
==== Front
EMBO J
EMBO J
The EMBO Journal
0261-4189
1460-2075
Nature Publishing Group UK London

39112803
187
10.1038/s44318-024-00187-1
Article
RNA helicase SKIV2L limits antiviral defense and autoinflammation elicited by the OAS-RNase L pathway
http://orcid.org/0000-0002-2011-7084
Yang Kun kun.yang@utsouthwestern.edu

1
Dong Beihua 2
Asthana Abhishek 2
http://orcid.org/0000-0003-2432-992X
Silverman Robert H 2
http://orcid.org/0000-0002-0637-3989
Yan Nan nan.yan@utsouthwestern.edu

1
1 grid.267313.2 0000 0000 9482 7121 Department of Immunology, UT Southwestern Medical Center, Dallas, TX USA
2 https://ror.org/03xjacd83 grid.239578.2 0000 0001 0675 4725 Department of Cancer Biology, Cleveland Clinic, Cleveland, OH USA
7 8 2024
7 8 2024
9 2024
43 18 38763894
7 3 2024
15 7 2024
16 7 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, 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 changes were made. 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/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.
The OAS-RNase L pathway is one of the oldest innate RNA sensing pathways that leads to interferon (IFN) signaling and cell death. OAS recognizes viral RNA and then activates RNase L, which subsequently cleaves both cellular and viral RNA, creating “processed RNA” as an endogenous ligand that further triggers RIG-I-like receptor signaling. However, the IFN response and antiviral activity of the OAS-RNase L pathway are weak compared to other RNA-sensing pathways. Here, we discover that the SKIV2L RNA exosome limits the antiviral capacity of the OAS-RNase L pathway. SKIV2L-deficient cells exhibit remarkably increased interferon responses to RNase L-processed RNA, resulting in heightened antiviral activity. The helicase activity of SKIV2L is indispensable for this function, acting downstream of RNase L. SKIV2L depletion increases the antiviral capacity of OAS-RNase L against RNA virus infection. Furthermore, SKIV2L loss exacerbates autoinflammation caused by human OAS1 gain-of-function mutations. Taken together, our results identify SKIV2L as a critical barrier to OAS-RNase L-mediated antiviral immunity that could be therapeutically targeted to enhance the activity of a basic antiviral pathway.

Synopsis

The antiviral activity of the OAS-RNase L pathway is weaker than that of other RNA-sensing pathways. This study shows a moderating effect of the SKIV2L cytoplasmic RNA exosome, which degrades the immunogenic RNA produced by RNase L.

The SKIV2L cytoplasmic RNA exosome limits the dsRNA-induced innate immune response.

The RNA helicase activity of SKIV2L is essential for this effect.

SKIV2L acts selectively downstream of RNase L in the OAS-RNase L pathway.

SKIV2L loss exacerbates autoinflammation caused by human OAS1 gain-of-function mutations.

The helicase activity of SKIV2L acts downstream of RNase L to reduce the immunogenic output of the OAS-RNase L pathway.

Keywords

SKIV2L
RNA Exosome
OAS
RNase L
IFN Response
Subject terms

Immunology
Microbiology, Virology & Host Pathogen Interaction
Signal Transduction
http://dx.doi.org/10.13039/100000060 HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) AI153576 AI135922 Silverman Robert H Yan Nan issue-copyright-statement© European Molecular Biology Organization 2024
==== Body
pmcIntroduction

The 2’,5’-oligoadenylate synthetase (OAS)-RNase L pathway is one of the first characterized innate immune pathway that senses cytoplasmic viral RNA (Kerr and Brown, 1978; Zhou et al, 1993). OAS family genes (OAS1-3 and OASL) are IFN-stimulated genes (ISGs) that encode pattern recognition receptors (PRRs). OAS1-3 sense dsRNA and produce 2’,5’ linked oligoadenylate (2–5A) from ATP (Kristiansen et al, 2011). 2–5A acts as a second messenger that activates constitutively expressed RNase L. Activated RNase L cleaves cellular and viral RNAs to produce processed RNA species with 2’,3’-cyclic phosphate at the 3’ end, which is biochemically compatible for activation of RIG-I (Jung et al, 2020; Malathi et al, 2007). RNase L is thought to be the final step of RNA processing before exerting antiviral effector functions.

