further research on them has been extremely limited98. In the past few years, sequencing efforts have led to hundreds or thousands of circRNAs being annotated in the genomes of the fungal pathogens M. oryzae, Ascosphaera apis, N. ceranae, and Ganoderma lucidum99–102. The ability of these circRNAs to act as “sponges” by competitively binding miRNAs was investigated and confirmed in all of these instances99–102. Interestingly, the expression patterns of different circRNAs often seemed to depend massively on the cell type or developmental stage of the fungus, though there has been a lack of characterization of individual circRNAs in specific cellular processes99,102,103. While intergenic, exonic, and intronic circRNAs exist, the proportion of circRNAs in either of the three groups seems to differ between fungal species101,103. Parallel efforts were also made in the human pathogenic T. rubrum, where researchers highlighted that one of the 4254 circRNAs they identified, Tru_circ07138_001, seemed to be highly conserved in ten other dermatophytic species analyzed, as well as in the distantly related red junglefowl Gallus gallus and C. elegans, indicating a shared role of this circRNA across the tree of life103.

The growing body of research on fungal regulatory ncRNAs serves as a compelling revelation of the importance of the hidden genome in fungi. New computational platforms, some of which leverage machine learning, that can identify regulatory ncRNAs from RNA sequencing data may be applied in fungi104,105. In addition, a diverse set of CRISPR-based screening platforms have already been demonstrated in human cells to study lncRNAs, miRNAs, and circRNAs en masse106–109. Indeed, harnessing new technologies developed for use in other species, along with continually improving techniques for functional genomic analysis in fungal taxa110,111, may greatly expand our understanding of these ncRNAs and their role in important facets of fungal biology.

Regulatory, repetitive, and canonically non-functional DNA

While many categories of ncRNAs and non-canonical proteins are beginning to be more clearly defined, there still exists parts of the genome that are more cryptic and underexplored. This involves the capacity of DNA to regulate gene expression independently of any transcribed or translated product, namely via non-coding regulatory elements (NCREs) such as promoters, silencers and enhancers, and insulators, that all contribute to cis-regulatory gene expression regulation112. However, it also includes regions of DNA that have historically been assumed to be non-functional, including repetitive regions and transposons, as well as pseudogenes and dubious ORFs, much of which has often been referred to as ‘junk’ DNA. ‘Junk’ DNA, has traditionally referred to any DNA sequence that does not play a known role in any cellular process, and has long been assumed to represent the vast majority of the DNA in the genome112,113. However, what constituent non-coding DNA is actually non-functional remains a contested topic, especially when considering the C-value paradox, which involves the discrepancy between the expectation that more complex organisms should tend to have larger genomes, and the reality that they often do not114. While the Encyclopedia of DNA Elements (ENCODE) project proposed that, in fact, 80% of the human genome is linked to biochemically active processes, criticisms of this claim involve the fact that their definition of biochemical activity does not extend to what is actually functional in the human cell, and that much of the DNA in a given genome remains to be assigned a true function114,115. Regardless, there are still many clear and validated examples of regions of ‘junk’ DNA contributing to diverse cellular phenomena113. These include pseudogenes, highly repetitive DNA, and transposable elements113. These groups of DNA have already been shown to be potential regulators of protein-coding DNA in some eukaryotes, as well as been demonstrated as effective therapeutic targets in cancer cells, but they remain relatively poorly studied in fungi116,117.

Non-coding regulatory elements

The identification and precise mapping of NCREs varies widely between different fungal taxa and often focuses on trans-acting regulatory components like transcription factors118,119. However, there have been important discoveries made on the topic of NCREs in fungi, and many efforts have been made, particularly in recent years, to employ methods that would allow for the mapping and functional analysis of these cis-regulatory components in Saccharomyces species120–122. Related phenomena have been identified in S. cerevisiae, in particular, where the genomic regions that encode tRNA seem to act as chromatin insulators with strong implications in preventing gene repression and activation123,124. Systematically categorizing fungal genetic circuits and NCREs may help uncover novel regulatory mechanisms of fungal pathogenesis and allow researchers to better harness fungal metabolism for industrial applications and for the production of important metabolites. Attempting to characterize cis-regulatory DNA is difficult, in part due to the complex nature of the interactions between transcription factors and a dynamic 3D DNA landscape125. However, CRISPR screens hold promise for functionally characterizing NCREs at scale126. In addition, the combination of improved machine learning models with new technologies that can produce long synthetic DNA molecules may help us to investigate transcription factor binding in the context of a locus large enough to account for high-level chromatin architecture and DNA-DNA interactions125.

Transposable elements and repetitive DNA

Another underexplored component of the fungal genome involves transposable elements (TEs). TEs encompass an array of different DNA sequences, some of which possess genes that enable them to change position in the genome, causing disruptions in gene function or alterations of local gene expression, and in some cases transport ‘cargo’ genes that are not required for propagation of the TE and may be of benefit to the host127,128. Additionally, the repetitive nature of non-mobile TE sequences scattered throughout the genome can facilitate structural rearrangements and shape genomic architecture127. The proportion of the genome that is made up of TEs varies from ~0% to upwards of 30% in fungi127. While the extent of their regulatory roles in shaping fungal phenotypes remains contested, they are linked to the expression of genes and their evolution in fungi129. Indeed, in M. oryzae, the presence of TEs appears to increase genetic diversity in neighboring genes, which in turn drives host specialization of the pathogen130. Studies done on the fungal plant pathogen Zymoseptoria tritici have revealed that TE insertions can directly regulate melanin biosynthesis, and therefore fungal virulence, as well as multi-drug resistance via fungicide efflux upregulation131,132. TE insertions also drive adaptation during host infection and contribute to the acquisition of drug resistance in Cryptococcus (Fig. 3)133,134. Transposon mobility in these cases displays a temperature-dependent pattern, underscoring their influence on the cell’s ability to respond to environmental changes (Fig. 3)133,135. Bioinformatic-based findings in other fungal pathogens also suggest that genes under TE influence are often repressed and that there tends to be a correlation between TE prevalence in the genome and symbiotic tendencies127,136.Fig. 3 The movement of transposable elements (TEs) within and between fungal genomes.

In the human fungal pathogen Cryptococcus deneoformans, the mobility of several different TEs is stress-responsive, including during infection and at the elevated (host-relevant) temperature of 37 °C. Insertion of mobilized TEs in this pathogen also appears to be biased depending on the type of TE, and has been associated with a decrease in susceptibility to antifungal drugs. The accumulation of the Cnl1 TE at subtelomeric regions, for example, can result in additional copies of Cnl1 being driven towards other sites in the genome, which may influence drug response and virulence phenotypes. The rate of other genomic changes, including from single nucleotide polymorphisms (SNPs) and insertion/deletion (indel) mutations, remain unchanged at elevated temperatures, suggesting TE mobility is a primary driver of genomic change during heat stress. In the plant fungal pathogen Pyrenophora tritici-repentis, the toxhAT TE contains the ToxA gene which can induce cell death in susceptible wheat strains during infection. The toxhAT TE has been observed to move between isolates of the same species, as well as between different species entirely. While toxhAT has been observed to exist within larger Starship TEs, there is also evidence to suggest that it is able to mobilize from the genome independently. Figure 3, created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

A role for fungal TEs in horizontal gene transfer has also recently emerged based on the identification of very large transposons named Starships, which can be hundreds of kilobases in size and carry genes important to fungal survival and adaptation137. Many different Starships have been identified with high sequence similarity in divergent fungal taxa, suggesting they are able to migrate between species independently of the fungal host’s cellular machinery138–140. For example, the Hephaestus Starship locus that confers heavy metal resistance to the emerging infectious mould Paecilomyces variotii appears to have been shared with the related species Paecilomyces lecythidis, as well as Penicillium fuscoglaucum138,141. The nested nature of these giant TEs also implies that they are highly diverse. The ToxA gene that confers virulence to fungal wheat pathogens has been transferred horizontally between several species, and appears to exist within a ~14 kb TE named ToxhAT (Fig. 3)128. However, the ToxhAT locus itself has been identified within two larger Starship elements named Horizon and Sanctuary (Fig. 3)142,143. Considering TEs and larger Starships may be involved in the regulation and active exchange of genes involved in stress adaptation and pathogenicity within and between fungal taxa, further research being done to mine fungal genomes for their presence will be imperative in functionally characterizing these genetic factors across the fungal kingdom144.

Analyzing large repetitive regions of DNA as a whole, some of which include both non-coding DNA and ORFs, can also be a strategy for broader genome characterization. In an attempt to understand the plastic and rapidly adapting nature of the C. albicans genome, one study found that all segmental aneuploidy events — which are a critical part of C. albicans pathogenesis and drug resistance145 — occurred at long repeat sequences (anywhere from 65–6499bp in length)146. Here, these long repeat regions of DNA seemed to be necessary for many of the adaptive traits that C. albicans can harness to survive in a diverse set of challenging environments146. This is not entirely surprising as it has been long understood that large repeat regions of this kind seem to be relatively more amenable to rapid evolution in filamentous fungal plant pathogens, and tend to harbor virulence genes147. Indeed, new telomere-to-telomere sequencing platforms that can accurately annotate highly repetitive DNA that have already been applied in some fungi and oomycetes is a promising avenue for accurately identifying transposons and functional repeat elements at a genome-wide level148,149.

Pseudogenes and Dubious ORFs

Another component of ‘junk’ DNA is pseudogenes. Pseudogenes are genes that share close sequence similarities with canonical ORFs but have a disruptive mutation that precludes their transcription or leads to products with either reduced or abolished function150. For over a decade, the idea of pseudogenes encoding important regulatory products and having divergent impacts on cellular fitness has been demonstrated, but this research has been predominantly focused on human cells116. However, pseudogenes have been identified in many different fungal species, and have been a valuable means of understanding evolution and loss of pathogenicity150–152. A landmark study in S. cerevisiae from over two decades ago found active expression of some pseudogenes and posited that some may have impacts on the fungal stress rseponse153. Despite the advancements being made in identifying functional pseudogenes, there remains much to be characterized about the impacts of pseudogenes in most fungal species153. However, comparative analysis of pseudogene profiles between closely related species may be used to help identify genes responsible for any divergent functional capacities between the species151. Further, methods for applying CRISPR-based tools to study pseudogenes have been outlined and could be applied in fungal species154.

