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

39227617
52034
10.1038/s41467-024-52034-w
Article
Sequestration of DBR1 to stress granules promotes lariat intronic RNAs accumulation for heat-stress tolerance
http://orcid.org/0000-0002-8202-5134
Wu Chengyun 123
http://orcid.org/0009-0005-8466-4852
Wang Xingsong 1
Li Yan 1
Zhen Weibo 1
Wang Chunfei 1
Wang Xiaoqing 1
http://orcid.org/0000-0001-6494-4949
Xie Zhouli 4
Xu Xiumei 12
http://orcid.org/0000-0003-2013-1772
Guo Siyi 12
http://orcid.org/0000-0002-4446-3432
Botella José Ramón 5
http://orcid.org/0000-0001-5650-5348
Zheng Binglian 6
http://orcid.org/0000-0002-3780-5158
Wang Wei 47
http://orcid.org/0000-0001-8774-4309
Song Chun-Peng songcp@henu.edu.cn

12
http://orcid.org/0000-0002-5394-9864
Hu Zhubing zhubinghu@henu.edu.cn

12
1 https://ror.org/003xyzq10 grid.256922.8 0000 0000 9139 560X The Zhongzhou Laboratory for Integrative Biology, State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, Kaifeng, 475004 China
2 https://ror.org/003xyzq10 grid.256922.8 0000 0000 9139 560X Sanya Institute, Henan University, Sanya, 572025 China
3 https://ror.org/0327f3359 grid.411389.6 0000 0004 1760 4804 The National Engineering Lab of Crop Stress Resistance Breeding, School of Life Sciences, Anhui Agricultural University, Hefei, 230036 China
4 grid.11135.37 0000 0001 2256 9319 State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China
5 https://ror.org/00rqy9422 grid.1003.2 0000 0000 9320 7537 Plant Genetic Engineering Laboratory, School of Agriculture and Food Sciences, The University of Queensland, Brisbane, QLD 4072 Australia
6 grid.8547.e 0000 0001 0125 2443 State Key Laboratory of Genetic Engineering, Ministry of Education Key Laboratory of Biodiversity Sciences and Ecological Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, 200438 China
7 https://ror.org/05kje8j93 grid.452723.5 0000 0004 7887 9190 Center for Life Sciences, Beijing, 100871 China
3 9 2024
3 9 2024
2024
15 769625 11 2023
22 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Heat stress (HS) poses a significant challenge to plant survival, necessitating sophisticated molecular mechanisms to maintain cellular homeostasis. Here, we identify SICKLE (SIC) as a key modulator of HS responses in Arabidopsis (Arabidopsis thaliana). SIC is required for the sequestration of RNA DEBRANCHING ENZYME 1 (DBR1), a rate-limiting enzyme of lariat intronic RNA (lariRNA) decay, into stress granules (SGs). The sequestration of DBR1 by SIC enhances the accumulation of lariRNAs, branched circular RNAs derived from excised introns during pre-mRNA splicing, which in turn promote the transcription of their parental genes. Our findings further demonstrate that SIC-mediated DBR1 sequestration in SGs is crucial for plant HS tolerance, as deletion of the N-terminus of SIC (SIC1–244) impairs DBR1 sequestration and compromises plant response to HS. Overall, our study unveils a mechanism of transcriptional regulation in the HS response, where lariRNAs are enriched through DBR1 sequestration, ultimately promoting the transcription of heat stress tolerance genes.

SICKLE sequestrates DBR1 into stress granules to promote the accumulation of lariRNAs, which upregulate heat stress-related genes and enhance heat-stress tolerance.

Subject terms

Heat
Plant molecular biology
Plant signalling
Transgenic plants
Plant physiology
https://doi.org/10.13039/501100001809 National Natural Science Foundation of China (National Science Foundation of China) No. 32370324 and 32070208 Hu Zhubing the National Natural Science Foundation of China (No. 32370324 and 32370320), the National Key R&D Program of China (No. 2023YFA0914600 and 2022YFF1001700), the Natural Science Foundation of Henan Province (222300420026), the Hainan Provincial Joint Project of Sanya Yazhou Bay Science and Technology City (No. 2021JJLH0044), the Guiding Funds of the Central Government for Supporting the Development of the Local Science and Technology in Henan Province (No. Z20221343006), the 111 Project (No. D16014)the Program for High‑level Talents Recruitment of Anhui Agricultural University (No. rc422305)State Key Laboratory for Protein and Plant Gene Research, School of Life Sciences, Peking University, National Natural Science Foundation of China (No. 31970641)issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Temperature is a major environmental factor that dramatically affects plant growth, development, fitness, and geographical distribution; abnormal temperatures outside an optimal range reduce crop yields and quality1. During the past 200 years, human activity has increased the release of greenhouse gases into the environment, resulting in global warming, which has exacerbated heat stress (HS) in a variety of crops2. In fact, each 1 °C increase in global mean temperature is predicted to decrease the global yield of wheat (Triticum sp.) by an average of 6.0%, that of rice (Oryza sativa) by 3.2%, maize (Zea mays) by 7.4%, and soybean (Glycine max) by 3.1%3. Therefore, exploring the mechanisms of the HS response in plants is crucial, as it will benefit the development of heat-tolerant crops to safeguard global food security.

In general, HS alters cellular homeostasis in plants, such as reducing the stability of mRNA, proteins, and membranes4. Plants have evolved diverse strategies to respond and acclimate to HS, including transcriptional/post-transcriptional regulation, to ensure their survival and reproductive success. HS-related transcription factors are the core players in transcriptional regulation1. Under HS, the expression of genes encoding HS-related transcription factors is activated or repressed, and their encoded proteins are finely regulated by post-translational modifications5. HS-related transcription factors specifically bind to heat-shock elements in the promoters of their target genes to activate the translation of heat-shock proteins (HSPs)6, which act as molecular chaperones to protect cellular proteins by preventing their denaturation and aggregation and facilitating the refolding of damaged proteins7.

Besides transcriptional regulation, HS can induce the formation of stress granules (SGs) to selectively recruit mRNAs and proteins for their storage, degradation, protection, and/or sequestration8–13. SGs are evolutionarily conserved, highly dynamic cytoplasmic membrane-less organelles10,14 that are enriched in polyadenylated (poly(A)) mRNAs, 40S ribosomal subunits, translation initiation factors, RNA-binding proteins, and other components10,15. Their assembly is driven by the collective interactions of a core protein–RNA network16. Initially, a dense, stable SG core structure is formed. Proteins containing intrinsically disordered regions, whose properties are dominated by weak interactions between proteins and RNAs, then accumulate into a dynamic peripheral shell by phase separation8,17–19. Increasing evidence suggests that the formation of SGs provides an elegant way to inhibit mRNA translation and protein activity within minutes upon perception of a stress stimulus, while SG disassembly allows rapid reactivation upon stress recovery10,11. Accordingly, SGs have a pro-survival function during stress. Defects in SG dynamics are associated with various diseases in humans (e.g., neurodegenerative disorders, cancers, enhanced susceptibility to viral infections, and autoimmune diseases), as well as disruptions of plant stress tolerance13,20–27.

Lariat intronic RNAs (lariRNAs) are circular, branched RNA products formed by excised introns during pre-mRNA splicing in eukaryotes28. LariRNAs are generally considered to be by-products and were traditionally thought to be degraded quickly by the dedicated RNA DEBRANCHING ENZYME 1 (DBR1), which recognizes the branchpoint and linearizes the lariats, leading to their turnover29–31. LariRNAs widely exist in eukaryotes. Some lariRNAs naturally escape debranching and stably accumulate in a circular form under certain physiological conditions in animals and plants32–38. Increasing evidence indicates that lariRNAs are not useless by-products of splicing but rather act as regulatory molecules that play essential roles during growth, development, and stress adaptation in eukaryotes33,37,39,40. LariRNA processing allows the biogenesis of rare so-called mirtrons (microRNAs produced from an intron) and the release of intron-derived small nucleolar RNAs in yeast (Saccharomyces cerevisiae) and animals41–43. As lariRNAs serve as a source of circular RNAs (circRNAs), which are widespread regulatory molecules with various functions, lariRNAs also function as circRNAs by regulating gene expression and splicing, binding to microRNAs or proteins, and being translated into proteins44–46.

SICKLE (SIC) is a highly proline-rich protein that participates in microRNA biogenesis, regulating plant development and abiotic stress tolerance47. SIC also plays a role in alternative splicing and the recycling of auxin transporters48,49. Recent studies have shown that SIC interacts with DBR1, directly modulating lariRNA homeostasis, which regulates root development and cell death in Arabidopsis (Arabidopsis thaliana)50,51. Here, we report the role of SIC in plant HS tolerance, which involves HS-induced DBR1 sequestration into SGs by SIC to accumulate lariRNAs. The enriched lariRNAs may enhance the expression of their parental mRNAs, ultimately improving plant HS tolerance. Thus, our work identifies a SIC-mediated regulatory mechanism linking lariRNAs to plant HS tolerance.

Results

HS induces the accumulation of lariRNAs

The production of lariRNAs requires the transcription of their parental genes, which are, in turn, regulated by the lariRNAs produce37,52. Previous studies have revealed that a crucial factor controlling lariRNAs levels, SIC was involved in abiotic stress tolerance47,50, indicating lariRNAs may play a role in adapting to such stresses. To determine whether lariRNAs are involved in plant heat stress (HS) tolerance, we compared lariRNA profiles by performing lariRNA sequencing in 5-d-old Col-0 seedlings under normal conditions (control, 22 °C) and HS (39 °C for 40 min) (Fig. 1a). Compared to control conditions, the abundance of 4488 lariRNAs derived from 3285 genes (parental genes) significantly increased after HS treatment (Supplementary Data 1) (Fig. 1b, c). Gene ontology (GO) enrichment analysis revealed that these 3285 genes are enriched in processes involved in heat-stress tolerance (Fig. 1d). To determine whether the enrichment of these lariRNAs is correlated with the increased expression of their corresponding parental mRNAs, we globally analyzed mRNA abundance by transcriptome deep sequencing (RNA-seq) (Supplementary Data 2). We observed no strict strong association between the levels of lariRNAs and their parental mRNAs under HS conditions (R value = 0.6, Fig. 1e and Supplementary Data 2). The mRNA levels of only ~29.6% (974 out of 3285) of parental genes that produced 1470 enriched lariRNAs were upregulated under HS, while we observed no significant change in the mRNA abundance of the remaining ~70.4% of genes that produced enriched lariRNAs (Fig. 1f and Supplementary Fig. 1). Moreover, when we compared the accumulation rate of each lariRNA with that of their parental mRNAs, calculated as fold-change (FC) values between HS and control conditions, 88% of introns (out of 1470 lariRNAs) showed a higher accumulation rate than their parental mRNAs (Fig. 1g). These results indicate that these HS-induced enriched lariRNAs may not be strictly correlated with the expression of their parental genes.Fig. 1 Heat stress induces lariRNA accumulation.

a Boxplot showing a genome-wide analysis of intron accumulation levels in the RNA-seq profiles of RNase R-treated samples under control vs. HS treatment (n = 3). >100 bp introns were examined. Boxplot boundaries represent the first and third quartiles; a horizontal line divides the interquartile range and median. Whiskers represent the horizontal line of the median in the Control. The average expression levels were compared using the Mann–Whitney U-test (P = 0). b Number of downregulated (blue) and upregulated (red) lariRNAs in HS-treated Col-0 RNase R (+) samples compared to Col-0 RNase R (+) samples under control conditions (using fold change >2, and P value <0.05 as cutoffs). Statistical significance was determined by a two-sided Wald test using Benjamini–Hochberg multiple testing adjustment. c The abundances of four chosen lariRNAs (highlighted in blue in b) in the RNA-seq profiles presented with the genome browser. The X-axis indicates the chromosomal location. The Y-axis indicates normalized peaks from the genomic region. The numbers at the top indicate the values of the normalized expression levels. d Gene ontology (GO) analysis of 4488 upregulated lariRNAs derived from 3285 genes. Statistical significance was determined by the Wallenius non-central hypergeometric distribution. e Correlation analysis of the abundance of enriched heat-induced lariRNAs and the expression of their corresponding parental mRNAs. Scatterplot comparing the log ratio of fold-change (FC) values of lariRNAs and their corresponding parental mRNAs. f Venn diagram showing the overlap between upregulated mRNAs and enriched heat-induced lariRNAs. g Pie chart showing the proportion and classification of 1470 lariRNAs from the 974 overlapping parental genes in (f). The log ratio of FC is presented as a change in relative multiples of mRNAs and lariRNAs and is hierarchically clustered into two groups. Percentages indicate the ratios of lariRNAs relative to the total number (1470) of lariRNAs.