Although both the OAS-RNase L and RIG-I/MDA5-MAVS pathways sense viral RNA and activate the IFN response, it has long been known that direct sensing of exogenous RNA by RIG-I/MDA5 contributes to most of the IFN production whereas the contribution from sensing RNase L-processed RNA is negligible or minimal. Consistent with this notion, Mavs-deficient cells and mice are highly susceptible to a broad spectrum of RNA viruses, whereas Rnasel-deficient cells and mice are only moderately susceptible to a few viruses (Chakrabarti et al, 2015; Drappier and Michiels, 2015; Zhou et al, 1997). The IFN-inducible nature of OAS expression and the viral antagonism of this pathway could be limiting factors at the immediate onset of a viral infection. However, IFN strongly induces the expression of OAS proteins that synthesize large amounts of 2–5A that activates a single target, RNase L, which should theoretically produce many more RNA ligands for RIG-I than that of the invading virus. This raises the speculation that the majority of RNase L-processed RNA may be lost before they can activate antiviral immunity.

The RNA exosome is an evolutionarily conserved cellular 3’-5’ RNA degradation machinery in eukaryotes, which is involved in RNA processing, maturation, surveillance and turnover (Houseley et al, 2006). In mammalian cells, different RNA exosomes are present in the nucleus, nucleolus or cytoplasm and are associated with different cofactor complexes to target different RNA substrates (Kilchert et al, 2016). The human super-killer complex (SKI), consisting of Ski2-like RNA helicase (SKIV2L), tetratricopeptide repeat domain 37 (TTC37) and WD repeat domain 61 (WDR61) forms the cofactor complex for the cytoplasmic RNA exosome (Halbach et al, 2013). SKIV2L is an RNA helicase that unwinds RNA substrates and threads them through RNA exosome for degradation, while TTC37 and WDR61 contribute to the structure and activity of the SKI complex. The role of SKIV2L and the cytoplasmic RNA exosome in antiviral immunity is unknown. Here, we identify SKIV2L as a critical barrier downstream of RNase L that severely limits the antiviral capacity of the OAS-RNase L pathway.

Results

The SKI complex limits dsRNA-induced innate immune response and apoptosis

To investigate whether SKIV2L plays a role in innate immune response to dsRNA, we generated SKIV2L knockout (SKIV2LKO) A549 cells and stimulated cells with dsRNA analog poly(I:C) (low molecular weight, throughout the study unless specified otherwise) (Fig. 1A). Two independent clones of SKIV2LKO cells showed drastically increased (> eightfold) IFN production to poly(I:C) compared to that in wild-type (WT) cells (Fig. 1B). Importantly, stable ectopic expression of wild-type SKIV2L reduced IFN response in SKIV2LKO cells to a similar level of WT cells (Fig. 1C,D), suggesting that the enhanced IFN response was indeed caused by loss of SKIV2L. As typical of the DExH family of RNA-dependent ATPase, SKIV2L has a helicase region containing an evolutionarily conserved catalytic core (Fig. 1C). We found that SKIV2L E424Q mutant of DExH core failed to reduce elevated IFN response of SKIV2LKO cells after poly(I:C) treatment (Fig. 1D), indicating that the helicase activity of SKIV2L is essential for suppression of dsRNA-induced IFN response. We further stimulated WT and SKIV2LKO cells with high molecular weight (HMW) poly(I:C) or 5’ triphosphate hairpin RNA (3p-hpRNA, in vitro transcription of influenza A (H1N1) virus sequence), and also found increased IFN expression in SKIV2LKO cells (Fig. EV1A,B). In addition to IFN mRNA, immunoblot revealed increased protein expression of IFN-stimulated gene (ISG) RSAD2 in SKIV2LKO cells (Fig. EV1C). To test whether SKIV2LKO cells have increased tonic IFN and ISGs (including RIG-I-like receptors) thus priming cells for enhanced RNA sensing, we measured the expression of molecules of RNA sensing pathway at both protein and mRNA levels and found no major difference between WT and SKIV2LKO cells (Fig. EV1D,E). We also measured the expression of a broad panel of ISGs using qPCR and did not observe increased tonic ISG in SKIV2LKO cells (Fig. EV1F). To further corroborate this, we generated IFNAR1KOSKIV2LKO double knockout cells (Fig. EV1G). Compared to IFNAR1KO cells, IFNAR1KOSKIV2LKO cells still exhibited enhanced IFN response after dsRNA stimulation (Fig. EV1H). Collectively, these results rule out the possibility that SKIV2LKO elevates baseline ISG therefore enhancing RNA sensing signaling.Figure 1 SKIV2L deficiency enhances dsRNA-induced type I IFN response.