While not ‘junk’ DNA per se, dubious ORFs also represent an interesting area of functional genomic research. Dubious ORFs are ORFs that were originally suspected to not encode for a functional product, often based on the criteria that they are not conserved in any related species from the same taxa and that there is no experimental evidence of a resulting gene product155. ORFs labeled as dubious also tend to overlap with microproteins and sORFs, since both have traditionally been considered to not generate anything useful to the cell, even if some are known to still be translated155,156. Despite this categorization, an evaluative study on the accuracy of dubious ORF classifications in S. cerevisiae showed that many of these ORFs, indeed, produced detectable transcripts and/or products of translation157. The implications of this study have become clear with the emergence of a few notable examples in S. cerevisiae where the specific deletion of dubious ORFs resulted in prominent phenotypes associated with protein burden regulation and mitochondrial DNA maintenance158,159. This notion may also extend into other fungal taxa as it was recently shown in the human pathogen C. albicans that many previously labeled dubious ORFs are actively transcribed and translated, and that the rates of which seem to be differentially regulated during C. albicans’ morphological transitions156.

It has also been demonstrated that non-coding DNA is positively selected for in certain fungi, and does not always represent genetic relics with simple coincidental influence on gene expression. One study done on the fungal plant pathogen genus Colletotrichum illustrated many instances of positive selection for non-coding DNA during infection, which they suggest implies a regulatory role of this non-coding DNA160. It has elsewhere been proposed that the persistence of non-coding DNA in fungi is due to its function in harboring regulatory ncRNAs, or its role in intragenic DNA methylation, like in other eukaryotes161,162.

Conclusions and future perspectives

Many facets of fungal biology remain underexplored. However, the recent discovery of novel components of the fungal hidden genome may help us develop a more holistic understanding of fungal genetics and genomics. New technologies, particularly CRISPR-based techniques, have proven to be powerful tools to characterize these hidden components of the genome in non-fungal cell systems8. Harnessing new platforms developed for functional genomics in fungi may similarly help characterize important processes regulated by the non-coding genome and products of non-canonical expression. Numerous iterations of CRISPR technologies with disparate mechanisms of action have been demonstrated in diverse fungal taxa, including those that may be amenable to specific interrogation of hidden genome components that are nested within larger canonical sequences via differential expression163–165, those with a sensitive resolution for editing very small sequences of DNA166, and those that can discriminately target RNA molecules167. Thus, as more of these technologies are applied in fungi, a more robust characterization of non-canonical fungal genomes will emerge.

Acknowledgements

N.C.G. is supported by an Ontario Graduate Scholarship and R.S.S. is supported by a Tier II Canada Research Chair from the Natural Sciences and Engineering Research Council of Canada (NSERC). The authors would like to acknowledge other important research that was not cited due to space limitations.

Author contributions

N.C.G. conceptualized this work and made the figures. N.C.G. and R.S.S. wrote and revised the manuscript together.