Lariats with a C or G branchpoint (BP) might be more stable than other introns53,54. To investigate whether lariRNAs that accumulated under HS conditions exhibit this specific BP feature, we analyzed the BPs of lariRNAs under control and HS conditions, finding that 73.2% of the introns under HS had canonical adenosine (A), and another 16.7% had uridine (U) as the BP (Supplementary Fig. 1a), which are typical BP sites under physiological conditions44. We detected a similar percentage of noncanonical guanine (G, 8.2%) and cytosine (C, 2.0%) BPs under HS and control conditions (Supplementary Fig. 1a). The enriched lariRNAs under HS conditions also showed a similar percentage of the different BPs (Supplementary Fig. 1a). We also analyzed other sequence features and observed that the 5′ and 3′ splice sites of lariRNAs that accumulated under HS had similar sequences to those under control conditions, which conform to the standard consensus sequences (Supplementary Fig. 1b). These data indicate that BP features may not determine the enrichment of specific lariRNAs under HS.

LariRNA enrichment promotes the expression of their parental genes

LariRNAs localize to the nucleus in plants55, and nucleus-enriched lariRNAs are involved in gene expression in mammalian cells37. To investigate whether lariRNAs that accumulate under HS conditions regulate the expression of their parental genes, we examined the enriched lariRNAs and differentially expressed genes under HS vs. control conditions. We identified a set of enriched lariRNAs derived from heat-responsive genes whose expression was positively correlated with the abundance of their corresponding parental transcripts (Fig. 2a), indicating that lariRNAs that are enriched under HS conditions may modulate heat acclimation via transcriptional regulation.Fig. 2 Increasing abundance of lariRNAs promotes the expression of the corresponding parental gene.

a Comparison of the log ratio of FC values of lariRNAs and their corresponding parental genes among representative heat-responsive genes detected by RNA-seq. b Strategy used to overexpress introns in Arabidopsis. The intron sequence from HSP70-4 or HSP90-1 (designed as lariat70-4 and lariat90-1, respectively) was inserted between the sequences encoding the N-terminal and C-terminal fragments of YFP and stably expressed in Col-0. c YFP signals in root cells of transgenic plants. Bright dots indicate YFP signals in the nucleus. Images are representative of individual lines from three independent experiments with similar results. Scale bar, 10 μm. d Accumulation of lariRNAs and expression of their corresponding parental mRNAs relative to UBQ5, as detected by RT-qPCR. Data were means ± SD (n = 3). n = 3 biologically independent samples. Two independent lariat70-4-OE and lariat90-1-OE lines were tested, respectively. Statistical significance was determined by one-way ANOVA followed by Tukey’s LSD test (P < 0.001). See Source data for P values.

To investigate this notion in detail, we selected two representative lariRNAs (designed as lariat70-4 and lariat90-1) derived from two genes encoding crucial heat-shock proteins (Hsp70 and Hsp90). We generated stable transgenic plants harboring the sequence encoding the N- and C-terminal halves of yellow fluorescent protein (YFP) separated by the intron-containing lariat70-4 or lariat90-1 and driven by the cauliflower mosaic virus (CaMV) 35S promoter (Fig. 2b). With this transgene design, intron splicing of the mRNAs would result in YFP fluorescence. We detected YFP signals in the root cells of lariat70-4-OE and lariat90-1-OE transgenic plants (Fig. 2c). Reverse-transcription quantitative PCR (RT-qPCR) revealed that the corresponding lariat intron accumulated in transgenic seedlings but not in control seedlings (Fig. 2d), demonstrating that proper splicing occurred and introns over-accumulated. Remarkably, the endogenous parental mRNA levels were substantially higher compared to control seedlings (Fig. 2d). However, overproduction of HSP70-4 and HSP90-1 mRNAs did not lead to increased expression of lariat70-4 and lariat90-1 (Supplementary Fig. 2). These findings suggest a positive correlation between lariRNA enrichment and the mRNA levels of their corresponding parental genes.

DBR1 is sequestered into cytoplasmic SGs under HS

DBR1 is currently the only characterized rate-limiting enzyme involved in lariRNA decay33,55–57. To investigate whether the enrichment of lariRNAs under HS conditions relies on DBR1 function, we assessed the total DBR1 protein abundance of Col-0 and pDBR1:DBR1-GFP plants under both control and HS conditions. Immunoblot analysis revealed no discernible difference in DBR1 abundance between control and HS conditions (Supplementary Fig. 3a, b). Next, we examined the subcellular distribution of DBR1-GFP of pDBR1:DBR1-GFP plants. Consistent with previous reports50,55, DBR1 localized to the nucleus and cytosol of root cells under control conditions (Fig. 3a). Notably, HS induced a subcellular translocation of DBR1-GFP and formed an association of the DBR1-GFP signal with numerous, discrete cytoplasmic granules (DBR1-GFP granules) in the cortex cells of the root meristematic zone (Fig. 3a), accompanied by an obvious reduction of DBR1-GFP in nuclei. To further explore the subcellular localization of DBR1 under HS conditions, we carried out nuclei isolation and immunoblotting experiments using antibodies against DBR1, the nuclear protein Histone 3 (H3), and the cytosolic protein actin in Col-0 plants. Consistent with the aforementioned subcellular localization of DBR1-GFP (Fig. 3a), DBR1 abundance was obviously decreased in the nuclei after heat treatment, compared to the control condition (Supplementary Fig. 3c, d). However, no difference in DBR1 levels was detected in the cytosol, which may be due to the approximately tenfold higher concentration of DBR1 used in the cytosol compared to that of the nuclei. Consequently, the increased amount of DBR1 translocation from the nuclei to the cytosol could not be significantly detected (Supplementary Fig. 3c, d).Fig. 3 DBR1 localizes to stress granules under heat stress.

a Fluorescence images showing DBR1 localization under control, HS, and CHX treatment plus HS. The arrow denotes an example of foci under HS. Scale bar, 10 μm. b, c Fluorescence images showing colocalization of DBR1 with the SG marker proteins PAB2 (b) (rs = −0.58 in Control and rs = 0.61 in HS) and PAB8 (c) (rs = −0.48 in Control and rs = 0.52 in HS). Correlation analysis using the Pearson-Spearman correlation (PSC) plugin for ImageJ. rs: PSC coefficients. Arrows denote examples of foci with co-localized mRFP and GFP signals. Scale bars, 10 μm. Images are representative of three independent biological replicates, and at least five roots were monitored per replicate.

HS is known to trigger SG formation in plant cells58. To examine whether the discrete cytoplasmic granules were SGs, we treated proDBR1:DBR1-GFP seedlings with cycloheximide (CHX), a protein translation inhibitor that blocks the formation of SGs in mammalian and plant cells58,59, during HS. We observed no DBR1-GFP granules in CHX-treated seedlings (Fig. 3a). Next, we introduced SG marker proteins POLY(A) BINDING PROTEIN 2 (PAB2) and/or PAB858,59 fused to monomeric red fluorescent protein (mRFP) into the proDBR1:DBR1-GFP background via genetic crossing. The DBR1-GFP granules co-localized with PAB2-mRFP or PAB8-mRFP under HS conditions (Fig. 3b, c), indicating that HS promotes the sequestration of DBR1 into SGs. To further examine whether DBR1 sequestration compromises the activity of lariRNAs degradation, we measured the levels of lariRNAs lariat70-1, lariat70-4, lariat90-1 and lariat90-3 in the nucleus and the cytosol. We found that higher levels of these lariRNAs were detected in both the nucleus and the cytosol under HS conditions (Supplementary Fig. 3e).

SICKLE is required for DBR1 sequestration into SGs

SIC is a direct interactor of DBR1 that facilitates its accumulation in the nucleus50. To investigate whether SIC is required for the sequestration of DBR1 into SGs, we examined SIC localization under HS conditions in proSIC:SIC-GFP transgenic lines. Similar to the subcellular localization of DBR1, HS also induced the association of SIC-GFP with cytoplasmic granules in the root cells of proSIC:SIC-GFP seedlings (Fig. 4a). Additionally, we introduced DBR1-mRFP, PAB2-mRFP, and PAB8-mRFP into proSIC:SIC-GFP seedings via genetic crossing. The SIC-GFP granules were strongly associated with DBR1-mRFP granules (Fig. 4b), as well as with the SG marker proteins PAB2-mRFP and PAB8-mRFP (Fig. 4c, d). SGs are mRNA-protein assemblies formed from nontranslating mRNAs60. To test the presence of polyadenylated mRNA in these cytoplasmic granules, we performed fluorescence in situ hybridization (FISH) using Cy3-oligo deoxythymidine (dT) as a probe. We observed a diffuse nucleocytoplasmic pattern for the Cy3 signal in the nuclei of seedlings under control conditions (Fig. 4e). However, after HS treatment, the Cy3 signal co-localized with SIC-GFP in cytoplasmic granules (Fig. 4e), suggesting that these SIC-GFP cytoplasmic granules contained polyadenylated mRNA.Fig. 4 SIC is required for the sequestration of DBR1 into stress granules.

a Fluorescence images showing SIC localization under control, HS, and CHX treatment plus HS. The arrow denotes an example of foci under HS. Scale bar, 10 μm. b Fluorescence images showing colocalization of DBR1-mRFP with SIC-GFP under HS (rs = 0.71). Arrows denote examples of foci with co-localized mRFP and GFP signals. Scale bar, 10 μm. c, d Fluorescence images showing colocalization of SIC with the SG marker proteins PAB2 (c) (rs = 0.29 in Control and rs = 0.53 in HS) and PAB8 (d) (rs = 0.11 in Control and rs = 0.52 in HS). Arrows denote examples of foci with co-localized mRFP and GFP signals. Scale bars, 5 μm. e Fluorescence images showing colocalization of SIC-GFP and poly(A) mRNAs in cytoplasmic granules under HS. Arabidopsis seedlings harboring proSIC:SIC-GFP were subjected to fluorescence in situ hybridization with Cy3-oligo-d(T) probes followed by fluorescence microscopy (rs = 0.63 in Control and rs = 0.95 in HS). Scale bars, 10 μm. f Fluorescence images showing DBR1-GFP in the root meristems of 5-day-old Col-0 or sic-4 seedlings under control and HS conditions. Scale bars, 10 μm. g Number (left, n = 19) and size (right, n = 30) of DBR1-GFP foci in (f). Boxplot boundaries represent the first and third quartiles; the horizontal line (median) divides the interquartile range. Whiskers represent the Y-axis at 0. Statistical significance was determined by Kruskal–Wallis test followed by two-sided Wilcoxon test (P < 0.0001). See Source data for P values. More than eight images were examined per experiment.

Next, we investigated the dynamics of HS-induced SIC-GFP granule formation. We subjected proSIC:SIC-GFP seedlings, grown under control conditions with 1.5 h of moderate HS at 38 °C (HS1), followed by a 2-h acclimation period at 22 °C (AC1) and severe HS at 45 °C for 1 h (HS2) and a variable-length recovery phase at 22 °C (Supplementary Fig. 4). Granules began to appear in the cytosol after HS1 and dissipated after AC1. However, granules reappeared after HS2 and disappeared again after a 16-h recovery at 22 °C (Supplementary Fig. 4). These results indicate that the formation of HS-induced SIC-GFP granules is dynamic and reversible.

Next, we compared DBR1 granules in wild-type Col-0 and sic-4 plants. Consistent with our previous finding50, the nuclear accumulation of DBR1-GFP was compromised in the sic-4 mutant under control conditions. Under HS conditions, we observed a significant reduction in the number of DBR1-GFP granules in sic-4 compared to Col-0, although their size remained unchanged (Fig. 4f, g). Subsequently, we introduced an SG marker protein PAB2-mRFP into sic-4 proDBR1:DBR1-GFP plant and found the DBR1 foci was co-localized with the fluorescently labeled marker, demonstrating that DBR1 foci formed in sic-4 mutant are within SGs (Supplementary Fig. 5). These data indicate that SIC mediates the sequestration of DBR1 into SGs. Additionally, we analyzed SIC-GFP granules in Col-0 and dbr1-2 plants and observed comparable numbers and sizes of SIC-GFP granules in both lines (Supplementary Fig. 6), suggesting that DBR1 is not required for the formation of SIC granules.