(A) Western blot analysis of SKIV2L and TTC37 proteins in WT and two independent lines of SKIV2LKO A549 cells. *, a non-specific band. (B) RT-qPCR analysis of IFNB1 and IL6 mRNA in WT and two independent lines of SKIV2LKO cells treated with increasing dose of poly(I:C) (0.1, 0.3, 1.0 μg/ml) for 4 h. Data are shown as mean ± SEM (n = 3 each genotype). Two-sided Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001. P values = 0.000144, 0.000069, 0.000041 (IFNB1, WT vs SKIV2LKO 1 from left to right); 0.005010, 0.000662, 0.012617 (IL6, WT vs SKIV2LKO 1 from left to right); 0.000220, 0.000007, 0.000712 (IFNB1, WT vs SKIV2LKO 2 from left to right); 0.001757, 0.000724, 0.000163 (IL6, WT vs SKIV2LKO 2 from left to right). (C) Western blot analysis of SKIV2L protein in SKIV2LKO cells reconstituted with WT or E424Q mutant SKIV2L. A schematic diagram showing SKIV2L domains and conserved DExH core (right). (D) RT-qPCR analysis of IFNB1 mRNA in WT, SKIV2LKO cells and SKIV2LKO cells reconstituted with WT or E424Q mutant SKIV2L after poly(I:C) (1.0 μg/ml) treatment for 4 h. Data are shown as mean ± SEM (n = 3 each genotype). Two-sided Student’s t test; **P < 0.01, ***P < 0.001. P values = 0.001366 (WT vs SKIV2LKO 1), 0.000396 (WT vs SKIV2LKO 2), 0.003549 (SKIV2LKO 1 vs SKIV2LKO 1 + WT-SKIV2L), 0.000487 (SKIV2LKO 2 vs SKIV2LKO 2 + WT-SKIV2L). (E) Western blot analysis of TTC37 and SKIV2L proteins in WT and two independent lines of TTC37KO cells. *, a non-specific band. (F) RT-qPCR analysis of IFNB1 mRNA in WT and two independent lines of TTC37KO cells treated with poly(I:C) (1.0 μg/ml) for 4 h. Data are shown as mean ± SEM (n = 4 each genotype). Two-sided Student’s t test; ***P < 0.001. P values = 0.000020 (IFNB1, WT vs TTC37KO 1), 0.000020 (IFNB1, WT vs TTC37KO 2), <0.000001 (IL6, WT vs TTC37KO 1), 0.000003 (IL6, WT vs TTC37KO 2). (G) A schematic diagram showing the generation of tamoxifen-inducible Skiv2l knockout mouse primary skin-derived fibroblast (MSFb). 4-OHT is a metabolite and the active component of tamoxifen. (H) Western blot analysis of SKIV2L and TTC37 proteins in control and iSkiv2l-/- MSFb cells. (I) RT-qPCR analysis of Ifnb and Il6 mRNA in control and iSkiv2l-/- MSFb cells treated with poly(I:C) (1.0 μg/ml) for 4 h. Data are shown as mean ± SEM (n = 4 each genotype). Two-sided Student’s t test; ***P < 0.001. P values = 0.000382 (Ifnb), 0.000243 (Il6). (J) Western blot analysis of SKIV2L protein in THES2 patient-derived fibroblasts (pt.) and derivative cells reconstituted with SKIV2L (pt. rescued). (K) RT-qPCR analysis of IFNB1 and IL6 mRNA in THES2 patient-derived fibroblasts (pt.) and derivative cells reconstituted with WT SKIV2L (pt. rescued) after poly(I:C) treatment (0.03, 0.1, 0.3 μg/ml) for 4 h. Data are shown as mean ± SEM (n = 4 each group). Two-sided Student’s t test; **P < 0.01, ***P < 0.001. P values = 0.000003, 0.000314, 0.000010 (IFNB1 from left to right); 0.003988, 0.000056, 0.000482 (IL6 from left to right). Source data are available online for this figure.