Peer review

Peer review information

Nature Communications thanks Neeraj Chauhan, Asiya Gusa and the other, anonymous, reviewer for their contribution to the peer review of this work.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Adhikari S A high-stringency blueprint of the human proteome Nat. Commun. 2020 11 5301 10.1038/s41467-020-19045-9 33067450
Adhikari, S. et al. A high-stringency blueprint of the human proteome. Nat. Commun. 11, 5301 (2020).33067450
2. Wright BW Yi Z Weissman JS Chen J The dark proteome: translation from noncanonical open reading frames Trends Cell Biol. 2022 32 243 258 10.1016/j.tcb.2021.10.010 34844857
Wright, B. W., Yi, Z., Weissman, J. S. & Chen, J. The dark proteome: translation from noncanonical open reading frames. Trends Cell Biol. 32, 243–258 (2022).34844857
3. Ruiz Cuevas MV Most non-canonical proteins uniquely populate the proteome or immunopeptidome Cell Rep. 2021 34 108815 10.1016/j.celrep.2021.108815 33691108
Ruiz Cuevas, M. V. et al. Most non-canonical proteins uniquely populate the proteome or immunopeptidome. Cell Rep. 34, 108815 (2021).33691108
4. Prensner JR Noncanonical open reading frames encode functional proteins essential for cancer cell survival Nat. Biotechnol. 2021 39 697 704 10.1038/s41587-020-00806-2 33510483
Prensner, J. R. et al. Noncanonical open reading frames encode functional proteins essential for cancer cell survival. Nat. Biotechnol. 39, 697–704 (2021).33510483
5. Orr MW Mao Y Storz G Qian S-B Alternative ORFs and small ORFs: shedding light on the dark proteome Nucleic Acids Res. 2020 48 1029 1042 10.1093/nar/gkz734 31504789
Orr, M. W., Mao, Y., Storz, G. & Qian, S.-B. Alternative ORFs and small ORFs: shedding light on the dark proteome. Nucleic Acids Res. 48, 1029–1042 (2020).31504789
6. Lu S A hidden human proteome encoded by ‘non-coding’ genes Nucleic Acids Res. 2019 47 8111 8125 10.1093/nar/gkz646 31340039
Lu, S. et al. A hidden human proteome encoded by ‘non-coding’ genes. Nucleic Acids Res. 47, 8111–8125 (2019).31340039
7. Statello L Guo C-J Chen L-L Huarte M Author Correction: Gene regulation by long non-coding RNAs and its biological functions Nat. Rev. Mol. Cell Biol. 2021 22 159 10.1038/s41580-021-00330-4 33420484
Statello, L., Guo, C.-J., Chen, L.-L. & Huarte, M. Author Correction: Gene regulation by long non-coding RNAs and its biological functions. Nat. Rev. Mol. Cell Biol. 22, 159 (2021).33420484
8. Montalbano A Canver MC Sanjana NE High-throughput approaches to pinpoint function within the noncoding genome Mol. Cell 2017 68 44 59 10.1016/j.molcel.2017.09.017 28985510
Montalbano, A., Canver, M. C. & Sanjana, N. E. High-throughput approaches to pinpoint function within the noncoding genome. Mol. Cell 68, 44–59 (2017).28985510
9. Sieber P Comparative study on alternative splicing in human fungal pathogens suggests its involvement during host invasion Front. Microbiol. 2018 9 2313 10.3389/fmicb.2018.02313 30333805
Sieber, P. et al. Comparative study on alternative splicing in human fungal pathogens suggests its involvement during host invasion. Front. Microbiol. 9, 2313 (2018).30333805
10. Balarezo-Cisneros LN Functional and transcriptional profiling of non-coding RNAs in yeast reveal context-dependent phenotypes and in trans effects on the protein regulatory network PLoS Genet 2021 17 e1008761 10.1371/journal.pgen.1008761 33493158
Balarezo-Cisneros, L. N. et al. Functional and transcriptional profiling of non-coding RNAs in yeast reveal context-dependent phenotypes and in trans effects on the protein regulatory network. PLoS Genet 17, e1008761 (2021).33493158
11. Huberman LB Developing functional genomics platforms for fungi mSystems 2021 6 e0073021 10.1128/msystems.00730-21 34427501
Huberman, L. B. Developing functional genomics platforms for fungi. mSystems 6, e0073021 (2021).34427501
12. Hassel KR Brito-Estrada O Makarewich CA Microproteins: overlooked regulators of physiology and disease iScience 2023 26 106781 10.1016/j.isci.2023.106781 37213226
Hassel, K. R., Brito-Estrada, O. & Makarewich, C. A. Microproteins: overlooked regulators of physiology and disease. iScience 26, 106781 (2023).37213226
13. Martinez TF Accurate annotation of human protein-coding small open reading frames Nat. Chem. Biol. 2020 16 458 468 10.1038/s41589-019-0425-0 31819274
Martinez, T. F. et al. Accurate annotation of human protein-coding small open reading frames. Nat. Chem. Biol. 16, 458–468 (2020).31819274
14. Chu Q Regulation of the ER stress response by a mitochondrial microprotein Nat. Commun. 2019 10 4883 10.1038/s41467-019-12816-z 31653868
Chu, Q. et al. Regulation of the ER stress response by a mitochondrial microprotein. Nat. Commun. 10, 4883 (2019).31653868
15. Garai P Blanc-Potard A Uncovering small membrane proteins in pathogenic bacteria: Regulatory functions and therapeutic potential Mol. Microbiol. 2020 114 710 720 10.1111/mmi.14564 32602138
Garai, P. & Blanc-Potard, A. Uncovering small membrane proteins in pathogenic bacteria: Regulatory functions and therapeutic potential. Mol. Microbiol. 114, 710–720 (2020).32602138
16. Erpf PE Fraser JA The long history of the diverse roles of short ORFs: sPEPs in fungi Proteomics 2018 18 e1700219 10.1002/pmic.201700219 29465163
Erpf, P. E. & Fraser, J. A. The long history of the diverse roles of short ORFs: sPEPs in fungi. Proteomics 18, e1700219 (2018).29465163
17. Mat-Sharani S Firdaus-Raih M Computational discovery and annotation of conserved small open reading frames in fungal genomes BMC Bioinforma. 2019 19 551 10.1186/s12859-018-2550-2
Mat-Sharani, S. & Firdaus-Raih, M. Computational discovery and annotation of conserved small open reading frames in fungal genomes. BMC Bioinforma. 19, 551 (2019).
18. Yang H Li Q Stroup EK Wang S Ji Z Widespread stable noncanonical peptides identified by integrated analyses of ribosome profiling and ORF features Nat. Commun. 2024 15 1932 10.1038/s41467-024-46240-9 38431639
Yang, H., Li, Q., Stroup, E. K., Wang, S. & Ji, Z. Widespread stable noncanonical peptides identified by integrated analyses of ribosome profiling and ORF features. Nat. Commun. 15, 1932 (2024).38431639
19. Huraiova B Proteomic analysis of meiosis and characterization of novel short open reading frames in the fission yeast Schizosaccharomyces pombe Cell Cycle 2020 19 1777 1785 10.1080/15384101.2020.1779470 32594847
Huraiova, B. et al. Proteomic analysis of meiosis and characterization of novel short open reading frames in the fission yeast Schizosaccharomyces pombe. Cell Cycle 19, 1777–1785 (2020).32594847
20. Tollis S The microprotein Nrs1 rewires the G1/S transcriptional machinery during nitrogen limitation in budding yeast PLoS Biol. 2022 20 e3001548 10.1371/journal.pbio.3001548 35239649
Tollis, S. et al. The microprotein Nrs1 rewires the G1/S transcriptional machinery during nitrogen limitation in budding yeast. PLoS Biol. 20, e3001548 (2022).35239649
21. Sun Y Huang J Wang Z Pan N Wan C Identification of microproteins in Saccharomyces cerevisiae under different stress conditions J. Proteome Res. 2022 21 1939 1947 10.1021/acs.jproteome.2c00212 35838590
Sun, Y., Huang, J., Wang, Z., Pan, N. & Wan, C. Identification of microproteins in Saccharomyces cerevisiae under different stress conditions. J. Proteome Res. 21, 1939–1947 (2022).35838590
22. Chen J Pervasive functional translation of noncanonical human open reading frames Science 2020 367 1140 1146 10.1126/science.aay0262 32139545
Chen, J. et al. Pervasive functional translation of noncanonical human open reading frames. Science 367, 1140–1146 (2020).32139545
23. Valdivia-Francia F Sendoel A No country for old methods: new tools for studying microproteins iScience 2024 27 108972 10.1016/j.isci.2024.108972 38333695
Valdivia-Francia, F. & Sendoel, A. No country for old methods: new tools for studying microproteins. iScience 27, 108972 (2024).38333695
24. Treichel AJ Bazzini AA Casting CRISPR-Cas13d to fish for microprotein functions in animal development iScience 2022 25 105547 10.1016/j.isci.2022.105547 36444300
Treichel, A. J. & Bazzini, A. A. Casting CRISPR-Cas13d to fish for microprotein functions in animal development. iScience 25, 105547 (2022).36444300
25. Nilsen TW Graveley BR Expansion of the eukaryotic proteome by alternative splicing Nature 2010 463 457 463 10.1038/nature08909 20110989
Nilsen, T. W. & Graveley, B. R. Expansion of the eukaryotic proteome by alternative splicing. Nature 463, 457–463 (2010).20110989
26. Yeoh LM Alternative splicing is required for stage differentiation in malaria parasites Genome Biol. 2019 20 151 10.1186/s13059-019-1756-6 31370870
Yeoh, L. M. et al. Alternative splicing is required for stage differentiation in malaria parasites. Genome Biol. 20, 151 (2019).31370870
27. Melnyk JE Targeting a splicing-mediated drug resistance mechanism in prostate cancer by inhibiting transcriptional regulation by PKCβ1 Oncogene 2022 41 1536 1549 10.1038/s41388-022-02179-z 35087237
Melnyk, J. E. et al. Targeting a splicing-mediated drug resistance mechanism in prostate cancer by inhibiting transcriptional regulation by PKCβ1. Oncogene 41, 1536–1549 (2022).35087237
28. Muzafar S Sharma RD Chauhan N Prasad R Intron distribution and emerging role of alternative splicing in fungi FEMS Microbiol. Lett. 2021 368 fnab135 10.1093/femsle/fnab135 34718529
Muzafar, S., Sharma, R. D., Chauhan, N. & Prasad, R. Intron distribution and emerging role of alternative splicing in fungi. FEMS Microbiol. Lett. 368, fnab135 (2021).34718529
29. Gehrmann T Schizophyllum commune has an extensive and functional alternative splicing repertoire Sci. Rep. 2016 6 33640 10.1038/srep33640 27659065