SIC is not involved in the assembly or disassembly of SGs

SGs are composed of a dynamic shell and a more stable core14,61. To determine whether SIC acts as a scaffold protein of SG components and facilitates the assembly or disassembly of SGs, we monitored the appearance and disappearance of granules in Col-0 and sic-4 seedlings expressing the SG marker PAB2-mRFP. The numbers and sizes of PAB2-mRFP granules in Col-0 and sic-4 were comparable (Fig. 5a, b). Time-lapse images of SGs after recovery from HS treatment showed that SG dissociation in Col-0 and sic-4 was also comparable (Fig. 5c, d). Taken together, these results indicate that SIC is not a scaffold protein of SG components and might not contribute to the assembly or disassembly of SG complexes.Fig. 5 SIC is not involved in the assembly or disassembly of stress granules.

a Fluorescence images showing the SG marker PAB2-mRFP in the root meristems of 5-day-old Col-0 or sic-4 seedlings under control and HS conditions. Scale bars, 10 μm. b Number (left, n = 18) and size (right, n = 401) of PAB2-mRFP foci in (a). Boxplot boundaries represent the first and third quartiles; the horizontal line (median) divides the interquartile range. Whiskers represent the Y-axis at 0. Statistical significance was determined by Kruskal–Wallis test followed by two-sided Wilcoxon test (P < 0.0001). See Source data for P values. More than eight images were examined per experiment. c Fluorescence images showing PAB2-mRFP foci in the root meristems of 5-day-old Col-0 or sic-4 seedlings during recovery from HS. The seedlings were subjected to HS at 39 °C for 40 min, transferred to 22 °C, and incubated for 1 or 2 h, and root tip cells photographed. Scale bars, 10 μm. d Number (left, n = 35) and size (right, n = 35) of PAB2-mRFP foci in (c). Boxplot boundaries represent the first and third quartiles; the horizontal line (median) divides the interquartile range. Statistical significance was determined by Kruskal–Wallis test followed by two-sided Wilcoxon test (P < 0.0001). See Source data for P values. More than eight images were examined per experiment.

SIC proteins undergo phase separation

Phase separation is an important mechanism underlying the formation of cellular foci, bodies, and granules62,63. The above findings suggest that phase separation may be involved in SIC-mediated DBR1 sequestration into SGs. We therefore evaluated the amino acid sequences and secondary structures of SIC using commonly used predictors of disordered regions in proteins: PONDR-VLXT, PONDR-VL3, PONDR-VSL2, IUpred_short, and IUpred_long64, and the online AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/). These analyses suggested that SIC is a largely unstructured and highly disordered protein with only two very small ordered sub-regions (Supplementary Fig. 7). Similar results were obtained using the Database of Disordered Protein Predictions (D2P2)65 (Supplementary Fig. 7).

Next, we tested whether SIC can readily diffuse into and out of stress granules to assess the role of phase separation in their formation66. Fluorescence recovery after photobleaching (FRAP) experiments showed that the SIC-GFP granules rapidly recover their fluorescence after photobleaching (t1/2 ≈ 25 s) (Fig. 6a, b). Furthermore, we produced and purified recombinant SIC fused to GFP and 6 × His tag in Escherichia coli (Supplementary Fig. 8) to determine the phase separation properties of SIC in vitro. Recombinant His6-SIC-GFP formed green puncta at various concentrations (2.75, 5.5, or 11 μmol/L protein in 20 mM Tris-HCl pH 8.0, 1 mM DDT, 50 mmol/L NaCl, 10% [v/v] glycerol), while His6-GFP protein did not (Fig. 6c, d and Supplementary Fig. 8). The number and size of SIC-GFP droplets increased with increasing protein concentration but decreased with increasing NaCl and 1,6-hexanediol concentration (Fig. 6c, d and Supplementary Fig. 8d). We then assessed the dynamics of SIC liquid droplets using FRAP. Time-lapse analysis of bleaching events indicated that SIC-GFP molecules were rapidly redistributed within droplets, with exchange between droplets and the surrounding solution (Fig. 6e, f). Fusion events of the in vitro droplets were also observed (Fig. 6g). Additionally, we also observed the in vitro droplets in SIC protein without tags (Supplementary Fig. 8e). Collectively, these data indicate that SIC undergoes phase separation in vitro, although further proof is needed to confirm the same mechanism in vivo. We also used purified recombinant His6-DBR1-mRuby. When added alone, His6-DBR1-mRuby failed to form droplets (Supplementary Fig. 8) (14.5 μmol/L protein in 20 mM Tris-HCl pH 8.0, 1 mM DDT, 50 mmol/L NaCl, 10% [v/v] glycerol). However, His6-DBR1-mRuby was incorporated into SIC-GFP droplets when added together with SIC-GFP (Fig. 6h). As a negative control, we used another protein, IMPA-8 (AT5G52000), which has a comparable molecular mass and pI value to DBR1. His6-IMPA-8-mRuby could not recruited into SIC-GFP droplets (Fig. 6h). Taken together, these results indicate that SIC undergoes phase separation, likely to sequester DBR1.Fig. 6 SIC undergoes phase separation to sequester DBR1.

a FRAP of SIC-GFP granules in the cytoplasm of root tip cells. Arrows indicate a bleached granule. Scale bar, 10 μm. Data were representative of four independent experiments. b FRAP curve of SIC-GFP cytoplasmic granules after photobleaching. Data were means ± SD (n = 4). n = 4 biologically independent samples. c Analysis of His6-SIC-GFP protein droplet formation in vitro. Different concentrations of SIC protein were used. Scale bar, 10 μm. d Analysis of His6-SIC-GFP protein droplet formation in the presence of different concentrations of NaCl. Scale bar, 10 μm. e FRAP of a His6-SIC-GFP protein droplet. Data were representative of four independent experiments. Scale bar, 1 μm. f FRAP curve of His6-SIC-GFP protein droplets after photobleaching. Data were means ± SD (n = 4). n = 4 biologically independent samples. g Fusion of His6-SIC-GFP droplets. Data were representative of three independent experiments. Scale bar, 5 μm. h Droplet formation of His6-DBR1-mRuby (7.25 μM) in the presence of His6-SIC-GFP (5.5 μM) (rs = 0.73). His6-IMPA-8-mRuby protein was used as a negative control. Scale bars, 5 μm. Images in c, d, h are representative of individual conditions from three independent experiments with similar results.

The N-terminus of SIC is essential for DBR1 sequestration into SGs

We previously demonstrated that SIC can be roughly divided into three regions: two regions important for nuclear localization (SIC1–244 and SIC252–319) and a DBR1-interacting region (SIC245–251)50. The stable expression of SIC1–251-GFP or SIC245–319-GFP, but not SIC1–244-GFP or SIC252–319-GFP, in the sic-4 background, rescued the developmental defects in leaves, roots, and flowering of the sic-4 mutant50 (Fig. 7a and Supplementary Fig. 9a, b) and recovered lariRNAs to wild-type levels (Supplementary Fig. 9c). This demonstrates that at least one nuclear localization region and the DBR1-interacting region must be present for proper SIC function. To investigate whether SIC contains a specific region responsible for SGs recruitment, we analyzed the presence of GFP granules in transgenic sic-4 mutants expressing the individual SIC1–244-GFP, SIC1–251-GFP, SIC245–319-GFP, or SIC252–319-GFP under HS conditions. Transgenic plants harboring SIC1–244-GFP or SIC1–251-GFP, but not SIC245–319-GFP or SIC252–319-GFP, formed SIC-GFP granules (Fig. 7b). Furthermore, we generated transgenic lines in which the N-terminus of SIC (SIC1–244) was replaced with LCD region of RBGD2 (AT2G33410, 182-404aa), which is required for RBGD2’s SGs localization26. The recombinant protein formed GFP foci under HS conditions (Supplementary Fig. 9d), indicating that phase separation caused by the N-terminus of SIC (SIC1–244) is required for SG recruitment under HS conditions.Fig. 7 The N-terminus of SIC is essential for phase separation and the sequestration of DBR1 into stress granules, and lariRNA accumulation is associated with the transcriptional machinery under HS.

a Phenotypes of 4-week-old sic-4 and transgenic plants accumulating truncated SIC proteins fused to GFP. Scale bar, 1 cm. b Fluorescence images showing the subcellular localization of different truncated SIC proteins under control and HS conditions. Images are representative of three independent biological replicates, and at least five roots were monitored for each replicate. Scale bar, 10 μm. c Fluorescence images showing DBR1-mRFP in the root meristems of 5-day-old Col-0 or sic-4 SIC245–319-GFP seedlings under control and HS conditions. Scale bars, 10 μm. d Number of DBR1-mRFP foci in (c). Boxplot boundaries represent the first and third quartiles; the horizontal line (median) divides the interquartile range. Whiskers represent the Y-axis at 0. Statistical significance was determined by Kruskal–Wallis test followed by two-sided Wilcoxon test (P < 0.0001). See source data for P values. More than eight images were examined per experiment (n = 10). e Diagram of the workflow for the nuclear run-on transcription assay. f Transcription rates of HSP70-4 and HSP90-1 relative to TUB6 detected by nuclear run-on assay and RT-qPCR under control and HS conditions. Data were means ± SD (n = 3). n = 3 biologically independent samples. Statistical significance was determined by one-way ANOVA followed by Tukey’s LSD test (P < 0.001). See Source Data for P values. g Diagrams of the HSP70-4 and HSP90-1 genomic loci. P1 and P2 indicate the target regions in Pol II ChIP-qPCR. h Quantification of Pol II enrichment in ChIP-qPCR. Pol II enrichment is presented as the percentage of Pol II antibodies coprecipitating DNAs along the HSP70-4 or HSP90-1 loci versus input under the indicated condition. Data were means ± SD (n = 3). n = 3 biologically independent samples. Statistical significance was determined by one-way ANOVA followed by Tukey’s LSD test (P < 0.001). ND not detected. See source data for P values.

To examine whether the N-terminus of SIC (SIC1–244) is required for DBR1 sequestration into SGs, we expressed pDBR1:DBR1-mRFP in sic-4 SIC245–319-GFP and sic-4 SIC1–251-GFP plants and analyzed the formation of DBR1 granules. Similar to the observations in the sic-4 mutant (Fig. 4f, g), the number of DBR1-mRFP granules was significantly reduced in sic-4 SIC245–319-GFP plants and was recovered in sic-4 SIC1–251-GFP compared to Col-0 (Fig. 7c, d and Supplementary Fig. 10), suggesting that N-terminus of SIC (SIC1–244) is required for DBR1 sequestration into SGs.

Enrichment of lariRNAs, mediated by SIC-mediated DBR1 sequestration, is associated with the transcriptional machinery under HS

The above results demonstrate that SIC1–244 is required for SIC-mediated DBR1 sequestration into SGs. To determine whether SIC-mediated DBR1 sequestration is required for the accumulation of lariRNAs, we measured the levels of lariat70-1, lariat70-4, lariat90-1, and lariat90-3 in Col-0 and sic-4 SIC245–319-GFP plants that are absent of SIC1–244 under control and HS conditions by RT-qPCR. These lariRNAs significantly accumulated in both genotypes after HS treatment, whereas their abundance was significantly reduced in sic-4 SIC245–319-GFP seedlings compared to Col-0 under HS conditions (Supplementary Fig. 11a). The corresponding parental mRNAs showed similar expression patterns to those of lariRNAs (Supplementary Fig. 11b). These results indicate that SIC-mediated DBR1 sequestration is required for the accumulation of lariRNAs. To further validate the reduction of DBR1 activity modulates the accumulation of lariRNAs and corresponding parental mRNAs, we examine the expression of lariat70-1, lariat70-4, lariat90-1, and lariat90-3 in Col-0, sic-4, and dbr1-255. Consistently, the lariRNAs significantly accumulated in Col-0, sic-4, and dbr1-2 after HS treatment (Supplementary Fig. 11c–f). We observed higher expressions in lariRNAs and corresponding parental mRNAs of lariat70-4 and lariat90-1 in sic-4 and dbr1-2 than in Col-0, likely due to the low DBR1 activity in the nucleus resulting from the dbr1-2 or sic-4 mutation (Supplementary Fig. 11c–f). Furthermore, we examined the expressions of lariat70-1, lariat70-4, lariat90-1, and lariat90-3 in Col-0, sic-4, and dbr1-2 by cell fractions. Higher expressions of lariat70-4, lariat90-1, and lariat90-3 were found in sic-4 and dbr1-2 compared to Col-0 under HS (Supplementary Fig. 12a). However, under control conditions, no accumulation of lariat70-1, lariat70-4, lariat90-1, and lariat90-3 was observed in sic-4 and dbr1-2 (Supplementary Fig. 12a). This may be explained by the fact that both sic-4 and dbr1-2 have lower activity in degrading DBR1-dependent RNAs compared to Col-050,55. The remaining activity in these mutants may be sufficient to degrade the amount of lariat70-1, lariat70-4, lariat90-1, and lariat90-3 produced under control conditions, but not under HS. To address how SIC-mediated DBR1 sequestration controls lariat RNAs levels. We further detected their levels of Col-0, sic-4 SIC1-251-GFP and sic-4 SIC245-319-GFP under control and HS conditions by cell fractions. Compared to Col-0, sic-4 SIC1-251-GFP showed higher expressions of lariat70-1, lariat70-4, lariat90-1, and lariat90-3 in both cytosol and nucleus, while these lariat RNAs were expressed lower in sic-4 SIC245-319-GFP (Supplementary Fig. 12b). These data indicate that SIC-mediated DBR1 sequestration affects DBR1 activity.