Knockout of TTC37, another component of the SKI complex, also significantly enhanced IFN response to poly(I:C) stimulation (Fig. 1E,F). To test whether SKIV2L regulates innate immune response to dsRNA in primary cells, we further generated tamoxifen-inducible Skiv2l knockout (iSkiv2l-/-) mouse primary skin-derived fibroblasts (MSFb) from Skiv2lfl/flUBC-Cre/ERT2 mice (Fig. 1G,H). We also observed elevated IFN response in Skiv2l-deficient MSFb after poly(I:C) stimulation (Fig. 1I). Interestingly, we noticed that loss of SKIV2L led to substantial decrease in TTC37 protein, and vice versa (Fig. 1A,E,H), suggesting both proteins are necessary for maintaining the stability of the SKI complex. Loss-of-function mutations in SKIV2L or TTC37 in humans are associated with a rare inherited disease, Trichohepatoenteric syndrome (THES), characterized by both primary B-cell immunodeficiency and autoinflammatory features (Yang et al, 2022a; Yang et al, 2022b). We restored SKIV2L expression in SKIV2L-deficiency patient-derived fibroblast with stable retroviral transduction (Fig. 1J). Compared with SKIV2L-rescued cells, SKIV2L-deficient patient cells showed increase IFN response after dsRNA stimulation (Fig. 1K).

While WT cells tolerated low-dose poly(I:C) stimulation with minimal cell death, we observed that both SKIV2LKO cell lines showed substantial increase in apoptosis, as evidenced by increased Caspase (Casp) 3 and PARP cleavage measured by western blots (Fig. 2A) as well as increased Annexin V staining measured by FACS (Fig. 2B,C). Ectopic expression of wild-type SKIV2L rescued cell death in dsRNA-treated SKIV2LKO cells (Fig. 2D), however E424Q mutant failed to rescue apoptosis of SKIV2LKO cells after dsRNA treatment (Fig. 2E), suggesting that the helicase activity of SKIV2L is essential for limiting dsRNA-induced cell death. Interestingly, the E424Q mutant also exerted a dominant negative effect and enhanced apoptosis in WT cells after dsRNA stimulation (Fig. 2E). Consistently, two independent lines of TTC37KO cells are also more sensitive to dsRNA-induced apoptosis (Fig. 2F). These data suggest that the SKI complex restricts innate immune response to dsRNA.Figure 2 SKIV2L deficiency enhances dsRNA-induced apoptosis.