Gehrmann, T. et al. Schizophyllum commune has an extensive and functional alternative splicing repertoire. Sci. Rep. 6, 33640 (2016).27659065
30. Fang S The occurrence and function of alternative splicing in fungi Fungal Biol. Rev. 2020 34 178 188 10.1016/j.fbr.2020.10.001
Fang, S. et al. The occurrence and function of alternative splicing in fungi. Fungal Biol. Rev. 34, 178–188 (2020).
31. Liu X-Y Fan L Gao J Shen X-Y Hou C-L Global identification of alternative splicing in Shiraia bambusicola and analysis of its regulation in hypocrellin biosynthesis Appl. Microbiol. Biotechnol. 2020 104 211 223 10.1007/s00253-019-10189-3 31768612
Liu, X.-Y., Fan, L., Gao, J., Shen, X.-Y. & Hou, C.-L. Global identification of alternative splicing in Shiraia bambusicola and analysis of its regulation in hypocrellin biosynthesis. Appl. Microbiol. Biotechnol. 104, 211–223 (2020).31768612
32. Leal J A splice variant of the Neurospora crassa hex-1 transcript, which encodes the major protein of the Woronin body, is modulated by extracellular phosphate and pH changes FEBS Lett. 2009 583 180 184 10.1016/j.febslet.2008.11.050 19071122
Leal, J. et al. A splice variant of the Neurospora crassa hex-1 transcript, which encodes the major protein of the Woronin body, is modulated by extracellular phosphate and pH changes. FEBS Lett. 583, 180–184 (2009).19071122
33. Trevisan GL Transcription of Aspergillus nidulans pacC is modulated by alternative RNA splicing of palB FEBS Lett. 2011 585 3442 3445 10.1016/j.febslet.2011.09.037 21985967
Trevisan, G. L. et al. Transcription of Aspergillus nidulans pacC is modulated by alternative RNA splicing of palB. FEBS Lett. 585, 3442–3445 (2011).21985967
34. Strijbis K van den Burg J Visser WF van den Berg M Distel B Alternative splicing directs dual localization of Candida albicans 6-phosphogluconate dehydrogenase to cytosol and peroxisomes FEMS Yeast Res. 2012 12 61 68 10.1111/j.1567-1364.2011.00761.x 22094058
Strijbis, K., van den Burg, J., Visser, W. F., van den Berg, M. & Distel, B. Alternative splicing directs dual localization of Candida albicans 6-phosphogluconate dehydrogenase to cytosol and peroxisomes. FEMS Yeast Res. 12, 61–68 (2012).22094058
35. Juneau K Nislow C Davis RW Alternative splicing of PTC7 in Saccharomyces cerevisiae determines protein localization Genetics 2009 183 185 194 10.1534/genetics.109.105155 19564484
Juneau, K., Nislow, C. & Davis, R. W. Alternative splicing of PTC7 in Saccharomyces cerevisiae determines protein localization. Genetics 183, 185–194 (2009).19564484
36. Parenteau J Introns are mediators of cell response to starvation Nature 2019 565 612 617 10.1038/s41586-018-0859-7 30651641
Parenteau, J. et al. Introns are mediators of cell response to starvation. Nature 565, 612–617 (2019).30651641
37. Morgan JT Fink GR Bartel DP Excised linear introns regulate growth in yeast Nature 2019 565 606 611 10.1038/s41586-018-0828-1 30651636
Morgan, J. T., Fink, G. R. & Bartel, D. P. Excised linear introns regulate growth in yeast. Nature 565, 606–611 (2019).30651636
38. Guo N Alternative transcription start site selection in Mr-OPY2 controls lifestyle transitions in the fungus Metarhizium robertsii Nat. Commun. 2017 8 1565 10.1038/s41467-017-01756-1 29146899
Guo, N. et al. Alternative transcription start site selection in Mr-OPY2 controls lifestyle transitions in the fungus Metarhizium robertsii. Nat. Commun. 8, 1565 (2017).29146899
39. Sibthorp C Transcriptome analysis of the filamentous fungus Aspergillus nidulans directed to the global identification of promoters BMC Genomics 2013 14 847 10.1186/1471-2164-14-847 24299161
Sibthorp, C. et al. Transcriptome analysis of the filamentous fungus Aspergillus nidulans directed to the global identification of promoters. BMC Genomics 14, 847 (2013).24299161
40. Dang TTV Alternative TSS use is widespread in Cryptococcus fungi in response to environmental cues and regulated genome-wide by the transcription factor Tur1 bioRxiv 2024 10.1101/2023.07.18.549460 39211176
Dang, T. T. V. et al. Alternative TSS use is widespread in Cryptococcus fungi in response to environmental cues and regulated genome-wide by the transcription factor Tur1. bioRxiv10.1101/2023.07.18.549460 (2024). 2023.07.18.549460.39211176
41. Grützmann K Fungal alternative splicing is associated with multicellular complexity and virulence: a genome-wide multi-species study DNA Res. 2014 21 27 39 10.1093/dnares/dst038 24122896
Grützmann, K. et al. Fungal alternative splicing is associated with multicellular complexity and virulence: a genome-wide multi-species study. DNA Res. 21, 27–39 (2014).24122896
42. Gao X A glycine-rich protein MoGrp1 functions as a novel splicing factor to regulate fungal virulence and growth in Magnaporthe oryzae Phytopathol. Res. 2019 1 1 15 10.1186/s42483-018-0007-1
Gao, X. et al. A glycine-rich protein MoGrp1 functions as a novel splicing factor to regulate fungal virulence and growth in Magnaporthe oryzae. Phytopathol. Res. 1, 1–15 (2019).
43. Jayaguru P Raghunathan M Group I intron renders differential susceptibility of Candida albicans to Bleomycin Mol. Biol. Rep. 2007 34 11 17 10.1007/s11033-006-9002-1 17115251
Jayaguru, P. & Raghunathan, M. Group I intron renders differential susceptibility of Candida albicans to Bleomycin. Mol. Biol. Rep. 34, 11–17 (2007).17115251
44. Mendes NS Silva PM Silva-Rocha R Martinez-Rossi NM Rossi A Pre-mRNA splicing is modulated by antifungal drugs in the filamentous fungus Neurospora crassa FEBS Open Bio 2016 6 358 368 10.1002/2211-5463.12047 27239448
Mendes, N. S., Silva, P. M., Silva-Rocha, R., Martinez-Rossi, N. M. & Rossi, A. Pre-mRNA splicing is modulated by antifungal drugs in the filamentous fungus Neurospora crassa. FEBS Open Bio 6, 358–368 (2016).27239448
45. Muzafar S Identification of genomewide alternative splicing events in sequential, isogenic clinical isolates of Candida albicans reveals a novel mechanism of drug resistance and tolerance to cellular stresses mSphere 2020 5 e00608 e00620 10.1128/msphere.00608-20 32817456
Muzafar, S. et al. Identification of genomewide alternative splicing events in sequential, isogenic clinical isolates of Candida albicans reveals a novel mechanism of drug resistance and tolerance to cellular stresses. mSphere 5, e00608–e00620 (2020).32817456
46. Tharappel AM Calcimycin inhibits Cryptococcus neoformans in vitro and in vivo by targeting the Prp8 intein splicing ACS Infect. Dis. 2022 8 1851 1868 10.1021/acsinfecdis.2c00137 35948057
Tharappel, A. M. et al. Calcimycin inhibits Cryptococcus neoformans in vitro and in vivo by targeting the Prp8 intein splicing. ACS Infect. Dis. 8, 1851–1868 (2022).35948057
47. Li Z Small-molecule inhibitors for the Prp8 intein as antifungal agents Proc. Natl. Acad. Sci. USA 2021 118 e2008815118 10.1073/pnas.2008815118 33397721
Li, Z. et al. Small-molecule inhibitors for the Prp8 intein as antifungal agents. Proc. Natl. Acad. Sci. USA 118, e2008815118 (2021).33397721
48. Su Y Comprehensive assessment of mRNA isoform detection methods for long-read sequencing data Nat. Commun. 2024 15 3972 10.1038/s41467-024-48117-3 38730241
Su, Y. et al. Comprehensive assessment of mRNA isoform detection methods for long-read sequencing data. Nat. Commun. 15, 3972 (2024).38730241
49. Xiao M-S Genome-scale exon perturbation screens uncover exons critical for cell fitness Mol. Cell 2024 84 2553 2572.e19 10.1016/j.molcel.2024.05.024 38917794
Xiao, M.-S. et al. Genome-scale exon perturbation screens uncover exons critical for cell fitness. Mol. Cell 84, 2553–2572.e19 (2024).38917794
50. Li JD Taipale M Blencowe BJ Efficient, specific, and combinatorial control of endogenous exon splicing with dCasRx-RBM25 Mol. Cell 2024 84 2573 2589.e5 10.1016/j.molcel.2024.05.028 38917795
Li, J. D., Taipale, M. & Blencowe, B. J. Efficient, specific, and combinatorial control of endogenous exon splicing with dCasRx-RBM25. Mol. Cell 84, 2573–2589.e5 (2024).38917795
51. Zhang P Wu W Chen Q Chen M Non-coding RNAs and their integrated networks J. Integr. Bioinform. 2019 16 20190027 10.1515/jib-2019-0027 31301674
Zhang, P., Wu, W., Chen, Q. & Chen, M. Non-coding RNAs and their integrated networks. J. Integr. Bioinform. 16, 20190027 (2019).31301674
52. Mattick JS Long non-coding RNAs: definitions, functions, challenges and recommendations Nat. Rev. Mol. Cell Biol. 2023 24 430 447 10.1038/s41580-022-00566-8 36596869
Mattick, J. S. et al. Long non-coding RNAs: definitions, functions, challenges and recommendations. Nat. Rev. Mol. Cell Biol. 24, 430–447 (2023).36596869
53. Shang R Lee S Senavirathne G Lai EC microRNAs in action: biogenesis, function and regulation Nat. Rev. Genet. 2023 24 816 833 10.1038/s41576-023-00611-y 37380761
Shang, R., Lee, S., Senavirathne, G. & Lai, E. C. microRNAs in action: biogenesis, function and regulation. Nat. Rev. Genet. 24, 816–833 (2023).37380761
54. Kristensen LS The biogenesis, biology and characterization of circular RNAs Nat. Rev. Genet. 2019 20 675 691 10.1038/s41576-019-0158-7 31395983
Kristensen, L. S. et al. The biogenesis, biology and characterization of circular RNAs. Nat. Rev. Genet. 20, 675–691 (2019).31395983
55. Yang Y The roles of miRNA, lncRNA and circRNA in the development of osteoporosis Biol. Res. 2020 53 40 10.1186/s40659-020-00309-z 32938500
Yang, Y. et al. The roles of miRNA, lncRNA and circRNA in the development of osteoporosis. Biol. Res. 53, 40 (2020).32938500
56. Li C Crosstalk of mRNA, miRNA, lncRNA, and circRNA and their regulatory pattern in pulmonary fibrosis Mol. Ther. Nucleic Acids 2019 18 204 218 10.1016/j.omtn.2019.08.018 31561125