In human cells, nucleus-enriched intron-derived circular RNAs associate with the RNA polymerase II (Pol II) machinery to regulate the expression of their parental genes, thereby positively regulating transcription by Pol II37. To investigate whether the enriched heat-induced lariRNAs are associated with the transcriptional machinery under HS conditions, we performed bromouridine immunocapture nuclear run-on RT-qPCR to quantify transcriptional activity67 (Fig. 7e) in Col-0 and sic-4 SIC245–319-GFP seedlings. We detected significantly reduced transcription rates for the nascent transcripts of HSP70-4 and HSP90-1 under HS in sic-4 SIC245–319-GFP seedlings compared to Col-0 (Fig. 7f). Next, we analyzed Pol II occupancy at the HSP70-4 and HSP90-1 loci in Col-0 and sic-4 SIC245–319-GFP seedlings via ChIP-qPCR using anti-RPB1 antibodies. We detected significantly decreased Pol II binding on the gene bodies in sic-4 SIC245–319-GFP seedlings compared to Col-0 under HS (Fig. 7g, h). Together, these results suggest that the enriched lariRNAs mediated by SIC-mediated DBR1 sequestration regulate the RNA Pol II-induced transcription of their parental genes and mRNA expression.

SIC-mediated DBR1 sequestration is required for HS tolerance in plants

Finally, we examined the physiological significance of SIC-mediated DBR1 sequestration into SGs under HS. We analyzed the heat sensitivity of 14-day-old Col-0, the functionally complemented sic-4 SIC1–251-GFP line, and the functionally deficient sic-4 SIC245–319-GFP line following exposure to 44 °C for 30 min before returning to normal growth conditions (22 °C) for 5 days (Fig. 8a). We observed a significant decrease in survival of sic-4 SIC245–319-GFP seedlings compared to Col-0 seedlings; ~60% of Col-0 seedlings survived, while only about 30% of sic-4 SIC245–319-GFP seedlings survived. Notably, sic-4 SIC1–251-GFP seedlings had a higher survival rate (~80%) than sic-4 SIC245–319-GFP seedlings (p < 0.05) (Fig. 8b) and Col-0, which may be caused by SIC1-251 overproduction (Supplementary Fig. 9b). However, SIC1-244 overproduction in Col-0 could not increase plants HS tolerance (Supplementary Fig. 13a, b). In line with this, the expression levels of HSP70-4 and HSP90-1 under HS were much higher in the sic-4 SIC1–251-GFP seedlings compared to Col-0, whereas sic-4 SIC245–319-GFP seedlings showed lower expression levels than Col-0 under HS (Supplementary Fig. 14a). Additionally, we examined the heat sensitivity of dbr1-2 and sic-4 and found that both dbr1-2 and sic-4 mutants showed a higher survival rate than Col-0 under HS conditions (Fig. 8c, d and Supplementary Fig. 13c, d). Since the dbr1-2 mutation reduces the DBR1 activity and the mutated dbr1-2 protein still interacts with SIC and is translocated from the nuclei into SGs, this explains that the comprised HS tolerance due to producing SIC245-319 was also observed in dbr1-2 mutant (Supplementary Fig. 13c, d).Fig. 8 Phenotypes of sic-4 SIC1–251-GFP and sic-4 SIC245–319-GFP seedlings under HS.

a Representative photographs of Col-0, sic-4 SIC1–251-GFP, and sic-4 SIC245–319-GFP seedlings under control and HS conditions. Seedlings in each horizontal plane were grown on the same plate. Scale bars, 2 cm. b Quantification of the data shown in (a). The percentage of seedlings in different phenotypic classes was determined for different genotypes. Data were means ± SD (n = 3). n = 3 biologically independent samples. At least 95 seedlings per genotype were examined in each biological replicate. Different letters indicate significant differences between the two groups, as determined by a two-sided Fisher’s exact test (P < 0.01). See source data for P values. c Representative photographs of Col-0, sic-4, and dbr1-2 seedlings under control and HS conditions. Seedlings in each horizontal plane were grown on the same plate. Scale bars, 2 cm. d Quantification of the data shown in (c). The percentage of seedlings in different phenotypic classes was determined for different genotypes. Data were means ± SD (n = 3). n = 3 biologically independent samples. At least 95 seedlings per genotype were examined in each biological replicate. Different letters indicate significant differences between the two groups, as determined by a two-sided Fisher’s exact test (P < 0.01). See source data for P values. e A working model for the role of phase separation of SIC in heat-stress tolerance in plants. Heat stress induces the accumulation of lariRNAs by sequestrating DBR1 into SGs via phase separation of SIC. The enriched heat-induced lariRNAs promote RNA Pol II transcription for heat-stress-response genes to enhance HS tolerance. RBPs RNA-binding proteins.

To further confirm the SIC-mediated DBR1 sequestration determines plant tolerance to HS, we analyzed DBR1 levels in the cytosol and nucleus of Col-0, sic-4 SIC245-319-GFP and sic-4 SIC1-251-GFP under control and HS conditions through cell fractionation. Compared with the nuclear DBR1 abundancy in Col-0, a lower level of nuclear DBR1 was detected in sic-4 SIC1-251-GFP and a higher level in sic-4 SIC245-319-GFP under HS conditions (Supplementary Fig. 14b, c). No significant difference was found among Col-0, sic-4 SIC245-319-GFP, and sic-4 SIC1-251-GFP under normal conditions (Supplementary Fig. 14b, c). Taken together, these data suggest that SIC-mediated DBR1 sequestration, which limits DBR1 activity under HS conditions, is required for HS tolerance in Arabidopsis.

Discussion

LariRNAs are widely present in eukaryotes33,68,69, but their roles remain poorly understood. In this study, we discovered a role for lariRNAs in plant HS tolerance. Under HS conditions, SIC-mediated sequestration of DBR1 into SGs prevents the DBR1-mediated degradation of lariRNAs, allowing them to accumulate (Fig. 8e). The enrichment of lariRNAs spliced from the mRNAs of HS-associated genes promotes the transcription of their corresponding parental genes, thereby enhancing HS tolerance (Fig. 8e).

While excised lariRNAs are usually linearized and degraded within seconds or minutes57,70,71, we showed that HS induces the accumulation of lariRNAs in plant cells (Fig. 1). RNA sequences near the 5’ splice site and BP can affect debranching in human cells3737, although in our study the BP sequence did not appear to influence lariRNAs accumulation, as we did not observe differential sequence preference for BP sites in the accumulating lariRNAs under HS conditions (Supplementary Fig. 1). Therefore, lariRNAs accumulation is most likely due to DBR1 sequestration. DBR1 is the only rate-limiting enzyme for lariRNAs degradation characterized to date, and reduced activity of DBR1 results in lariRNAs accumulation55–57,72. In this study, we observed that DBR1 is sequestered into cytoplasmic SGs under HS conditions (Fig. 3), making this enzyme unavailable for lariRNAs degradation, thus allowing lariRNAs to accumulate. This may be a general strategy for plants to combat stress through SGs. Increasing evidence supports that SGs play a conserved, essential role in coping with acute stress by sequestering proteins or translationally inactive mRNAs for degradation or protection12,13,73. This mechanism is also observed in plants facing HS, as SGs protect mRNAs encoding HS transcription factors from degradation, thereby conferring HS tolerance13.

SIC is a shell component of SGs (Fig. 4), as supported by the finding that depletion of SIC did not disrupt the assembly and disassembly of SGs (Fig. 5). SIC has not been identified in the heat SG proteomes published to date9,23,26. Generally, SGs are composed of a dynamic shell and a more stable core14,61, and the shell proteins may not be efficiently isolated and detected. Supporting this, several known shell proteins, such as acetylation lowers binding affinity proteins (ALBAs), vascular plant one-zinc-finger protein 2 (VOZ2), metacaspase 1 (MC1) m6A RNA demethylase ALKBH9B and suppressor of gene silencing 3 (SGS3), have also not been detected in the heat SG proteomes13,74–76.

SIC acts as a promoting factor for DBR1 sequestration into SGs, rather than an essential protein. Supporting this, under HS condition, deficiency of SIC did not completely abolish SGs localization of DBR1, but significantly reduced the efficiency of DBR1 sequestration into SGs (Fig. 4f, g). We previously demonstrated that SIC contains two functionally redundant regions for nuclear localization (SIC1–244 and SIC252–319) and a DBR1-interacting region (SIC246–251) and that SIC directly interacts with DBR1 through the DBR1-interacting region (SIC245–251)50. The promoting effect of SIC relies on its N-terminal region (SIC1-244), as evidenced by the fact that DBR1 abundance in SGs was not recovered in the sic-4 SIC245–319-GFP plants (Fig. 7c, d), but was recovered to WT levels in the sic-4 SIC1-251-GFP plants (Supplementary Fig. 10). Moreover, the promoting power may originate from the phase separation property of SIC (Fig. 6), specifically from SIC1-244 (Fig. 7b).

The sequestration of DBR1 into SGs through SIC1-244 is crucial for plant HS tolerance. sic-4 SIC245–319-GFP seedlings, which lack the N-terminal region of SIC necessary for promoting DBR1 sequestration into SGs, were more sensitive to HS compared to Col-0 (Fig. 8a, b). Conversely, sic-4 SIC1-251-GFP seedlings showed increased tolerance to HS (Fig. 8a, b). The hypersensitivity of sic-4 SIC245–319-GFP seedlings to HS may result from the reduced efficiency of DBR1 out of nuclei (Supplementary Fig. 14b, c). In contrast, the increased tolerance of sic-4 SIC1-251-GFP seedlings to HS could be attributed to the enhanced efficiency of DBR1 out of nuclei due to the overproduction of SIC1-251 (Supplementary Fig. 14b, c). LariRNAs seem to play a role in the downstream regulation of SIC-mediated HS responses in plants. The enhanced accumulation of lariat70-1, lariat70-4, lariat90-3, and lariat90-1, along with their corresponding parental mRNAs under HS, was more pronounced in Col-0 than in sic-4 SIC245–319-GFP (Supplementary Fig. 11). While we cannot entirely exclude additional or non-transcriptional mechanisms, it is possible that the enriched lariRNAs enhance the expression of parental mRNAs by increasing Pol II occupancy at their parental loci during transcription. Consistent with this idea, we showed that sic-4 SIC245–319-GFP seedlings exhibited lower Pol II occupancy at the genomic loci of two critical HSP genes compared to Col-0 under HS (Fig. 7e–h), which aligns with findings in human cells where lariRNAs influence gene expression by modulating Pol II dynamics37. However, the exact mechanism by which lariRNAs increase Pol II occupancy to regulate gene expression remains unknown. It is likely that nucleus-localized lariRNAs function similarly to exon-intron circRNAs (EIciRNAs) in human cells. EIciRNAs associate with Pol II in the nucleus, interact with U1 small nuclear ribonucleoproteins, and enhance the expression of their parental genes in cis via specific RNA–RNA interactions77. This suggests that lariRNAs might play a similar role in plants by facilitating interactions that enhance transcriptional activity at their parental gene loci. Further research is needed to elucidate the precise mechanisms involved.

SIC functions in microRNA biogenesis, mRNA decay, and pre-mRNA splicing50,51. Additionally, SIC promotes the accumulation of DBR1 in the nucleus through its two redundant nuclear localization regions, which in turn promote lariRNA decay50. In this study, we propose a regulatory mechanism by which lariRNAs mediate HS tolerance in Arabidopsis. The SIC facilitates the sequestration of DBR1 into SGs, thereby limiting its ability to degrade lariRNAs. This allows the lariRNAs to upregulate the transcription of their HS-associated parental genes, enhancing HS tolerance (Fig. 8e). Intriguingly, the accumulation of DBR1-dependent debranched lariRNAs leads to developmental defects and even lethality31,39,50,55. This suggests that the homeostasis of DBR1-dependent lariRNAs, similar to other plant regulators, such as miRNAs, abscisic acid, and salicylic acid, which display dual roles with both positive and negative effects, need to be spatially and temporally controlled in plants. A recent study reports that the levels of lariat RNAs reveal tissue-specific regulation and have the potential to protect the expression of miRNA target genes78. In the future, it will be intriguing to investigate how the homeostasis of lariRNAs is controlled and whether other stresses also induce the accumulation of lariRNAs and to explore the potential of using lariRNAs to enhance stress tolerance in crops. This opens up new avenues for research into stress tolerance mechanisms and the possible biotechnological applications in agriculture to improve crop resilience under various stress conditions.