(A) Western blot analysis of apoptosis and PKR-eIF2α pathways in WT and two independent lines of SKIV2LKO cells after increasing dose of poly(I:C) treatment (0.1, 0.3, 1.0 μg/ml) for 4 h. (B, C) Cell death analysis of WT and two independent lines of SKIV2LKO cells after poly(I:C) treatment (1.0 μg/ml for 4 h) using Annexin V staining followed by FACS. Representative flow cytometry plot (B) and statistics of three independent experiments (C) were shown. Two-sided Student’s t test; *P < 0.05, ***P < 0.001. P values = 0.015950, 0.000065 (WT vs SKIV2LKO 1 from left to right); 0.000201, 0.000757 (WT vs SKIV2LKO 2 from left to right). (D) Western blot analysis of apoptosis and PKR-eIF2α pathways in WT and SKIV2LKO cells stably expressing vector or WT SKIV2L after poly(I:C) treatment (1.0 μg/ml for 4 h). (E) Western blot analysis of apoptosis and PKR-eIF2α pathways in WT and SKIV2LKO cells stably expressing vector or SKIV2L E424Q mutant after poly(I:C) treatment (1.0 μg/ml for 4 h). (F) Western blot analysis of apoptosis and PKR-eIF2α pathways in WT and two independent lines of TTC37KO cells treated with increasing dose of poly(I:C) treatment (0.3, 1.0 μg/ml) for 4 h. *, a non-specific band. Data are representative of at least three independent experiments. Source data are available online for this figure.

SKIV2L restricts the OAS-RNase L pathway

We next investigated the three major cytosolic RNA sensing pathways, RLR-MAVS, OAS-RNase L and PKR-eIF2α, each of which has been implicated in IFN response and/or cell death through distinct mechanisms. We found no major difference in activation of the PKR-eIF2α pathway (measured by phosphorylation of PKR and eIF2α) between WT and SKIV2LKO cells before or after dsRNA stimulation (Figs. 2A and 3A, compare lane 5 to lane 6). This data suggest that SKIV2L does not act on the PKR-eIF2α pathway or directly degrades exogenous dsRNA (which would impact all three RNA sensing pathways).Figure 3 SKIV2L negatively regulates the OAS-RNase L pathway.

(A–C) Western blot analysis of apoptosis and PKR-eIF2α pathways in single or double gene knockout cells (as indicated on top) treated with mock or poly(I:C) (1.0 μg/ml) for 4 h. (D, E) RT-qPCR analysis of IFNB1 mRNA in WT and indicated gene knockout cells after poly(I:C) (1.0 μg/ml) for 4 h treatment. Fold change (1000×) of IFNB1 mRNA compared to mock-treated WT cells is shown. Data are shown as mean ± SEM of three independent experiments. Two-sided Student’s t test; **P < 0.01, ***P < 0.001. (D) P values = 0.009380 (WT vs RNASELKO), 0.000116 (WT vs OAS3KO), 0.005046 (WT vs SKIV2LKO), 0.003540 (SKIV2LKO vs SKIV2LKORNASELKO), 0.002554 (SKIV2LKO vs SKIV2LKOOAS3KO). (E) P values = 0.000001 (WT vs SKIV2LKO), <0.000001 (WT vs MAVSKO), 0.000001 (SKIV2LKO vs SKIV2LKOMAVSKO). (F) Schematic diagram of RLR-MAVS pathway sensing both incoming exogenous RNA and RNase L-processed RNA. Data are representative of at least three independent experiments. Source data are available online for this figure.

We next knocked out RNase L or MAVS in SKIV2LKO cells as well as in WT cells to generate a panel of single and double knockouts A549 cells. Ablation of RNase L fully blocked dsRNA-induced apoptosis in SKIV2LKO cells, suggesting that SKIV2L restricts OAS-RNaseL-mediated apoptosis (Fig. 3A, compare lane 8 to lane 6). There are three catalytically active OAS proteins in humans, and OAS3 is reported to be the major enzyme responsible for RNase L activation (Li et al, 2016). We found that knocking out OAS3 largely reduced dsRNA-induced apoptosis in SKIV2LKO cells, but with some residual cell death (Fig. 3B). Recent studies, particularly of SARS-CoV-2, have suggested OAS1 to be another contributor of 2–5A production that activates RNase L antiviral pathway (Banday et al, 2022; Soveg et al, 2021; Wickenhagen et al, 2021). We further generated SKIV2LKOOAS3KOOAS1KO triple knockout cells to assess the role of OAS1. Compared to SKIV2LKOOAS3KO, SKIV2LKOOAS3KOOAS1KO further decreased dsRNA-induced cell death (Fig. EV2A), suggesting a role for OAS1 in RNase L pathway. In contrast to OAS-RNase L pathway, loss of MAVS did not block cell death in SKIV2LKO cells (Fig. 3C, compare lane 8 to lane 6).