Li, C. et al. Crosstalk of mRNA, miRNA, lncRNA, and circRNA and their regulatory pattern in pulmonary fibrosis. Mol. Ther. Nucleic Acids 18, 204–218 (2019).31561125
57. Eichner H Karlsson J Loh E The emerging role of bacterial regulatory RNAs in disease Trends Microbiol. 2022 30 959 972 10.1016/j.tim.2022.03.007 35379550
Eichner, H., Karlsson, J. & Loh, E. The emerging role of bacterial regulatory RNAs in disease. Trends Microbiol. 30, 959–972 (2022).35379550
58. Li J Liu X Yin Z Hu Z Zhang K-Q An overview on identification and regulatory mechanisms of long non-coding RNAs in fungi Front. Microbiol. 2021 12 638617 10.3389/fmicb.2021.638617 33995298
Li, J., Liu, X., Yin, Z., Hu, Z. & Zhang, K.-Q. An overview on identification and regulatory mechanisms of long non-coding RNAs in fungi. Front. Microbiol. 12, 638617 (2021).33995298
59. Johnsson P Lipovich L Grandér D Morris KV Evolutionary conservation of long non-coding RNAs; sequence, structure, function Biochim. Biophys. Acta 2014 1840 1063 1071 10.1016/j.bbagen.2013.10.035 24184936
Johnsson, P., Lipovich, L., Grandér, D. & Morris, K. V. Evolutionary conservation of long non-coding RNAs; sequence, structure, function. Biochim. Biophys. Acta 1840, 1063–1071 (2014).24184936
60. Dhingra S Role of non-coding RNAs in fungal pathogenesis and antifungal drug responses Curr. Clin. Microbiol. Rep. 2020 7 133 141 10.1007/s40588-020-00151-7
Dhingra, S. Role of non-coding RNAs in fungal pathogenesis and antifungal drug responses. Curr. Clin. Microbiol. Rep. 7, 133–141 (2020).
61. Wilkinson D Long noncoding RNAs in yeast cells and differentiated subpopulations of yeast colonies and biofilms Oxid. Med. Cell. Longev. 2018 2018 4950591 10.1155/2018/4950591 29765496
Wilkinson, D. et al. Long noncoding RNAs in yeast cells and differentiated subpopulations of yeast colonies and biofilms. Oxid. Med. Cell. Longev. 2018, 4950591 (2018).29765496
62. Parker S Large-scale profiling of noncoding RNA function in yeast PLoS Genet 2018 14 e1007253 10.1371/journal.pgen.1007253 29529031
Parker, S. et al. Large-scale profiling of noncoding RNA function in yeast. PLoS Genet 14, e1007253 (2018).29529031
63. Kyriakou D Functional characterisation of long intergenic non-coding RNAs through genetic interaction profiling in Saccharomyces cerevisiae BMC Biol. 2016 14 106 10.1186/s12915-016-0325-7 27927215
Kyriakou, D. et al. Functional characterisation of long intergenic non-coding RNAs through genetic interaction profiling in Saccharomyces cerevisiae. BMC Biol. 14, 106 (2016).27927215
64. Novačić A Vučenović I Primig M Stuparević I Non-coding RNAs as cell wall regulators in Saccharomyces cerevisiae Crit. Rev. Microbiol. 2020 46 15 25 10.1080/1040841X.2020.1715340 31994960
Novačić, A., Vučenović, I., Primig, M. & Stuparević, I. Non-coding RNAs as cell wall regulators in Saccharomyces cerevisiae. Crit. Rev. Microbiol. 46, 15–25 (2020).31994960
65. Atkinson SR Long noncoding RNA repertoire and targeting by nuclear exosome, cytoplasmic exonuclease, and RNAi in fission yeast RNA 2018 24 1195 1213 10.1261/rna.065524.118 29914874
Atkinson, S. R. et al. Long noncoding RNA repertoire and targeting by nuclear exosome, cytoplasmic exonuclease, and RNAi in fission yeast. RNA 24, 1195–1213 (2018).29914874
66. Ard R Tong P Allshire RC Long non-coding RNA-mediated transcriptional interference of a permease gene confers drug tolerance in fission yeast Nat. Commun. 2014 5 5576 10.1038/ncomms6576 25428589
Ard, R., Tong, P. & Allshire, R. C. Long non-coding RNA-mediated transcriptional interference of a permease gene confers drug tolerance in fission yeast. Nat. Commun. 5, 5576 (2014).25428589
67. Sun W-H Wang Y-Z Xu Y Yu X-W Genome-wide analysis of long non-coding RNAs in Pichia pastoris during stress by RNA sequencing Genomics 2019 111 398 406 10.1016/j.ygeno.2018.02.016 29496514
Sun, W.-H., Wang, Y.-Z., Xu, Y. & Yu, X.-W. Genome-wide analysis of long non-coding RNAs in Pichia pastoris during stress by RNA sequencing. Genomics 111, 398–406 (2019).29496514
68. Cemel IA Ha N Schermann G Yonekawa S Brunner M The coding and noncoding transcriptome of Neurospora crassa BMC Genomics 2017 18 978 10.1186/s12864-017-4360-8 29258423
Cemel, I. A., Ha, N., Schermann, G., Yonekawa, S. & Brunner, M. The coding and noncoding transcriptome of Neurospora crassa. BMC Genomics 18, 978 (2017).29258423
69. Linde J Defining the transcriptomic landscape of Candida glabrata by RNA-Seq Nucleic Acids Res. 2015 43 1392 1406 10.1093/nar/gku1357 25586221
Linde, J. et al. Defining the transcriptomic landscape of Candida glabrata by RNA-Seq. Nucleic Acids Res. 43, 1392–1406 (2015).25586221
70. Donovan PD Schröder MS Higgins DG Butler G Identification of non-coding RNAs in the Candida parapsilosis species group PLoS One 2016 11 e0163235 10.1371/journal.pone.0163235 27658249
Donovan, P. D., Schröder, M. S., Higgins, D. G. & Butler, G. Identification of non-coding RNAs in the Candida parapsilosis species group. PLoS One 11, e0163235 (2016).27658249
71. Sellam A Experimental annotation of the human pathogen Candida albicans coding and noncoding transcribed regions using high-resolution tiling arrays Genome Biol. 2010 11 R71 10.1186/gb-2010-11-7-r71 20618945
Sellam, A. et al. Experimental annotation of the human pathogen Candida albicans coding and noncoding transcribed regions using high-resolution tiling arrays. Genome Biol. 11, R71 (2010).20618945
72. Mathur K Singh B Puria R Nain V In silico genome wide identification of long non-coding RNAs differentially expressed during Candida auris host pathogenesis Arch. Microbiol. 2024 206 253 10.1007/s00203-024-03969-7 38727738
Mathur, K., Singh, B., Puria, R. & Nain, V. In silico genome wide identification of long non-coding RNAs differentially expressed during Candida auris host pathogenesis. Arch. Microbiol. 206, 253 (2024).38727738
73. Gao J LncRNA DINOR is a virulence factor and global regulator of stress responses in Candida auris Nat. Microbiol 2021 6 842 851 10.1038/s41564-021-00915-x 34083769
Gao, J. et al. LncRNA DINOR is a virulence factor and global regulator of stress responses in Candida auris. Nat. Microbiol 6, 842–851 (2021).34083769
74. Hovhannisyan H Gabaldón T The long non-coding RNA landscape of Candida yeast pathogens Nat. Commun. 2021 12 7317 10.1038/s41467-021-27635-4 34916523
Hovhannisyan, H. & Gabaldón, T. The long non-coding RNA landscape of Candida yeast pathogens. Nat. Commun. 12, 7317 (2021).34916523
75. Chacko N The lncRNA RZE1 controls cryptococcal morphological transition PLoS Genet 2015 11 e1005692 10.1371/journal.pgen.1005692 26588844
Chacko, N. et al. The lncRNA RZE1 controls cryptococcal morphological transition. PLoS Genet 11, e1005692 (2015).26588844
76. Jiménez-Gómez I Surviving in the brine: a multi-omics approach for understanding the physiology of the halophile fungus Aspergillus sydowii at saturated NaCl concentration Front. Microbiol. 2022 13 840408 10.3389/fmicb.2022.840408 35586858
Jiménez-Gómez, I. et al. Surviving in the brine: a multi-omics approach for understanding the physiology of the halophile fungus Aspergillus sydowii at saturated NaCl concentration. Front. Microbiol. 13, 840408 (2022).35586858
77. Davati N Ghorbani A Discovery of long non-coding RNAs in Aspergillus flavus response to water activity, CO2 concentration, and temperature changes Sci. Rep. 2023 13 10330 10.1038/s41598-023-37236-4 37365206
Davati, N. & Ghorbani, A. Discovery of long non-coding RNAs in Aspergillus flavus response to water activity, CO2 concentration, and temperature changes. Sci. Rep. 13, 10330 (2023).37365206
78. Kim W Miguel-Rojas C Wang J Townsend JP Trail F Developmental dynamics of long noncoding RNA expression during sexual fruiting body formation in Fusarium graminearum MBio 2018 9 e01292 1 10.1128/mBio.01292-18 30108170
Kim, W., Miguel-Rojas, C., Wang, J., Townsend, J. P. & Trail, F. Developmental dynamics of long noncoding RNA expression during sexual fruiting body formation in Fusarium graminearum. MBio 9, e01292–1 (2018).30108170
79. Wang Z Jiang Y Wu H Xie X Huang B Genome-wide identification and functional prediction of long non-coding RNAs involved in the heat stress response in Metarhizium robertsii Front. Microbiol. 2019 10 2336 10.3389/fmicb.2019.02336 31649657
Wang, Z., Jiang, Y., Wu, H., Xie, X. & Huang, B. Genome-wide identification and functional prediction of long non-coding RNAs involved in the heat stress response in Metarhizium robertsii. Front. Microbiol. 10, 2336 (2019).31649657
80. Guo R First identification of long non-coding RNAs in fungal parasite Nosema ceranae Apidologie 2018 49 660 670 10.1007/s13592-018-0593-z
Guo, R. et al. First identification of long non-coding RNAs in fungal parasite Nosema ceranae. Apidologie 49, 660–670 (2018).
81. Wang Y XRN1-associated long non-coding RNAs may contribute to fungal virulence and sexual development in entomopathogenic fungus Cordyceps militaris Pest Manag. Sci. 2019 75 3302 3311 10.1002/ps.5453 31025499
Wang, Y. et al. XRN1-associated long non-coding RNAs may contribute to fungal virulence and sexual development in entomopathogenic fungus Cordyceps militaris. Pest Manag. Sci. 75, 3302–3311 (2019).31025499
82. Dang Y Yang Q Xue Z Liu Y RNA interference in fungi: pathways, functions, and applications Eukaryot. Cell 2011 10 1148 1155 10.1128/EC.05109-11 21724934
Dang, Y., Yang, Q., Xue, Z. & Liu, Y. RNA interference in fungi: pathways, functions, and applications. Eukaryot. Cell 10, 1148–1155 (2011).21724934
83. Zhou J Identification of microRNA-like RNAs in a plant pathogenic fungus Sclerotinia sclerotiorum by high-throughput sequencing Mol. Genet. Genomics 2012 287 275 282 10.1007/s00438-012-0678-8 22314800