Methods

Plant materials and growth conditions

All Arabidopsis (Arabidopsis thaliana) plants used in this study were in the accession Columbia-0 (Col-0) background. Surface-sterilized, stratified seeds were plated on half-strength Murashige and Skoog (MS) solid medium containing 1% (w/v) sucrose and 1.2% (w/v) agar and incubated in a growth chamber at 22 °C under a 16-h light/8-h dark photoperiod. Seven-day-old seedlings were harvested for experiments or transferred to soil. All primers used in this study are listed in Supplementary Data 3.

Bioinformatics

Prion-like domains were identified using Prion-Like Amino Acid Composition (PLAAC)79, with a minimum length for prion domains Lcore = 60, organism background Arabidopsis, and the parameter α = 1. Per-residue disorder content was evaluated using PONDR predictors, including PONDR-FIT80 and PONDR-VSL281. The intrinsic disorder propensities of SIC were evaluated as previously described82. Disorder evaluations, together with disorder-related functional information, were retrieved from the Database of Disordered Protein Predictions D2P2 (http://d2p2.pro/)65. Intrinsically disordered regions were predicted using Iupred2A83. phase separation predisposition was evaluated using the PSPredictor tool84. Protein structure prediction was conducted with the online AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/)85. Image analysis was performed using ImageJ software.

Plasmid construction and plant materials

The plasmids used in this study were generated using Gateway cloning. The full-length coding sequences of SIC, PAB2, and PAB8 amplified from Col-0 cDNA were cloned into pDONR™207. The individual genes in pDONR207 were recombined into the different destination vectors via LR reaction (LR Clonase II Plus, Thermo Fisher). For the PAB2- and PAB8-mRFP fusion constructs, the individual genes in pDONR207 were transferred into the plant expression vector pB7RWG2. For the His6-SIC-GFP construct, the full-length coding sequence of SIC was cloned into pET28a-GFP by In-Fusion cloning (ClonExpress II One Step Cloning Kit, Vazyme). For the His6-DBR1-mRuby construct, the full-length coding sequence of DBR1 was cloned into pET28a-mRuby by In-Fusion cloning (ClonExpress II One Step Cloning Kit, Vazyme). To construct the lariat70-4-OE and lariat90-1-OE expression vectors, the intron sequence of HSP70-4 or HSP90-1 was amplified and inserted between nYFP and cYFP by overlap PCR and transferred into pDONR207, followed by pB7WG2. All primers used for cloning are listed in Supplementary Data 3.

Arabidopsis Columbia (Col-0) was used as WT in this study. To obtain transgenic plants, the binary constructs were transformed into Agrobacterium (Agrobacterium tumefaciens) strain GV3101 by the freeze-thaw method and transformed into Col-0 or the indicated mutant plants using the floral dip method86. The sic-4, dbr1-2, proSIC:SIC-GFP, proDBR1:DBR1-GFP, proDBR1:DBR1-mRFP, sic-4 SIC1–244-GFP, sic‐4 SIC1–251-GFP, SIC245–319-GFP, and sic-4 SIC252–319-GFP lines were as described previously50. The lariat70-4-OE, lariat90-1-OE, Hsp70-4-OE, Hsp90-1-OE, proPAB2:PAB2-GFP, proPAB2:PAB2-mRFP, proPAB8:PAB8-GFP, and proPAB8:PAB8-mRFP lines were generated by transgenic transformation. Double fluorescence lines, dbr1-2 proSIC:SIC-GFP, sic-4 proDBR1:DBR1-GFP, and sic-4 proPAB2:PAB2-mRFP lines were generated by genetic crosses and combined phenotyping and genotyping of F2 progeny.

Protein production and purification

All proteins were produced in Escherichia coli BL21 (DE3) cells (Tiangen) in the presence of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). Cells were induced overnight at 18 °C, collected by centrifugation, and resuspended in lysis buffer (20 mM HEPES pH 7.4, 500 mM KCl, 1 mM PMSF). The cells were lysed using a high-pressure homogenizer (ATS Engineering) and centrifuged at 10,000 × g for 30 min. The supernatants were first purified with Ni-NTA or amylose resin (GenScript), followed by purification on a Superdex 200 increase 10/300 column (SD200) (GE Healthcare). The proteins were flash-frozen in liquid nitrogen and stored in a storage buffer (20 mM Tris-HCl pH 8.0, 1 mM DDT, 50 mM NaCl, 10% [v/v] glycerol) at −80 °C.

Microscopy analysis

Microscopy images were acquired under a Zeiss LSM880 laser scanning confocal microscope (LSCM) or a Nikon A1 LSCM. Root meristem cells from 5-day-old vertically grown seedlings were used to determine the subcellular localization of proteins. For HS treatment, 5-day-old seedlings were transferred to a growth chamber set to 39 °C and incubated for 40 min. For cycloheximide (CHX) treatment, 5-day-old seedlings were transferred to a half-strength MS liquid medium with 50 μM CHX and incubated for 30 min at room temperature, after which they were subjected to HS at 39 °C for 40 min. GFP fluorescence was observed after excitation using a 488-nm laser and detected using the bandpass 505–530-nm emission filter setting. mRFP was observed after excitation using a 561-nm laser and detected using the bandpass 605–630-nm emission filter setting. For granules analysis, the meristem of the root and the same cell layer and more than eight images (about 20 cells per image) from eight different roots were examined. The number of granules was quantified with ImageJ. The Pearson-Spearman correlation (PSC) plugin for ImageJ was used for correlation analysis87.

For in vivo time-lapse microscopy of SIC cytoplasmic granules, 5-day-old proSIC:SIC-GFP seedlings were treated at 39 °C for 40 min. The seedlings were transferred into glass-bottom dishes (Cellvis, D35-20-1-N) containing half-strength MS liquid medium with 1% (w/v) sucrose and 0.3% (w/v) electrophoresis-grade agarose. High-resolution images of root tip cells were obtained for the indicated time periods. At least four cells from different roots were analyzed. For the in vitro experiments, at least five droplets were analyzed. The recovery curves were generated with ZEN software.

In vitro droplet assay

In vitro droplet assays were performed in a buffer containing 20 mM Tris-HCl pH 8.0, 1 mM DDT, 50 mmol/L NaCl, and 10% (v/v) glycerol. Droplets were observed under a Zeiss LSM880 microscope equipped with a ×63/1.4 oil immersion objective.

RNA extraction and RNA-seq library preparation

RNA isolation, RNA-seq, and data analysis were performed as previously described88. Briefly, total RNA was isolated from the different genotypes using TRIzol (Ambion). Total RNA was treated with a Ribo Zero kit (Epicenter) to obtain rRNA-depleted RNA, incubated with or without RNase R (Epicenter), and subjected to phenol:chloroform purification. Purified RNAs were used for library preparation with a NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420), and the libraries were sequenced on an Illumina HiSeq 2500 instrument. Three replicates were performed per sample.

RNA FISH

RNA FISH was performed as previously described59. Seven-day-old transgenic seedlings were fixed in 50% (v/v) fixation buffer (120 mM NaCl, 7 mM disodium phosphate, 3 mM monosodium phosphate pH 7.5, 2.7 mM KCl, 0.1% [v/v] Tween-20, 80 mM EGTA pH 8.0, 5% [w/v] formaldehyde, 10% [v/v] DMSO, 50% [v/v] heptane) for 30 min at room temperature with shaking, dehydrated in 100% methanol twice (5 min each time), and dehydrated in 100% ethanol three times (5 min each). The seedlings were incubated in 1:1 (v/v) ethanol:xylene for 30 min, dehydrated in 100% ethanol twice (5 min each), and dehydrated in 100% methanol twice (5 min each). The seedlings were postfixed in 1:1 (v/v) methanol:fixation buffer without formaldehyde for 30 min at room temperature and rinsed twice in fixation buffer without formaldehyde. The seedlings were rinsed in 1 mL of PerfectHyb Plus hybridization buffer (Sigma-Aldrich; H-7033) and prehybridized in 1 mL of hybridization buffer for 1 h at 50 °C. After adding 1 μL of 10 μM 5′-Cy3-labeled probes, the samples were incubated in the dark at 50 °C for 12 h. After hybridization, the samples were washed for 60 min in 2 × SSC (300 mM NaCl, 30 mM sodium citrate pH 7.0) containing 0.1% (w/v) SDS, followed by a 20 min wash in 0.2 × SSC containing 0.1% (w/v) SDS at 50 °C. The samples were stored in 0.2 × SSC containing 0.1% (w/v) SDS until imaging.

Expression analysis of genes and lariRNAs from RNA-seq data

Expression analysis of genes and lariRNAs from RNA-seq data was performed as previously described50. Briefly, the raw data of RNA-seq profiles were filtered out using Fastp with parameters of default values to output clean reads, which were then aligned to the reference genome of A. thaliana (TAIR10) using Hisat2. Samtools and HTSeq-count were used to count the reads of a number of genes or introns and their expression level was normalized as FPKM by a custom program. Differential gene expression analysis was performed using DESeq2, based on the negative binomial distribution. A threshold value of P-adjusted value (FDR) <0.05 was used to obtain differentially expressed introns between two replicates for each sample and introns of chloroplast and mitochondrion genes that not changed in gene expression level were used as spike-in for Deseq2 normalization, as chloroplasts and mitochondrion lack the intron lariat debranching enzyme in Arabidopsis31.

Reverse-transcription quantitative PCR (RT-qPCR)

Total RNA was isolated from 7-day-old Arabidopsis seedlings grown on liquid half-strength MS medium with or without HS (39 °C for 40 min) using TRIzol reagent. First-strand cDNA was generated using a PrimeScript II 1st Strand cDNA Synthesis Kit (Takara) according to the manufacturer’s instructions. qPCR was performed using a Roche LightCycler480 with LightCycler 480 SYBR Green I Master kit (Roche) following the manufacturer’s protocol. The data were normalized to TUB6 expression as an internal control. Three biological replicates (different seedlings sampled at different times) were performed per gene. Primers are listed in Supplementary Data 3.

Detection of lariRNAs

LariRNAs were detected by RT-PCR as previously described50. Total RNA with or without RNase R treatment was used as templates. Reverse transcriptional reaction was performed using SuperScript III (Invitrogen) with a random hexamer. The reaction mixtures were incubated at 30 °C for 10 min, at 42 °C for 120 min, at 50 °C for 30 min, at 60 °C for 30 min, and at 99 °C for 5 min. A divergent primer set was used to detect lariRNAs by RT-qPCR. TUBULIN ALPHA-6 (TUB6) or UBQ5 was used as a reference gene. Primer sequences used are listed in Supplementary Data 3.

Chromatin immunoprecipitation (ChIP)-qPCR

ChIP experiments were performed as previously described89. Nuclei were purified from 2 g of tissue from 7-day-old seedlings. An anti-phospho-RPB1 CTD (Ser2) (E1Z3G) antibody (Cell Signaling Technology, Cat. #13499) was used for ChIP assays. Rabbit immunoglobulin G (IgG) was used as a negative control. The genomic fragments were enriched and eluted into 100 µL of TE buffer. A 2 µL sample was used for each qPCR. ACTIN7 was used as a reference gene. Primers are listed in Supplementary Data 3.

Nuclear run-on assay

The nuclear run-on assay was performed as previously described67. Briefly, 0.5 g of tissue was collected from 7-day-old seedlings grown at 22 °C in a growth chamber with or without HS (39 °C for 40 min). The tissue was ground to a fine powder in liquid nitrogen and resuspended in 30 mL of lysis buffer (20 mM Tris-HCl pH 7.5, 20 mM KCl, 2 mM EDTA pH 8.0, 2.5 mM MgCl2, 25% [v/v] glycerol, 250 mM sucrose, 5 mM DTT, 1 tablet per 50 mL of cOmplete EDTA-free protease inhibitor). Following centrifugation at 2000 × g for 10 min at 4 °C, the precipitates were washed with 10 mL NRBT buffer (20 mM Tris-HCl pH 7.4, 25% [v/v] glycerol, 2.5 mM MgCl2, 0.2% [v/v] Triton X-100, 4 mM DTT). The precipitates were resuspended in 50 µL Nuclei Storage buffer (50 mM Tris-HCl pH 7.8, 1 mM DTT, 20% [v/v] glycerol, 5 mM MgCl2, 0.44 M sucrose) and added to the transcription system: 10 µL 10 × Transcription buffer (50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 150 mM KCl, 0.2% [v/v] Sarkosyl, 20 U/mL RNase inhibitor, 1 mM DTT), 5 µL NTP mixture (100 mM ATP, 100 mM CTP, 100 mM GTP, 100 mM BrUTP), and 35 µL RNase-free H2O. The run-on reaction was performed at 30 °C for 30 min. RNAs were extracted with TRIzol reagent followed by DNase I treatment to remove genomic DNAs. The purified RNAs were incubated with 60 µL anti-BrdU beads (Santa Cruz) at 4 °C for 2 h. After immunoprecipitation, the beads were washed twice with low-salt buffer (0.2 × SSPE [0.03 M NaCl, 2 mM NaH2PO4, 0.2 mM EDTA, pH 7.4], 1 mM EDTA pH 8.0, 0.05% [v/v] Tween-20) at 4 °C for 10 min each time, followed by two washes with high-salt buffer (0.5 × SSPE [0.075 M NaCl, 5 mM NaH2PO4, 0.5 mM EDTA, pH 7.4], 0.05% Tween-20, 37.5 mM NaCl, 1 mM EDTA pH 8.0) at 4 °C for 10 min each time. The precipitated RNAs were extracted with TRIzol reagent and used for cDNA synthesis and qPCR analysis. All measured samples were normalized to TUB6 transcript levels.