We next measured dsRNA-induced IFNB1 mRNA expression and grouped single and double knockout cell lines by the presence or absence of SKIV2L. In the SKIV2LWT group, RNASELKO and OAS3KO showed a slight decrease in poly(I:C)-induced IFNB1 mRNA expression whereas MAVSKO completely eliminated IFNB1 mRNA expression (Fig. 3D,E). This is consistent with the long-standing notion that IFN response to exogenous RNA mostly comes from direct sensing of incoming exogenous RNA (‘direct RNA sensing’ in Fig. 3D) rather than RNase L-processed RNA (‘processed RNA sensing’ in Fig. 3D). Surprisingly, in the SKIV2LKO group, SKIV2LKO alone produced very high levels of IFN response after poly(I:C) stimulation, which was largely eliminated in RNASELKOSKIV2LKO and OAS3KOSKIV2LKO cells (Fig. 3D). In other words, SKIV2LKO significantly boosted IFN response to exogenous RNA (compared to WT) by expanding the portion of innate immune response from RNase L-processed RNA (Fig. 3F). Of note, MAVSKO cells still have residual IFN response to poly(I:C), probably through RLR-MAVS-independent RNA sensing pathway, such as TLR3 (Fig. 3E). We further knocked out RIG-I or MDA5 and found that loss of RIG-I abolished enhanced IFN response in SKIV2LKO cells (Fig. EV2B,C,D). These data demonstrate that the OAS-RNase L pathway has an underappreciated capability to amplify RNA-induced IFN response, much higher than direct sensing of exogenous RNA (as in WT cells), but most of that capability is restricted by SKIV2L.

SKIV2L acts downstream of RNase L

We next determined where SKIV2L acts in the OAS-RNase L pathway. OAS senses exogenous RNA, then produces 2–5A, which activates RNase L and subsequent cleavage of cellular and viral RNA. The SKIV2L RNA exosome could act on the RNA species either up- or downstream the OAS-RNase L pathway. We first measured 2–5A production that directly indicates OAS activity and found similar amount of 2–5A in WT and SKIV2LKO cells after poly(I:C) stimulation (Fig. 4A), suggesting that SKIV2L acts downstream of OAS3. We also performed the complementary experiment to stimulate WT and SKIV2LKO cells with authentic trimeric 2–5A (2’,5’p3A3) to directly activate RNase L, and we found that 2’,5’p3A3 stimulated higher apoptosis (Fig. 4B) and IFN response (Fig. 4C) in SKIV2LKO cells compared to WT, similar to that of poly(I:C) stimulation (Figs. 1 and 2), confirming SKIV2L acts downstream of OAS. Cleavage of ribosomal RNA, a readout of RNase L enzymatic activity after 2–5A binding, was similar in SKIV2LKO and WT cells after either poly(I:C) (Fig. 4D) or 2–5A stimulation (Fig. 4E), suggesting that SKIV2L does not directly affect RNase L enzymatic activity. Therefore, these data suggest that SKIV2L acts downstream of RNase L likely by removing RNase L-processed RNA products with 2’–3’ cyclic phosphate moiety that would be ideal substrates for the 3’–5’ exoribonuclease activity in the SKIV2L RNA exosome (Zinder et al, 2016).Figure 4 SKIV2L acts downstream of RNase L.

(A) Quantification of 2–5A production in WT and knockout cells after poly(I:C) (1 μg/ml) treatment for 4 h. Bars are mean of two independent experiments. (B) Western blot analysis of apoptosis in WT, SKIV2LKO and RNASELKO cells transfected with trimer 2–5A, 2’-5’p3A3 (20 μM) for 4 h. (C) RT-qPCR analysis of IFNB1 mRNA in WT, SKIV2LKO and RNASELKO cells transfected with 2’-5’p3A3 (20 μM) for 4 h. Data are mean ± SEM of three independent experiments. Two-sided Student’s t test; *P < 0.05, ns, not significant. P values = 0.022367 (WT vs SKIV2LKO), 0.292054 (RNASELKO, mock vs 2–5A). (D, E) rRNA cleavage analysis of WT and SKIV2LKO cells after increasing dose of poly(I:C) (0.06, 0.12, 0.25, 0.5, 1.0 μg/ml) (D), or 2’-5’p3A3 (10, 20 μM) (E) treatment using Bioanalyzer Nanochip. Data are representative of at least three independent experiments. Source data are available online for this figure.