Zhou, J. et al. Identification of microRNA-like RNAs in a plant pathogenic fungus Sclerotinia sclerotiorum by high-throughput sequencing. Mol. Genet. Genomics 287, 275–282 (2012).22314800
84. Lee H-C Diverse pathways generate microRNA-like RNAs and Dicer-independent small interfering RNAs in fungi Mol. Cell 2010 38 803 814 10.1016/j.molcel.2010.04.005 20417140
Lee, H.-C. et al. Diverse pathways generate microRNA-like RNAs and Dicer-independent small interfering RNAs in fungi. Mol. Cell 38, 803–814 (2010).20417140
85. Lau SKP Identification of microRNA-like RNAs in mycelial and yeast phases of the thermal dimorphic fungus Penicillium marneffei PLoS Negl. Trop. Dis. 2013 7 e2398 10.1371/journal.pntd.0002398 23991243
Lau, S. K. P. et al. Identification of microRNA-like RNAs in mycelial and yeast phases of the thermal dimorphic fungus Penicillium marneffei. PLoS Negl. Trop. Dis. 7, e2398 (2013).23991243
86. Özkan S Mohorianu I Xu P Dalmay T Coutts RHA Profile and functional analysis of small RNAs derived from Aspergillus fumigatus infected with double-stranded RNA mycoviruses BMC Genomics 2017 18 416 10.1186/s12864-017-3773-8 28558690
Özkan, S., Mohorianu, I., Xu, P., Dalmay, T. & Coutts, R. H. A. Profile and functional analysis of small RNAs derived from Aspergillus fumigatus infected with double-stranded RNA mycoviruses. BMC Genomics 18, 416 (2017).28558690
87. Kang K Identification of microRNA-Like RNAs in the filamentous fungus Trichoderma reesei by solexa sequencing PLoS One 2013 8 e76288 10.1371/journal.pone.0076288 24098464
Kang, K. et al. Identification of microRNA-Like RNAs in the filamentous fungus Trichoderma reesei by solexa sequencing. PLoS One 8, e76288 (2013).24098464
88. Zhou Q Wang Z Zhang J Meng H Huang B Genome-wide identification and profiling of microRNA-like RNAs from Metarhizium anisopliae during development Fungal Biol. 2012 116 1156 1162 10.1016/j.funbio.2012.09.001 23153806
Zhou, Q., Wang, Z., Zhang, J., Meng, H. & Huang, B. Genome-wide identification and profiling of microRNA-like RNAs from Metarhizium anisopliae during development. Fungal Biol. 116, 1156–1162 (2012).23153806
89. Wang L Integrated microRNA and mRNA analysis in the pathogenic filamentous fungus Trichophyton rubrum BMC Genomics 2018 19 933 10.1186/s12864-018-5316-3 30547762
Wang, L. et al. Integrated microRNA and mRNA analysis in the pathogenic filamentous fungus Trichophyton rubrum. BMC Genomics 19, 933 (2018).30547762
90. Iracane E Identification of an active RNAi pathway in Candida albicans Proc. Natl Acad. Sci. USA 2024 121 e2315926121 10.1073/pnas.2315926121 38625945
Iracane, E. et al. Identification of an active RNAi pathway in Candida albicans. Proc. Natl Acad. Sci. USA 121, e2315926121 (2024).38625945
91. Jiang N Yang Y Janbon G Pan J Zhu X Identification and functional demonstration of miRNAs in the fungus Cryptococcus neoformans PLoS One 2012 7 e52734 10.1371/journal.pone.0052734 23300755
Jiang, N., Yang, Y., Janbon, G., Pan, J. & Zhu, X. Identification and functional demonstration of miRNAs in the fungus Cryptococcus neoformans. PLoS One 7, e52734 (2012).23300755
92. Calo S Antifungal drug resistance evoked via RNAi-dependent epimutations Nature 2014 513 555 558 10.1038/nature13575 25079329
Calo, S. et al. Antifungal drug resistance evoked via RNAi-dependent epimutations. Nature 513, 555–558 (2014).25079329
93. Mathur M Nair A Kadoo N Plant-pathogen interactions: microRNA-mediated trans-kingdom gene regulation in fungi and their host plants Genomics 2020 112 3021 3035 10.1016/j.ygeno.2020.05.021 32454170
Mathur, M., Nair, A. & Kadoo, N. Plant-pathogen interactions: microRNA-mediated trans-kingdom gene regulation in fungi and their host plants. Genomics 112, 3021–3035 (2020).32454170
94. Xu M A fungal microRNA-like RNA subverts host immunity and facilitates pathogen infection by silencing two host receptor-like kinase genes N. Phytol. 2022 233 2503 2519 10.1111/nph.17945
Xu, M. et al. A fungal microRNA-like RNA subverts host immunity and facilitates pathogen infection by silencing two host receptor-like kinase genes. N. Phytol. 233, 2503–2519 (2022).
95. He B Fungal small RNAs ride in extracellular vesicles to enter plant cells through clathrin-mediated endocytosis Nat. Commun. 2023 14 4383 10.1038/s41467-023-40093-4 37474601
He, B. et al. Fungal small RNAs ride in extracellular vesicles to enter plant cells through clathrin-mediated endocytosis. Nat. Commun. 14, 4383 (2023).37474601
96. Chen R Exploring microRNA-like small RNAs in the filamentous fungus Fusarium oxysporum PLoS One 2014 9 e104956 10.1371/journal.pone.0104956 25141304
Chen, R. et al. Exploring microRNA-like small RNAs in the filamentous fungus Fusarium oxysporum. PLoS One 9, e104956 (2014).25141304
97. Johnson NR Larrondo LF Álvarez JM Vidal EA Comprehensive re-analysis of hairpin small RNAs in fungi reveals loci with conserved links Elife 2022 11 e83691 10.7554/eLife.83691 36484778
Johnson, N. R., Larrondo, L. F., Álvarez, J. M. & Vidal, E. A. Comprehensive re-analysis of hairpin small RNAs in fungi reveals loci with conserved links. Elife 11, e83691 (2022).36484778
98. Wang PL Circular RNA is expressed across the eukaryotic tree of life PLoS One 2014 9 e90859 10.1371/journal.pone.0090859 24609083
Wang, P. L. et al. Circular RNA is expressed across the eukaryotic tree of life. PLoS One 9, e90859 (2014).24609083
99. Yuan J Wang Z Xing J Yang Q Chen X-L Genome-wide Identification and characterization of circular RNAs in the rice blast fungus Magnaporthe oryzae Sci. Rep. 2018 8 6757 10.1038/s41598-018-25242-w 29713025
Yuan, J., Wang, Z., Xing, J., Yang, Q. & Chen, X.-L. Genome-wide Identification and characterization of circular RNAs in the rice blast fungus Magnaporthe oryzae. Sci. Rep. 8, 6757 (2018).29713025
100. Guo R Systematic investigation of circular RNAs in Ascosphaera apis, a fungal pathogen of honeybee larvae Gene 2018 678 17 22 10.1016/j.gene.2018.07.076 30077766
Guo, R. et al. Systematic investigation of circular RNAs in Ascosphaera apis, a fungal pathogen of honeybee larvae. Gene 678, 17–22 (2018).30077766
101. Guo R Genome-wide identification of circular RNAs in fungal parasite Nosema ceranae Curr. Microbiol. 2018 75 1655 1660 10.1007/s00284-018-1576-z 30269253
Guo, R. et al. Genome-wide identification of circular RNAs in fungal parasite Nosema ceranae. Curr. Microbiol. 75, 1655–1660 (2018).30269253
102. Shao J Identification and characterization of circular RNAs in Ganoderma lucidum Sci. Rep. 2019 9 16522 10.1038/s41598-019-52932-w 31712736
Shao, J. et al. Identification and characterization of circular RNAs in Ganoderma lucidum. Sci. Rep. 9, 16522 (2019).31712736
103. Cao X Genome-wide identification and functional analysis of circRNAs in Trichophyton rubrum conidial and mycelial stages BMC Genomics 2022 23 21 10.1186/s12864-021-08184-y 34983376
Cao, X. et al. Genome-wide identification and functional analysis of circRNAs in Trichophyton rubrum conidial and mycelial stages. BMC Genomics 23, 21 (2022).34983376
104. Vromman M Large-scale benchmarking of circRNA detection tools reveals large differences in sensitivity but not in precision Nat. Methods 2023 20 1159 1169 10.1038/s41592-023-01944-6 37443337
Vromman, M. et al. Large-scale benchmarking of circRNA detection tools reveals large differences in sensitivity but not in precision. Nat. Methods 20, 1159–1169 (2023).37443337
105. Li M Liang C LncDC: a machine learning-based tool for long non-coding RNA detection from RNA-Seq data Sci. Rep. 2022 12 19083 10.1038/s41598-022-22082-7 36351980
Li, M. & Liang, C. LncDC: a machine learning-based tool for long non-coding RNA detection from RNA-Seq data. Sci. Rep. 12, 19083 (2022).36351980
106. Bester AC An integrated genome-wide CRISPRa approach to functionalize lncRNAs in drug resistance Cell 2018 173 649 664.e20 10.1016/j.cell.2018.03.052 29677511
Bester, A. C. et al. An integrated genome-wide CRISPRa approach to functionalize lncRNAs in drug resistance. Cell 173, 649–664.e20 (2018).29677511
107. Wallace J Genome-wide CRISPR-Cas9 screen identifies MicroRNAs that regulate myeloid leukemia cell growth PLoS One 2016 11 e0153689 10.1371/journal.pone.0153689 27081855
Wallace, J. et al. Genome-wide CRISPR-Cas9 screen identifies MicroRNAs that regulate myeloid leukemia cell growth. PLoS One 11, e0153689 (2016).27081855
108. Li S Screening for functional circular RNAs using the CRISPR-Cas13 system Nat. Methods 2021 18 51 59 10.1038/s41592-020-01011-4 33288960
Li, S. et al. Screening for functional circular RNAs using the CRISPR-Cas13 system. Nat. Methods 18, 51–59 (2021).33288960
109. Montero JJ Genome-scale pan-cancer interrogation of lncRNA dependencies using CasRx Nat. Methods 2024 21 584 596 10.1038/s41592-024-02190-0 38409225
Montero, J. J. et al. Genome-scale pan-cancer interrogation of lncRNA dependencies using CasRx. Nat. Methods 21, 584–596 (2024).38409225
110. Uthayakumar D Sharma J Wensing L Shapiro RS CRISPR-based genetic manipulation of Candida species: historical perspectives and current approaches Front Genome Ed. 2020 2 606281 10.3389/fgeed.2020.606281 34713231
Uthayakumar, D., Sharma, J., Wensing, L. & Shapiro, R. S. CRISPR-based genetic manipulation of Candida species: historical perspectives and current approaches. Front Genome Ed. 2, 606281 (2020).34713231
111. Ross RL Santiago-Tirado FH Advanced genetic techniques in fungal pathogen research mSphere 2024 9 e0064323 10.1128/msphere.00643-23 38470131