Cell fractionation

Cell fraction was performed as described before with some modifications90. Briefly, about 1.0 g of the 10-day-old Arabidopsis seedlings was ground into fine powder in liquid nitrogen and suspended in 1.5-mL fractionation buffer (20 mM Tris-HCl pH 7.4, 25% Glycerol, 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl2, 250 mM Sucrose, 1 mM DTT, 1 mM PMSF, 0.7% Triton X-100, 1 x of Roche protease inhibitor cocktail). The homogenate was sequentially filtered through 100-μm and 40-μm mesh nylon, and 100-μL flow-through was aliquoted as the total protein sample. The rest flow-through was centrifuged at 1500 × g for 10 min at 4 °C. The supernatant was centrifuged at 3000 × g for 5 min at 4 °C, and then collected as the cytosolic fraction. The pellet was washed four times with 5 mL of NRBT buffer (20 mM Tris-HCl pH 7.4, 25% Glycerol, 2.5 mM MgCl2, 0.2% Triton X-100, 4 mM DTT, 1 x of Roche protease inhibitor cocktail) and centrifuged at 1500 × g for 10 min at 4 °C to pellet the nuclei. The nuclear pellet was resuspended in 300 μL of Lysis buffer (20 mM Tris-HCl pH 7.4, 25% Glycerol, 2.5 mM MgCl2, 0.2% Triton X-100, 4 mM DTT, 250 mM NaCl, 1 x of Roche protease inhibitor cocktail). Then, the mixture was vortexed and incubated for 1 h on ice to obtain the lysis of the nucleus as the nucleus fraction. Protein samples were mixed with 5 x SDS loading buffer and were boiled at 95 °C for 10 min. We loaded 10 μL (~1%) of the total and the cytosol sample and 30 μL (~10%) of the nuclear sample on the SDS-PAGE gel. Anti-DBR1 (obtained from Prof. Binglian Zheng’s laboratory), anti-Actin (Abiocode, USA, Cat. #R3772-1P), and anti-Histone H3 (Abcam, USA, Cat. #ab18521) antibodies were used for immunoblotting. For lariRNAs detection in cell fractions, the cytosol and the nucleus fractions were used for RNA extraction, and then lariRNAs were detected by RT-PCR.

Statistics and reproducibility

Statistical parameters are reported in the figures and figure legends. All data were presented herein as the mean ± standard deviation and were compared using one-way ANOVA, two-way ANOVA, or two-tailed, two-sample Student’s t-tests whenever appropriate using GraphPad Prism 8 software. The statistical analysis for each experiment is described in the figure legends. The P value < 0.05 was considered to indicate statistical significance.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary information

Peer Review File

Description of Additional Supplementary Files

Supplementary Data 1

Supplementary Data 2

Supplementary Data 3

Reporting Summary

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-024-52034-w.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 32370324 and 32370320), the National Key R&D Program of China (No. 2023YFA0914600 and 2022YFF1001700), the Natural Science Foundation of Henan Province (222300420026), the Hainan Provincial Joint Project of Sanya Yazhou Bay Science and Technology City (No. 2021JJLH0044), the Guiding Funds of the Central Government for Supporting the Development of the Local Science and Technology in Henan Province (No. Z20221343006), the 111 Project (No. D16014), the Program for High‑level Talents Recruitment of Anhui Agricultural University (No. rc422305), State Key Laboratory for Protein and Plant Gene Research, School of Life Sciences, Peking University, National Natural Science Foundation of China (No. 31970641), the Center for Life Sciences to W.W., the International Postdoctoral Exchange Fellowship Program and Postdoctoral Fellowship of Center for Life Sciences, and the National Natural Science Foundation of China (No. 3220050423) to Z.X.

Author contributions

Z.H. and C.Y.W. designed all experiments, analyzed the data, and wrote the manuscript. C.Y.W. and X.S.W. performed most of the experiments and prepared the data; C.F.W. and X.Q.W. contributed to the generation of transgenic plants and helped with the genotyping and phenotyping, RT-qPCR, and ChIP-qPCR analysis; Z.X. and W.W. helped with the FISH analysis; Y.L. and W.Z. performed the bioinformatics analysis; X.X., S.G., J.R.B., B.Z., W.W., and C.-P.S. edited and revised the manuscript.

Peer review

Peer review information

Nature Communications thanks Peter Bozhkov, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

The RNA-seq and lariRNA-seq data generated in this study have been deposited in the SRA database under accession code PRJCA019776. Source data are provided with this paper.

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.

These authors contributed equally: Chengyun Wu, Xingsong Wang.
==== Refs
References