Ross, R. L. & Santiago-Tirado, F. H. Advanced genetic techniques in fungal pathogen research. mSphere 9, e0064323 (2024).38470131
112. Pang B van Weerd JH Hamoen FL Snyder MP Identification of non-coding silencer elements and their regulation of gene expression Nat. Rev. Mol. Cell Biol. 2023 24 383 395 10.1038/s41580-022-00549-9 36344659
Pang, B., van Weerd, J. H., Hamoen, F. L. & Snyder, M. P. Identification of non-coding silencer elements and their regulation of gene expression. Nat. Rev. Mol. Cell Biol. 24, 383–395 (2023).36344659
113. Palazzo AF Gregory TR The case for junk DNA PLoS Genet 2014 10 e1004351 10.1371/journal.pgen.1004351 24809441
Palazzo, A. F. & Gregory, T. R. The case for junk DNA. PLoS Genet 10, e1004351 (2014).24809441
114. Eddy SR The C-value paradox, junk DNA and ENCODE Curr. Biol. 2012 22 R898 R899 10.1016/j.cub.2012.10.002 23137679
Eddy, S. R. The C-value paradox, junk DNA and ENCODE. Curr. Biol. 22, R898–R899 (2012).23137679
115. Ecker JR Genomics: ENCODE explained Nature 2012 489 52 55 10.1038/489052a 22955614
Ecker, J. R. et al. Genomics: ENCODE explained. Nature 489, 52–55 (2012).22955614
116. Pink RC Pseudogenes: pseudo-functional or key regulators in health and disease? RNA 2011 17 792 798 10.1261/rna.2658311 21398401
Pink, R. C. et al. Pseudogenes: pseudo-functional or key regulators in health and disease? RNA 17, 792–798 (2011).21398401
117. Brocks D DNMT and HDAC inhibitors induce cryptic transcription start sites encoded in long terminal repeats Nat. Genet. 2017 49 1052 1060 10.1038/ng.3889 28604729
Brocks, D. et al. DNMT and HDAC inhibitors induce cryptic transcription start sites encoded in long terminal repeats. Nat. Genet. 49, 1052–1060 (2017).28604729
118. Hughes TR de Boer CG Mapping yeast transcriptional networks Genetics 2013 195 9 36 10.1534/genetics.113.153262 24018767
Hughes, T. R. & de Boer, C. G. Mapping yeast transcriptional networks. Genetics 195, 9–36 (2013).24018767
119. Daguerre Y Regulatory networks underlying mycorrhizal development delineated by genome-wide expression profiling and functional analysis of the transcription factor repertoire of the plant symbiotic fungus Laccaria bicolor BMC Genomics 2017 18 737 10.1186/s12864-017-4114-7 28923004
Daguerre, Y. et al. Regulatory networks underlying mycorrhizal development delineated by genome-wide expression profiling and functional analysis of the transcription factor repertoire of the plant symbiotic fungus Laccaria bicolor. BMC Genomics 18, 737 (2017).28923004
120. Kita R Venkataram S Zhou Y Fraser HB High-resolution mapping of cis-regulatory variation in budding yeast Proc. Natl. Acad. Sci. USA 2017 114 E10736 E10744 10.1073/pnas.1717421114 29183975
Kita, R., Venkataram, S., Zhou, Y. & Fraser, H. B. High-resolution mapping of cis-regulatory variation in budding yeast. Proc. Natl. Acad. Sci. USA 114, E10736–E10744 (2017).29183975
121. Renganaath K Systematic identification of cis-regulatory variants that cause gene expression differences in a yeast cross Elife 2020 9 e62669 10.7554/eLife.62669 33179598
Renganaath, K. et al. Systematic identification of cis-regulatory variants that cause gene expression differences in a yeast cross. Elife 9, e62669 (2020).33179598
122. Shih C-H Fay J Cis-regulatory variants affect gene expression dynamics in yeast Elife 2021 10 e68469 10.7554/eLife.68469 34369376
Shih, C.-H. & Fay, J. Cis-regulatory variants affect gene expression dynamics in yeast. Elife 10, e68469 (2021).34369376
123. Simms TA TFIIIC binding sites function as both heterochromatin barriers and chromatin insulators in Saccharomyces cerevisiae Eukaryot. Cell 2008 7 2078 2086 10.1128/EC.00128-08 18849469
Simms, T. A. et al. TFIIIC binding sites function as both heterochromatin barriers and chromatin insulators in Saccharomyces cerevisiae. Eukaryot. Cell 7, 2078–2086 (2008).18849469
124. Valenzuela L Dhillon N Kamakaka RT Transcription independent insulation at TFIIIC-dependent insulators Genetics 2009 183 131 148 10.1534/genetics.109.106203 19596900
Valenzuela, L., Dhillon, N. & Kamakaka, R. T. Transcription independent insulation at TFIIIC-dependent insulators. Genetics 183, 131–148 (2009).19596900
125. de Boer CG Taipale J Hold out the genome: a roadmap to solving the cis-regulatory code Nature 2024 625 41 50 10.1038/s41586-023-06661-w 38093018
de Boer, C. G. & Taipale, J. Hold out the genome: a roadmap to solving the cis-regulatory code. Nature 625, 41–50 (2024).38093018
126. Li Y Genome-wide Cas9-mediated screening of essential non-coding regulatory elements via libraries of paired single-guide RNAs Nat. Biomed. Eng. 2024 8 890 908 10.1038/s41551-024-01204-8 38778183
Li, Y. et al. Genome-wide Cas9-mediated screening of essential non-coding regulatory elements via libraries of paired single-guide RNAs. Nat. Biomed. Eng. 8, 890–908 (2024).38778183
127. Castanera R Transposable elements versus the fungal genome: impact on whole-genome architecture and transcriptional profiles PLoS Genet 2016 12 e1006108 10.1371/journal.pgen.1006108 27294409
Castanera, R. et al. Transposable elements versus the fungal genome: impact on whole-genome architecture and transcriptional profiles. PLoS Genet 12, e1006108 (2016).27294409
128. McDonald MC Transposon-mediated horizontal transfer of the host-specific virulence protein ToxA between three fungal wheat pathogens MBio 2019 10 e01515 e01519 10.1128/mBio.01515-19 31506307
McDonald, M. C. et al. Transposon-mediated horizontal transfer of the host-specific virulence protein ToxA between three fungal wheat pathogens. MBio 10, e01515–e01519 (2019).31506307
129. Muszewska A Steczkiewicz K Stepniewska-Dziubinska M Ginalski K Transposable elements contribute to fungal genes and impact fungal lifestyle Sci. Rep. 2019 9 4307 10.1038/s41598-019-40965-0 30867521
Muszewska, A., Steczkiewicz, K., Stepniewska-Dziubinska, M. & Ginalski, K. Transposable elements contribute to fungal genes and impact fungal lifestyle. Sci. Rep. 9, 4307 (2019).30867521
130. Yoshida K Host specialization of the blast fungus Magnaporthe oryzae is associated with dynamic gain and loss of genes linked to transposable elements BMC Genomics 2016 17 370 10.1186/s12864-016-2690-6 27194050
Yoshida, K. et al. Host specialization of the blast fungus Magnaporthe oryzae is associated with dynamic gain and loss of genes linked to transposable elements. BMC Genomics 17, 370 (2016).27194050
131. Krishnan P Transposable element insertions shape gene regulation and melanin production in a fungal pathogen of wheat BMC Biol. 2018 16 78 10.1186/s12915-018-0543-2 30012138
Krishnan, P. et al. Transposable element insertions shape gene regulation and melanin production in a fungal pathogen of wheat. BMC Biol. 16, 78 (2018).30012138
132. Omrane S Plasticity of the MFS1 promoter leads to multidrug resistance in the wheat pathogen Zymoseptoria tritici mSphere 2017 2 e00393 17 10.1128/mSphere.00393-17 29085913
Omrane, S. et al. Plasticity of the MFS1 promoter leads to multidrug resistance in the wheat pathogen Zymoseptoria tritici. mSphere 2, e00393–17 (2017).29085913
133. Gusa A Transposon mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance in vitro Proc. Natl. Acad. Sci. USA 2020 117 9973 9980 10.1073/pnas.2001451117 32303657
Gusa, A. et al. Transposon mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance in vitro. Proc. Natl. Acad. Sci. USA 117, 9973–9980 (2020).32303657
134. Priest SJ Uncontrolled transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of Cryptococcus neoformans Nat. Microbiol. 2022 7 1239 1251 10.1038/s41564-022-01183-z 35918426
Priest, S. J. et al. Uncontrolled transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of Cryptococcus neoformans. Nat. Microbiol. 7, 1239–1251 (2022).35918426
135. Gusa A Genome-wide analysis of heat stress-stimulated transposon mobility in the human fungal pathogen Cryptococcus deneoformans Proc. Natl. Acad. Sci. USA 2023 120 e2209831120 10.1073/pnas.2209831120 36669112
Gusa, A. et al. Genome-wide analysis of heat stress-stimulated transposon mobility in the human fungal pathogen Cryptococcus deneoformans. Proc. Natl. Acad. Sci. USA 120, e2209831120 (2023).36669112
136. Hess J Transposable element dynamics among asymbiotic and ectomycorrhizal Amanita fungi Genome Biol. Evol. 2014 6 1564 1578 10.1093/gbe/evu121 24923322
Hess, J. et al. Transposable element dynamics among asymbiotic and ectomycorrhizal Amanita fungi. Genome Biol. Evol. 6, 1564–1578 (2014).24923322
137. Bucknell AH McDonald MC That’s no moon, it’s a Starship: giant transposons driving fungal horizontal gene transfer Mol. Microbiol. 2023 120 555 563 10.1111/mmi.15118 37434470
Bucknell, A. H. & McDonald, M. C. That’s no moon, it’s a Starship: giant transposons driving fungal horizontal gene transfer. Mol. Microbiol. 120, 555–563 (2023).37434470
138. Urquhart AS Vogan AA Gardiner DM Idnurm A Starships are active eukaryotic transposable elements mobilized by a new family of tyrosine recombinases Proc. Natl. Acad. Sci. USA 2023 120 e2214521120 10.1073/pnas.2214521120 37023132
Urquhart, A. S., Vogan, A. A., Gardiner, D. M. & Idnurm, A. Starships are active eukaryotic transposable elements mobilized by a new family of tyrosine recombinases. Proc. Natl. Acad. Sci. USA 120, e2214521120 (2023).37023132
139. Gluck-Thaler E Giant Starship elements mobilize accessory genes in fungal genomes Mol. Biol. Evol. 2022 39 msac109 10.1093/molbev/msac109 35588244
Gluck-Thaler, E. et al. Giant Starship elements mobilize accessory genes in fungal genomes. Mol. Biol. Evol. 39, msac109 (2022).35588244
140. Vogan AA The Enterprise, a massive transposon carrying Spok meiotic drive genes Genome Res. 2021 31 789 798 10.1101/gr.267609.120 33875482