1. Ohama N Sato H Shinozaki K Yamaguchi-Shinozaki K Transcriptional regulatory network of plant heat stress response Trends Plant Sci. 2017 22 53 65 10.1016/j.tplants.2016.08.015 27666516
Ohama, N., Sato, H., Shinozaki, K. & Yamaguchi-Shinozaki, K. Transcriptional regulatory network of plant heat stress response. Trends Plant Sci. 22, 53–65 (2017).27666516 10.1016/j.tplants.2016.08.015
2. Battisti DS Naylor RL Historical warnings of future food insecurity with unprecedented seasonal heat Science 2009 323 240 244 10.1126/science.1164363 19131626
Battisti, D. S. & Naylor, R. L. Historical warnings of future food insecurity with unprecedented seasonal heat. Science 323, 240–244 (2009).19131626 10.1126/science.1164363
3. Zhao C Temperature increase reduces global yields of major crops in four independent estimates Proc. Natl Acad. Sci. USA 2017 114 9326 9331 10.1073/pnas.1701762114 28811375
Zhao, C. et al. Temperature increase reduces global yields of major crops in four independent estimates. Proc. Natl Acad. Sci. USA 114, 9326–9331 (2017).28811375 10.1073/pnas.1701762114
4. Zhao, J., Lu, Z., Wang, L. & Jin, B. Plant responses to heat stress: physiology, transcription, noncoding RNAs, and epigenetics. Int. J. Mol. Sci. 22, 117 (2020).
5. Guo M The plant heat stress transcription factors (HSFs): structure, regulation, and function in response to abiotic stresses Front. Plant Sci. 2016 7 114 10.3389/fpls.2016.00114 26904076
Guo, M. et al. The plant heat stress transcription factors (HSFs): structure, regulation, and function in response to abiotic stresses. Front. Plant Sci. 7, 114 (2016).26904076 10.3389/fpls.2016.00114
6. Charng YY A heat-inducible transcription factor, HsfA2, is required for extension of acquired thermotolerance in Arabidopsis Plant Physiol. 2007 143 251 262 10.1104/pp.106.091322 17085506
Charng, Y. Y. et al. A heat-inducible transcription factor, HsfA2, is required for extension of acquired thermotolerance in Arabidopsis. Plant Physiol. 143, 251–262 (2007).17085506 10.1104/pp.106.091322
7. Wang WX Vinocur B Shoseyov O Altman A Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response Trends Plant Sci. 2004 9 244 252 10.1016/j.tplants.2004.03.006 15130550
Wang, W. X., Vinocur, B., Shoseyov, O. & Altman, A. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 9, 244–252 (2004).15130550 10.1016/j.tplants.2004.03.006
8. Protter DSW Parker R Principles and properties of stress granules Trends Cell Biol. 2016 26 668 679 10.1016/j.tcb.2016.05.004 27289443
Protter, D. S. W. & Parker, R. Principles and properties of stress granules. Trends Cell Biol. 26, 668–679 (2016).27289443 10.1016/j.tcb.2016.05.004
9. Kosmacz M Protein and metabolite composition of Arabidopsis stress granules N. Phytol. 2019 222 1420 1433 10.1111/nph.15690
Kosmacz, M. et al. Protein and metabolite composition of Arabidopsis stress granules. N. Phytol. 222, 1420–1433 (2019).10.1111/nph.15690
10. Chantarachot T Bailey-Serres J Polysomes, stress granules, and processing bodies: a dynamic triumvirate controlling cytoplasmic mRNA fate and function Plant Physiol. 2018 176 254 269 10.1104/pp.17.01468 29158329
Chantarachot, T. & Bailey-Serres, J. Polysomes, stress granules, and processing bodies: a dynamic triumvirate controlling cytoplasmic mRNA fate and function. Plant Physiol. 176, 254–269 (2018).29158329 10.1104/pp.17.01468
11. Decker CJ Parker R P-bodies and stress granules: possible roles in the control of translation and mRNA degradation Cold Spring Harb. Perspect. Biol. 2012 4 a012286 10.1101/cshperspect.a012286 22763747
Decker, C. J. & Parker, R. P-bodies and stress granules: possible roles in the control of translation and mRNA degradation. Cold Spring Harb. Perspect. Biol. 4, a012286 (2012).22763747 10.1101/cshperspect.a012286
12. Anderson P Kedersha N RNA granules: post-transcriptional and epigenetic modulators of gene expression Nat. Rev. Mol. Cell Biol. 2009 10 430 436 10.1038/nrm2694 19461665
Anderson, P. & Kedersha, N. RNA granules: post-transcriptional and epigenetic modulators of gene expression. Nat. Rev. Mol. Cell Biol. 10, 430–436 (2009).19461665 10.1038/nrm2694
13. Tong J ALBA proteins confer thermotolerance through stabilizing HSF messenger RNAs in cytoplasmic granules Nat. Plants 2022 8 778 791 10.1038/s41477-022-01175-1 35817823
Tong, J. et al. ALBA proteins confer thermotolerance through stabilizing HSF messenger RNAs in cytoplasmic granules. Nat. Plants 8, 778–791 (2022).35817823 10.1038/s41477-022-01175-1
14. Maruri-Lopez I Figueroa NE Hernandez-Sanchez IE Chodasiewicz M Plant stress granules: trends and beyond Front. Plant Sci. 2021 12 722643 10.3389/fpls.2021.722643 34434210
Maruri-Lopez, I., Figueroa, N. E., Hernandez-Sanchez, I. E. & Chodasiewicz, M. Plant stress granules: trends and beyond. Front. Plant Sci. 12, 722643 (2021).34434210 10.3389/fpls.2021.722643
15. Thomas MG Loschi M Desbats MA Boccaccio GL RNA granules: the good, the bad and the ugly Cell Signal 2011 23 324 334 10.1016/j.cellsig.2010.08.011 20813183
Thomas, M. G., Loschi, M., Desbats, M. A. & Boccaccio, G. L. RNA granules: the good, the bad and the ugly. Cell Signal 23, 324–334 (2011).20813183 10.1016/j.cellsig.2010.08.011
16. Loschi M Leishman CC Berardone N Boccaccio GL Dynein and kinesin regulate stress-granule and P-body dynamics J. Cell Sci. 2009 122 3973 3982 10.1242/jcs.051383 19825938
Loschi, M., Leishman, C. C., Berardone, N. & Boccaccio, G. L. Dynein and kinesin regulate stress-granule and P-body dynamics. J. Cell Sci. 122, 3973–3982 (2009).19825938 10.1242/jcs.051383
17. Kato M Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels Cell 2012 149 753 767 10.1016/j.cell.2012.04.017 22579281
Kato, M. et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753–767 (2012).22579281 10.1016/j.cell.2012.04.017
18. Molliex A Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization Cell 2015 163 123 133 10.1016/j.cell.2015.09.015 26406374
Molliex, A. et al. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163, 123–133 (2015).26406374 10.1016/j.cell.2015.09.015
19. Van Treeck B RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome Proc. Natl Acad. Sci. USA 2018 115 2734 2739 10.1073/pnas.1800038115 29483269
Van Treeck, B. et al. RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome. Proc. Natl Acad. Sci. USA 115, 2734–2739 (2018).29483269 10.1073/pnas.1800038115
20. Wolozin B Ivanov P Stress granules and neurodegeneration Nat. Rev. Neurosci. 2019 20 649 666 10.1038/s41583-019-0222-5 31582840
Wolozin, B. & Ivanov, P. Stress granules and neurodegeneration. Nat. Rev. Neurosci. 20, 649–666 (2019).31582840 10.1038/s41583-019-0222-5
21. Anderson P Kedersha N Ivanov P Stress granules, P-bodies and cancer Biochim. Biophys. Acta 2015 1849 861 870 10.1016/j.bbagrm.2014.11.009 25482014
Anderson, P., Kedersha, N. & Ivanov, P. Stress granules, P-bodies and cancer. Biochim. Biophys. Acta 1849, 861–870 (2015).25482014 10.1016/j.bbagrm.2014.11.009
22. Tian, X. et al. Stress granule associated TaMBF1c confers thermotolerance through regulating specific mRNA translation in wheat (Triticum aestivum). New Phytol. 233, 1719–1731 (2022).
23. Gutierrez-Beltran, E. et al. Tudor staphylococcal nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis. EMBO J. 40, e105043 (2021).
24. Frei dit Frey N The RNA binding protein Tudor-SN is essential for stress tolerance and stabilizes levels of stress-responsive mRNAs encoding secreted proteins in Arabidopsis Plant Cell 2010 22 1575 1591 10.1105/tpc.109.070680 20484005
Frei dit Frey, N. et al. The RNA binding protein Tudor-SN is essential for stress tolerance and stabilizes levels of stress-responsive mRNAs encoding secreted proteins in Arabidopsis. Plant Cell 22, 1575–1591 (2010).20484005 10.1105/tpc.109.070680
25. Nguyen CC Oligouridylate binding protein 1b plays an integral role in plant heat stress tolerance Front. Plant Sci. 2016 7 853 10.3389/fpls.2016.00853 27379136
Nguyen, C. C. et al. Oligouridylate binding protein 1b plays an integral role in plant heat stress tolerance. Front. Plant Sci. 7, 853 (2016).27379136 10.3389/fpls.2016.00853
26. Zhu S Liquid-liquid phase separation of RBGD2/4 is required for heat stress resistance in Arabidopsis Dev. Cell 2022 57 583 597.e586 10.1016/j.devcel.2022.02.005 35231447
Zhu, S. et al. Liquid-liquid phase separation of RBGD2/4 is required for heat stress resistance in Arabidopsis. Dev. Cell 57, 583–597.e586 (2022).35231447 10.1016/j.devcel.2022.02.005
27. Ruiz-Solani, N. et al. Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates and delays senescence. Plant Cell 35, 3325–3344 (2023).
28. Domdey H Lariat structures are in vivo intermediates in yeast pre-mRNA splicing Cell 1984 39 611 621 10.1016/0092-8674(84)90468-9 6096014
Domdey, H. et al. Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell 39, 611–621 (1984).6096014 10.1016/0092-8674(84)90468-9
29. Ruskin B Green MR An RNA processing activity that debranches RNA lariats Science 1985 229 135 140 10.1126/science.2990042 2990042
Ruskin, B. & Green, M. R. An RNA processing activity that debranches RNA lariats. Science 229, 135–140 (1985).2990042 10.1126/science.2990042
30. Nam K Lee G Trambley J Devine SE Boeke JD Severe growth defect in a Schizosaccharomyces pombe mutant defective in intron lariat degradation Mol. Cell Biol. 1997 17 809 818 10.1128/MCB.17.2.809 9001235
Nam, K., Lee, G., Trambley, J., Devine, S. E. & Boeke, J. D. Severe growth defect in a Schizosaccharomyces pombe mutant defective in intron lariat degradation. Mol. Cell Biol. 17, 809–818 (1997).9001235 10.1128/MCB.17.2.809
31. Wang H Hill K Perry SE An Arabidopsis RNA lariat debranching enzyme is essential for embryogenesis J. Biol. Chem. 2004 279 1468 1473 10.1074/jbc.M309106200 14570879
Wang, H., Hill, K. & Perry, S. E. An Arabidopsis RNA lariat debranching enzyme is essential for embryogenesis. J. Biol. Chem. 279, 1468–1473 (2004).14570879 10.1074/jbc.M309106200
32. Talhouarne GJS Gall JG Lariat intronic RNAs in the cytoplasm of vertebrate cells Proc. Natl Acad. Sci. USA 2018 115 E7970 E7977 10.1073/pnas.1808816115 30082412
Talhouarne, G. J. S. & Gall, J. G. Lariat intronic RNAs in the cytoplasm of vertebrate cells. Proc. Natl Acad. Sci. USA 115, E7970–E7977 (2018).30082412 10.1073/pnas.1808816115
33. Zhang X A comprehensive map of intron branchpoints and lariat RNAs in plants Plant Cell 2019 31 956 973 10.1105/tpc.18.00711 30894459
Zhang, X. et al. A comprehensive map of intron branchpoints and lariat RNAs in plants. Plant Cell 31, 956–973 (2019).30894459 10.1105/tpc.18.00711
34. Tay ML Pek JW Maternally inherited stable intronic sequence RNA triggers a self-reinforcing feedback loop during development Curr. Biol. 2017 27 1062 1067 10.1016/j.cub.2017.02.040 28343963
Tay, M. L. & Pek, J. W. Maternally inherited stable intronic sequence RNA triggers a self-reinforcing feedback loop during development. Curr. Biol. 27, 1062–1067 (2017).28343963 10.1016/j.cub.2017.02.040
35. Wong JT DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1 Nat. Commun. 2017 8 759 10.1038/s41467-017-00684-4 28970471
Wong, J. T. et al. DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1. Nat. Commun. 8, 759 (2017).28970471 10.1038/s41467-017-00684-4
36. Suzuki H Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing Nucleic Acids Res. 2006 34 e63 10.1093/nar/gkl151 16682442
Suzuki, H. et al. Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing. Nucleic Acids Res. 34, e63 (2006).16682442 10.1093/nar/gkl151
37. Zhang Y Circular intronic long noncoding RNAs Mol. Cell 2013 51 792 806 10.1016/j.molcel.2013.08.017 24035497
Zhang, Y. et al. Circular intronic long noncoding RNAs. Mol. Cell 51, 792–806 (2013).24035497 10.1016/j.molcel.2013.08.017
38. Talhouarne GJ Gall JG Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes RNA 2014 20 1476 1487 10.1261/rna.045781.114 25051970
Talhouarne, G. J. & Gall, J. G. Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes. RNA 20, 1476–1487 (2014).25051970 10.1261/rna.045781.114
39. Cheng J A lariat-derived circular RNA is required for plant development in Arabidopsis Sci. China Life Sci. 2018 61 204 213 10.1007/s11427-017-9182-3 29101586
Cheng, J. et al. A lariat-derived circular RNA is required for plant development in Arabidopsis. Sci. China Life Sci. 61, 204–213 (2018).29101586 10.1007/s11427-017-9182-3
40. Mohanta A Chakrabarti K Dbr1 functions in mRNA processing, intron turnover and human diseases Biochim. 2021 180 134 142 10.1016/j.biochi.2020.10.003
Mohanta, A. & Chakrabarti, K. Dbr1 functions in mRNA processing, intron turnover and human diseases. Biochim. 180, 134–142 (2021).10.1016/j.biochi.2020.10.003
41. Ooi SL Samarsky DA Fournier MJ Boeke JD Intronic snoRNA biosynthesis in Saccharomyces cerevisiae depends on the lariat-debranching enzyme: intron length effects and activity of a precursor snoRNA RNA 1998 4 1096 1110 10.1017/S1355838298980785 9740128
Ooi, S. L., Samarsky, D. A., Fournier, M. J. & Boeke, J. D. Intronic snoRNA biosynthesis in Saccharomyces cerevisiae depends on the lariat-debranching enzyme: intron length effects and activity of a precursor snoRNA. RNA 4, 1096–1110 (1998).9740128 10.1017/S1355838298980785
42. Okamura K Hagen JW Duan H Tyler DM Lai EC The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila Cell 2007 130 89 100 10.1016/j.cell.2007.06.028 17599402
Okamura, K., Hagen, J. W., Duan, H., Tyler, D. M. & Lai, E. C. The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila. Cell 130, 89–100 (2007).17599402 10.1016/j.cell.2007.06.028
43. Ruby JG Jan CH Bartel DP Intronic microRNA precursors that bypass Drosha processing Nature 2007 448 83 86 10.1038/nature05983 17589500
Ruby, J. G., Jan, C. H. & Bartel, D. P. Intronic microRNA precursors that bypass Drosha processing. Nature 448, 83–86 (2007).17589500 10.1038/nature05983
44. Chen LL The biogenesis and emerging roles of circular RNAs Nat. Rev. Mol. Cell Biol. 2016 17 205 211 10.1038/nrm.2015.32 26908011
Chen, L. L. The biogenesis and emerging roles of circular RNAs. Nat. Rev. Mol. Cell Biol. 17, 205–211 (2016).26908011 10.1038/nrm.2015.32
45. Zhang, P. & Dai, M. CircRNA: a rising star in plant biology. J. Genet. Genomics 49, 1081–1092 (2022).
46. Lai X CircRNAs in plants Adv. Exp. Med. Biol. 2018 1087 329 343 10.1007/978-981-13-1426-1_26 30259378
Lai, X. et al. CircRNAs in plants. Adv. Exp. Med. Biol. 1087, 329–343 (2018).30259378 10.1007/978-981-13-1426-1_26
47. Zhan X Arabidopsis proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis Proc. Natl Acad. Sci. USA 2012 109 18198 18203 10.1073/pnas.1216199109 23071326
Zhan, X. et al. Arabidopsis proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis. Proc. Natl Acad. Sci. USA 109, 18198–18203 (2012).23071326 10.1073/pnas.1216199109
48. Karampelias M ROTUNDA3 function in plant development by phosphatase 2A-mediated regulation of auxin transporter recycling Proc. Natl Acad. Sci. USA 2016 113 2768 2773 10.1073/pnas.1501343112 26888284
Karampelias, M. et al. ROTUNDA3 function in plant development by phosphatase 2A-mediated regulation of auxin transporter recycling. Proc. Natl Acad. Sci. USA 113, 2768–2773 (2016).26888284 10.1073/pnas.1501343112
49. Marshall CM Tartaglio V Duarte M Harmon FG The Arabidopsis sickle mutant exhibits altered circadian clock responses to cool temperatures and temperature-dependent alternative splicing Plant Cell 2016 28 2560 2575 10.1105/tpc.16.00223 27624757
Marshall, C. M., Tartaglio, V., Duarte, M. & Harmon, F. G. The Arabidopsis sickle mutant exhibits altered circadian clock responses to cool temperatures and temperature-dependent alternative splicing. Plant Cell 28, 2560–2575 (2016).27624757 10.1105/tpc.16.00223
50. Wu C SICKLE modulates lateral root development by promoting degradation of lariat intronic RNA Plant Physiol. 2022 190 548 561 10.1093/plphys/kiac301 35788403
Wu, C. et al. SICKLE modulates lateral root development by promoting degradation of lariat intronic RNA. Plant Physiol. 190, 548–561 (2022).35788403 10.1093/plphys/kiac301
51. Wu, C. et al. Absence of SICKLE triggers programmed cell death by disturbing alternative splicing and decay of mRNAs. Plant Physiol. 192, 2523–2536 (2023).
52. Chen LL The expanding regulatory mechanisms and cellular functions of circular RNAs Nat. Rev. Mol. Cell Biol. 2020 21 475 490 10.1038/s41580-020-0243-y 32366901
Chen, L. L. The expanding regulatory mechanisms and cellular functions of circular RNAs. Nat. Rev. Mol. Cell Biol. 21, 475–490 (2020).32366901 10.1038/s41580-020-0243-y
53. Saini, H., Bicknell, A. A., Eddy, S. R. & Moore, M. J. Free circular introns with an unusual branchpoint in neuronal projections. Elife 8, e47809 (2019).
54. Jacquier A Rosbash M RNA splicing and intron turnover are greatly diminished by a mutant yeast branch point Proc. Natl Acad. Sci. USA 1986 83 5835 5839 10.1073/pnas.83.16.5835 3090547
Jacquier, A. & Rosbash, M. RNA splicing and intron turnover are greatly diminished by a mutant yeast branch point. Proc. Natl Acad. Sci. USA 83, 5835–5839 (1986).3090547 10.1073/pnas.83.16.5835
55. Li Z Intron Lariat RNA inhibits microRNA biogenesis by sequestering the dicing complex in Arabidopsis PLoS Genet. 2016 12 e1006422 10.1371/journal.pgen.1006422 27870853
Li, Z. et al. Intron Lariat RNA inhibits microRNA biogenesis by sequestering the dicing complex in Arabidopsis. PLoS Genet. 12, e1006422 (2016).27870853 10.1371/journal.pgen.1006422
56. Arenas J Hurwitz J Purification of a RNA debranching activity from HeLa cells J. Biol. Chem. 1987 262 4274 4279 10.1016/S0021-9258(18)61343-2 2435736
Arenas, J. & Hurwitz, J. Purification of a RNA debranching activity from HeLa cells. J. Biol. Chem. 262, 4274–4279 (1987).2435736 10.1016/S0021-9258(18)61343-2
57. Chapman KB Boeke JD Isolation and characterization of the gene encoding yeast debranching enzyme Cell 1991 65 483 492 10.1016/0092-8674(91)90466-C 1850323
Chapman, K. B. & Boeke, J. D. Isolation and characterization of the gene encoding yeast debranching enzyme. Cell 65, 483–492 (1991).1850323 10.1016/0092-8674(91)90466-C
58. Gutierrez-Beltran E Moschou PN Smertenko AP Bozhkov PV Tudor staphylococcal nuclease links formation of stress granules and processing bodies with mRNA catabolism in Arabidopsis Plant Cell 2015 27 926 943 10.1105/tpc.114.134494 25736060
Gutierrez-Beltran, E., Moschou, P. N., Smertenko, A. P. & Bozhkov, P. V. Tudor staphylococcal nuclease links formation of stress granules and processing bodies with mRNA catabolism in Arabidopsis. Plant Cell 27, 926–943 (2015).25736060 10.1105/tpc.114.134494
59. Sorenson R Bailey-Serres J Selective mRNA sequestration by OLIGOURIDYLATE-BINDING PROTEIN 1 contributes to translational control during hypoxia in Arabidopsis Proc. Natl Acad. Sci. USA 2014 111 2373 2378 10.1073/pnas.1314851111 24469793
Sorenson, R. & Bailey-Serres, J. Selective mRNA sequestration by OLIGOURIDYLATE-BINDING PROTEIN 1 contributes to translational control during hypoxia in Arabidopsis. Proc. Natl Acad. Sci. USA 111, 2373–2378 (2014).24469793 10.1073/pnas.1314851111
60. Khong A The stress granule transcriptome reveals principles of mRNA accumulation in stress granules Mol. Cell 2017 68 808 820.e805 10.1016/j.molcel.2017.10.015 29129640
Khong, A. et al. The stress granule transcriptome reveals principles of mRNA accumulation in stress granules. Mol. Cell 68, 808–820.e805 (2017).29129640 10.1016/j.molcel.2017.10.015
61. Jain S ATPase-modulated stress granules contain a diverse proteome and substructure Cell 2016 164 487 498 10.1016/j.cell.2015.12.038 26777405
Jain, S. et al. ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164, 487–498 (2016).26777405 10.1016/j.cell.2015.12.038
62. Banani SF Lee HO Hyman AA Rosen MK Biomolecular condensates: organizers of cellular biochemistry Nat. Rev. Mol. Cell Biol. 2017 18 285 298 10.1038/nrm.2017.7 28225081
Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).28225081 10.1038/nrm.2017.7
63. Fan P Exploring the frontier of plant phase separation: current insights and future prospects N. Crops 2024 1 100026 10.1016/j.ncrops.2024.100026
Fan, P. et al. Exploring the frontier of plant phase separation: current insights and future prospects. N. Crops 1, 100026 (2024).10.1016/j.ncrops.2024.100026
64. Meng, F., Na, I., Kurgan, L. & Uversky, V. N. Compartmentalization and functionality of nuclear disorder: intrinsic disorder and protein-protein interactions in intra-nucclear compartments. Int. J. Mol. Sci. 17, 24 (2015).
65. Oates ME D(2)P(2): database of disordered protein predictions Nucleic Acids Res. 2013 41 D508 516, 10.1093/nar/gks1226 23203878
Oates, M. E. et al. D(2)P(2): database of disordered protein predictions. Nucleic Acids Res. 41, D508–516, (2013).23203878 10.1093/nar/gks1226
66. McSwiggen DT Mir M Darzacq X Tjian R Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences Genes Dev. 2019 33 1619 1634 10.1101/gad.331520.119 31594803
McSwiggen, D. T., Mir, M., Darzacq, X. & Tjian, R. Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences. Genes Dev. 33, 1619–1634 (2019).31594803 10.1101/gad.331520.119
67. Roberts TC Quantification of nascent transcription by bromouridine immunocapture nuclear run-on RT-qPCR Nat. Protoc. 2015 10 1198 1211 10.1038/nprot.2015.076 26182239
Roberts, T. C. et al. Quantification of nascent transcription by bromouridine immunocapture nuclear run-on RT-qPCR. Nat. Protoc. 10, 1198–1211 (2015).26182239 10.1038/nprot.2015.076
68. Lee Y Rio DC Mechanisms and regulation of alternative pre-mRNA splicing Annu. Rev. Biochem. 2015 84 291 323 10.1146/annurev-biochem-060614-034316 25784052
Lee, Y. & Rio, D. C. Mechanisms and regulation of alternative pre-mRNA splicing. Annu. Rev. Biochem. 84, 291–323 (2015).25784052 10.1146/annurev-biochem-060614-034316
69. Jo, B.-S. & Choi, S. S. Introns: the functional benefits of introns in genomes. Genomics Inform. 13, 112–118 (2015).
70. 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 10.1038/s41586-018-0859-7
71. Pai, A. A. et al. The kinetics of pre-mRNA splicing in the Drosophila genome and the influence of gene architecture. Elife 6, e32537 (2017).
72. Armakola M Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models Nat. Genet 2012 44 1302 1309 10.1038/ng.2434 23104007
Armakola, M. et al. Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models. Nat. Genet. 44, 1302–1309 (2012).23104007 10.1038/ng.2434
73. Vanderweyde T Youmans K Liu-Yesucevitz L Wolozin B Role of stress granules and RNA-binding proteins in neurodegeneration: a mini-review Gerontology 2013 59 524 533 10.1159/000354170 24008580
Vanderweyde, T., Youmans, K., Liu-Yesucevitz, L. & Wolozin, B. Role of stress granules and RNA-binding proteins in neurodegeneration: a mini-review. Gerontology 59, 524–533 (2013).24008580 10.1159/000354170
74. Koguchi M Yamasaki K Hirano T Sato MH Vascular plant one-zinc-finger protein 2 is localized both to the nucleus and stress granules under heat stress in Arabidopsis Plant Signal Behav. 2017 12 e1295907 10.1080/15592324.2017.1295907 28277968
Koguchi, M., Yamasaki, K., Hirano, T. & Sato, M. H. Vascular plant one-zinc-finger protein 2 is localized both to the nucleus and stress granules under heat stress in Arabidopsis. Plant Signal Behav. 12, e1295907 (2017).28277968 10.1080/15592324.2017.1295907
75. Fan W m6A RNA demethylase AtALKBH9B promotes mobilization of a heat-activated long terminal repeat retrotransposon in Arabidopsis Sci. Adv. 2023 9 eadf3292 10.1126/sciadv.adf3292 38019921
Fan, W. et al. m6A RNA demethylase AtALKBH9B promotes mobilization of a heat-activated long terminal repeat retrotransposon in Arabidopsis. Sci. Adv. 9, eadf3292 (2023).38019921 10.1126/sciadv.adf3292
76. Tan H Phase separation of SGS3 drives siRNA body formation and promotes endogenous gene silencing Cell Rep. 2023 42 111985 10.1016/j.celrep.2022.111985 36640363
Tan, H. et al. Phase separation of SGS3 drives siRNA body formation and promotes endogenous gene silencing. Cell Rep. 42, 111985 (2023).36640363 10.1016/j.celrep.2022.111985
77. Li Z Exon-intron circular RNAs regulate transcription in the nucleus Nat. Struct. Mol. Biol. 2015 22 256 264 10.1038/nsmb.2959 25664725
Li, Z. et al. Exon-intron circular RNAs regulate transcription in the nucleus. Nat. Struct. Mol. Biol. 22, 256–264 (2015).25664725 10.1038/nsmb.2959
78. Zhang Y A repertoire of intronic lariat RNAs reveals tissue-specific regulation and target mimicry potential in plants Sci. China Life Sci. 2024 67 1280 1291 10.1007/s11427-023-2466-7 38489006
Zhang, Y. et al. A repertoire of intronic lariat RNAs reveals tissue-specific regulation and target mimicry potential in plants. Sci. China Life Sci. 67, 1280–1291 (2024).38489006 10.1007/s11427-023-2466-7
79. Lancaster AK Nutter-Upham A Lindquist S King OD PLAAC: a web and command-line application to identify proteins with prion-like amino acid composition Bioinformatics 2014 30 2501 2502 10.1093/bioinformatics/btu310 24825614
Lancaster, A. K., Nutter-Upham, A., Lindquist, S. & King, O. D. PLAAC: a web and command-line application to identify proteins with prion-like amino acid composition. Bioinformatics 30, 2501–2502 (2014).24825614 10.1093/bioinformatics/btu310
80. Xue B Dunbrack RL Williams RW Dunker AK Uversky VN PONDR-FIT: a meta-predictor of intrinsically disordered amino acids Biochim. Biophys. Acta 2010 1804 996 1010 10.1016/j.bbapap.2010.01.011 20100603
Xue, B., Dunbrack, R. L., Williams, R. W., Dunker, A. K. & Uversky, V. N. PONDR-FIT: a meta-predictor of intrinsically disordered amino acids. Biochim. Biophys. Acta 1804, 996–1010 (2010).20100603 10.1016/j.bbapap.2010.01.011
81. Peng K Optimizing long intrinsic disorder predictors with protein evolutionary information J. Bioinform. Comput. Biol. 2005 3 35 60 10.1142/S0219720005000886 15751111
Peng, K. et al. Optimizing long intrinsic disorder predictors with protein evolutionary information. J. Bioinform. Comput. Biol. 3, 35–60 (2005).15751111 10.1142/S0219720005000886
82. Uversky VN How to predict disorder in a protein of interest Methods Mol. Biol. 2017 1484 137 158 10.1007/978-1-4939-6406-2_11 27787825
Uversky, V. N. How to predict disorder in a protein of interest. Methods Mol. Biol. 1484, 137–158 (2017).27787825 10.1007/978-1-4939-6406-2_11
83. Erdos G Dosztanyi Z Analyzing protein disorder with IUPred2A Curr. Protoc. Bioinformatics 2020 70 e99 10.1002/cpbi.99 32237272
Erdos, G. & Dosztanyi, Z. Analyzing protein disorder with IUPred2A. Curr. Protoc. Bioinformatics 70, e99 (2020).32237272 10.1002/cpbi.99
84. Chu X Prediction of liquid-liquid phase separating proteins using machine learning BMC Bioinformatics 2022 23 72 10.1186/s12859-022-04599-w 35168563
Chu, X. et al. Prediction of liquid-liquid phase separating proteins using machine learning. BMC Bioinformatics 23, 72 (2022).35168563 10.1186/s12859-022-04599-w
85. Jumper J Highly accurate protein structure prediction with AlphaFold Nature 2021 596 583 589 10.1038/s41586-021-03819-2 34265844
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).34265844 10.1038/s41586-021-03819-2
86. Clough SJ Bent AF Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana Plant J. 1998 16 735 743 10.1046/j.1365-313x.1998.00343.x 10069079
Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).10069079 10.1046/j.1365-313x.1998.00343.x
87. French AP Mills S Swarup R Bennett MJ Pridmore TP Colocalization of fluorescent markers in confocal microscope images of plant cells Nat. Protoc. 2008 3 619 628 10.1038/nprot.2008.31 18388944
French, A. P., Mills, S., Swarup, R., Bennett, M. J. & Pridmore, T. P. Colocalization of fluorescent markers in confocal microscope images of plant cells. Nat. Protoc. 3, 619–628 (2008).18388944 10.1038/nprot.2008.31
88. Li B ZmMYB56 regulates stomatal closure and drought tolerance in maize seedlings through the transcriptional regulation of ZmTOM7 N. Crops 2024 1 100012 10.1016/j.ncrops.2024.100012
Li, B. et al. ZmMYB56 regulates stomatal closure and drought tolerance in maize seedlings through the transcriptional regulation of ZmTOM7. N. Crops 1, 100012 (2024).10.1016/j.ncrops.2024.100012
89. Saleh A Alvarez-Venegas R Avramova Z An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants Nat. Protoc. 2008 3 1018 1025 10.1038/nprot.2008.66 18536649
Saleh, A., Alvarez-Venegas, R. & Avramova, Z. An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat. Protoc. 3, 1018–1025 (2008).18536649 10.1038/nprot.2008.66
90. Huang S A phase-separated nuclear GBPL circuit controls immunity in plants Nature 2021 594 424 429 10.1038/s41586-021-03572-6 34040255
Huang, S. et al. A phase-separated nuclear GBPL circuit controls immunity in plants. Nature 594, 424–429 (2021).34040255 10.1038/s41586-021-03572-6