Vogan, A. A. et al. The Enterprise, a massive transposon carrying Spok meiotic drive genes. Genome Res. 31, 789–798 (2021).33875482
141. Urquhart AS Chong NF Yang Y Idnurm A A large transposable element mediates metal resistance in the fungus Paecilomyces variotii Curr. Biol. 2022 32 937 950.e5 10.1016/j.cub.2021.12.048 35063120
Urquhart, A. S., Chong, N. F., Yang, Y. & Idnurm, A. A large transposable element mediates metal resistance in the fungus Paecilomyces variotii. Curr. Biol. 32, 937–950.e5 (2022).35063120
142. Gourlie R The pangenome of the wheat pathogen Pyrenophora tritici-repentis reveals novel transposons associated with necrotrophic effectors ToxA and ToxB BMC Biol. 2022 20 239 10.1186/s12915-022-01433-w 36280878
Gourlie, R. et al. The pangenome of the wheat pathogen Pyrenophora tritici-repentis reveals novel transposons associated with necrotrophic effectors ToxA and ToxB. BMC Biol. 20, 239 (2022).36280878
143. Bucknell, A. et al. Sanctuary: A Starship transposon facilitating the movement of the virulence factor ToxA in fungal wheat pathogens. bioRxiv10.1101/2024.03.04.583430 (2024).
144. Gluck-Thaler E Vogan AA Systematic identification of cargo-mobilizing genetic elements reveals new dimensions of eukaryotic diversity Nucleic Acids Res. 2024 52 5496 5513 10.1093/nar/gkae327 38686785
Gluck-Thaler, E. & Vogan, A. A. Systematic identification of cargo-mobilizing genetic elements reveals new dimensions of eukaryotic diversity. Nucleic Acids Res. 52, 5496–5513 (2024).38686785
145. Vande Zande P Zhou X Selmecki A The dynamic fungal genome: polyploidy, aneuploidy and copy number variation in response to stress Annu. Rev. Microbiol. 2023 77 341 361 10.1146/annurev-micro-041320-112443 37307856
Vande Zande, P., Zhou, X. & Selmecki, A. The dynamic fungal genome: polyploidy, aneuploidy and copy number variation in response to stress. Annu. Rev. Microbiol. 77, 341–361 (2023).37307856
146. Todd RT Wikoff TD Forche A Selmecki A Genome plasticity in Candida albicans is driven by long repeat sequences Elife 2019 8 e45954 10.7554/eLife.45954 31172944
Todd, R. T., Wikoff, T. D., Forche, A. & Selmecki, A. Genome plasticity in Candida albicans is driven by long repeat sequences. Elife 8, e45954 (2019).31172944
147. Raffaele S Kamoun S Genome evolution in filamentous plant pathogens: why bigger can be better Nat. Rev. Microbiol. 2012 10 417 430 10.1038/nrmicro2790 22565130
Raffaele, S. & Kamoun, S. Genome evolution in filamentous plant pathogens: why bigger can be better. Nat. Rev. Microbiol. 10, 417–430 (2012).22565130
148. Bowyer P Currin A Delneri D Fraczek MG Telomere-to-telomere genome sequence of the model mould pathogen Aspergillus fumigatus Nat. Commun. 2022 13 5394 10.1038/s41467-022-32924-7 36104328
Bowyer, P., Currin, A., Delneri, D. & Fraczek, M. G. Telomere-to-telomere genome sequence of the model mould pathogen Aspergillus fumigatus. Nat. Commun. 13, 5394 (2022).36104328
149. Zhang Z Complete telomere-to-telomere genomes uncover virulence evolution conferred by chromosome fusion in oomycete plant pathogens Nat. Commun. 2024 15 4624 10.1038/s41467-024-49061-y 38816389
Zhang, Z. et al. Complete telomere-to-telomere genomes uncover virulence evolution conferred by chromosome fusion in oomycete plant pathogens. Nat. Commun. 15, 4624 (2024).38816389
150. van der Burgt A Karimi Jashni M Bahkali AH de Wit PJGM Pseudogenization in pathogenic fungi with different host plants and lifestyles might reflect their evolutionary past Mol. Plant Pathol. 2014 15 133 144 10.1111/mpp.12072 24393451
van der Burgt, A., Karimi Jashni, M., Bahkali, A. H. & de Wit, P. J. G. M. Pseudogenization in pathogenic fungi with different host plants and lifestyles might reflect their evolutionary past. Mol. Plant Pathol. 15, 133–144 (2014).24393451
151. Jackson AP Comparative genomics of the fungal pathogens Candida dubliniensis and Candida albicans Genome Res. 2009 19 2231 2244 10.1101/gr.097501.109 19745113
Jackson, A. P. et al. Comparative genomics of the fungal pathogens Candida dubliniensis and Candida albicans. Genome Res. 19, 2231–2244 (2009).19745113
152. Lafontaine I Dujon B Origin and fate of pseudogenes in Hemiascomycetes: a comparative analysis BMC Genomics 2010 11 260 10.1186/1471-2164-11-260 20412590
Lafontaine, I. & Dujon, B. Origin and fate of pseudogenes in Hemiascomycetes: a comparative analysis. BMC Genomics 11, 260 (2010).20412590
153. Harrison P A small reservoir of disabled ORFs in the yeast genome and its implications for the dynamics of proteome evolution J. Mol. Biol. 2002 316 409 419 10.1006/jmbi.2001.5343 11866506
Harrison, P. et al. A small reservoir of disabled ORFs in the yeast genome and its implications for the dynamics of proteome evolution. J. Mol. Biol. 316, 409–419 (2002).11866506
154. Vitiello, M. & Poliseno, L. CRISPR/Cas technologies applied to pseudogenes. in Pseudogenes: Functions and Protocols (ed. Poliseno, L.) 265–284 (Springer, 2021).
155. Cheng H Small open reading frames: current prediction techniques and future prospect Curr. Protein Pept. Sci. 2011 12 503 507 10.2174/138920311796957667 21787300
Cheng, H. et al. Small open reading frames: current prediction techniques and future prospect. Curr. Protein Pept. Sci. 12, 503–507 (2011).21787300
156. Mundodi V Choudhary S Smith AD Kadosh D Global translational landscape of the Candida albicans morphological transition G3 2021 11 jkaa043 10.1093/g3journal/jkaa043 33585865
Mundodi, V., Choudhary, S., Smith, A. D. & Kadosh, D. Global translational landscape of the Candida albicans morphological transition. G3 11, jkaa043 (2021).33585865
157. Li Q-R Revisiting the Saccharomyces cerevisiae predicted ORFeome Genome Res. 2008 18 1294 1303 10.1101/gr.076661.108 18502943
Li, Q.-R. et al. Revisiting the Saccharomyces cerevisiae predicted ORFeome. Genome Res. 18, 1294–1303 (2008).18502943
158. Saeki N N-terminal deletion of Swi3 created by the deletion of a dubious ORF YJL175W mitigates protein burden effect in S. cerevisiae Sci. Rep. 2020 10 9500 10.1038/s41598-020-66307-z 32528012
Saeki, N. et al. N-terminal deletion of Swi3 created by the deletion of a dubious ORF YJL175W mitigates protein burden effect in S. cerevisiae. Sci. Rep. 10, 9500 (2020).32528012
159. Sahu PK Salim S Pp M Chauhan S Tomar RS Reverse genetic analysis of yeast YPR099C/MRPL51 reveals a critical role of both overlapping ORFs in respiratory growth and MRPL51 in mitochondrial DNA maintenance FEMS Yeast Res. 2019 19 foz056 10.1093/femsyr/foz056 31374566
Sahu, P. K., Salim, S., Pp, M., Chauhan, S. & Tomar, R. S. Reverse genetic analysis of yeast YPR099C/MRPL51 reveals a critical role of both overlapping ORFs in respiratory growth and MRPL51 in mitochondrial DNA maintenance. FEMS Yeast Res. 19, foz056 (2019).31374566
160. Rech GE Sanz-Martín JM Anisimova M Sukno SA Thon MR Natural selection on coding and noncoding DNA sequences is associated with virulence genes in a plant pathogenic fungus Genome Biol. Evol. 2014 6 2368 2379 10.1093/gbe/evu192 25193312
Rech, G. E., Sanz-Martín, J. M., Anisimova, M., Sukno, S. A. & Thon, M. R. Natural selection on coding and noncoding DNA sequences is associated with virulence genes in a plant pathogenic fungus. Genome Biol. Evol. 6, 2368–2379 (2014).25193312
161. Ellwood SR Syme RA Moffat CS Oliver RP Evolution of three Pyrenophora cereal pathogens: recent divergence, speciation and evolution of non-coding DNA Fungal Genet. Biol. 2012 49 825 829 10.1016/j.fgb.2012.07.003 22850609
Ellwood, S. R., Syme, R. A., Moffat, C. S. & Oliver, R. P. Evolution of three Pyrenophora cereal pathogens: recent divergence, speciation and evolution of non-coding DNA. Fungal Genet. Biol. 49, 825–829 (2012).22850609
162. Maunakea AK Conserved role of intragenic DNA methylation in regulating alternative promoters Nature 2010 466 253 257 10.1038/nature09165 20613842
Maunakea, A. K. et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466, 253–257 (2010).20613842
163. Maroc L Shaker H Shapiro RS Functional genetic characterization of stress tolerance and biofilm formation in Nakaseomyces (Candida) glabrata via a novel CRISPR activation system mSphere 2024 9 e0076123 10.1128/msphere.00761-23 38265239
Maroc, L., Shaker, H. & Shapiro, R. S. Functional genetic characterization of stress tolerance and biofilm formation in Nakaseomyces (Candida) glabrata via a novel CRISPR activation system. mSphere 9, e0076123 (2024).38265239
164. Gervais NC Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans G3 2023 13 jkac301 10.1093/g3journal/jkac301 36450451
Gervais, N. C. et al. Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans. G3 13, jkac301 (2023).36450451
165. Ciurkot K Gorochowski TE Roubos JA Verwaal R Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a Nucleic Acids Res. 2021 49 7775 7790 10.1093/nar/gkab529 34197613
Ciurkot, K., Gorochowski, T. E., Roubos, J. A. & Verwaal, R. Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a. Nucleic Acids Res. 49, 7775–7790 (2021).34197613
166. Després PC Dubé AK Seki M Yachie N Landry CR Perturbing proteomes at single residue resolution using base editing Nat. Commun. 2020 11 1871 10.1038/s41467-020-15796-7 32313011
Després, P. C., Dubé, A. K., Seki, M., Yachie, N. & Landry, C. R. Perturbing proteomes at single residue resolution using base editing. Nat. Commun. 11, 1871 (2020).32313011
167. Jing X Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing Nucleic Acids Res. 2018 46 e90 10.1093/nar/gky433 29860393
Jing, X. et al. Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing. Nucleic Acids Res. 46, e90 (2018).29860393