SKIV2L restricts antiviral capacity of the OAS-RNase L pathway

To further examine the physiological importance of SKIV2L restriction of the OAS-RNase L pathway during viral infection, we challenged WT, SKIV2LKO, RNASELKO, RNASELKOSKIV2LKO cells with Sindbis virus (SINV) that is known to activate OAS-RNase L innate immune pathway (Li et al, 2016). Comparing WT to RNASELKO cells, SINV induced a similar amount of IFN response (Fig. 5A), again confirming the long-standing notion of innate sensing of RNase L-processed RNA is largely negligible. RNASELKO cells did show sevenfold higher viral titer at 24 h post-infection than that in WT cells, likely due to cleavage of viral RNA as shown previously (Fig. 5B). In contrast, SKIV2LKO cells expressed significantly higher IFNB mRNA than WT cells after SINV infection, which was abolished by removing RNase L (RNASELKOSKIV2LKO, Fig. 5A). SINV titer was also significantly decreased in SKIV2LKO cells by 20- and 66-fold at 24 h and 36 h post-infection, respectively, and this antiviral ‘power’ was completely lost in RNASELKOSKIV2LKO cells (Fig. 5B). We also observed enhanced antiviral activity of SKIV2L-deficient patient’s fibroblasts compared to SKIV2L-rescued cells after SINV infection (Fig. 5C). These data suggest that SKIV2L limits the antiviral capacity of the OAS-RNase L pathway and removing or inhibiting SKIV2L could potentially release substantial antiviral power from the OAS-RNase L pathway.Figure 5 Loss of SKIV2L enhances RNase L-mediated antiviral activity.

(A) RT-qPCR analysis of IFNB1 mRNA in WT, SKIV2LKO, RNASELKO and RNASELKO SKIV2LKO cells infected with increasing SINV (MOI 0.1, 0.3) for 24 h. Data are mean ± SEM of three independent experiments. Two-sided Student’s t test; ***P < 0.001; ns, not significant. P values = 0.000014 (MOI 0.1, WT vs SKIV2LKO), 0.000010 (MOI 0.3, WT vs SKIV2LKO), 0.179827 (MOI 0.1, RNASELKO vs RNASELKOSKIV2LKO), 0.221373 (MOI 0.3, RNASELKO vs RNASELKOSKIV2LKO). (B) Viral production was assessed by plaque assay (PFU, plaque-forming unit) in cells infected with SINV (MOI 0.1) for the indicated time. Data are mean ± SEM of three independent experiments. Two-sided Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. P values = 0.006833 (24 h, WT vs RNASELKO), 0.036438 (36 h, WT vs RNASELKO), 0.000689 (24 h, WT vs SKIV2LKO), 0.000044 (36 h, WT vs SKIV2LKO), 0.201542 (24 h, RNASELKO vs RNASELKOSKIV2LKO), 0.571109 (36 h, RNASELKO vs RNASELKOSKIV2LKO). (C) Viral production in THES2 patient-derived fibroblasts (pt.) and derivative cells reconstituted with WT SKIV2L (pt. rescued) after SINV (MOI 0.3) for 24 h. Data are mean ± SEM of four independent experiments. Two-sided Student’s t test; ***P < 0.001. P value = 0.000579. Source data are available online for this figure.

SKIV2L limits OAS1-mediated autoinflammation

Aberrant activation of innate immune RNA sensing pathway, such as gain-of-function (GoF) mutation in RNA sensor OAS1 and loss-of-function (LoF) mutation in RNA editing enzyme ADAR1, has been associated with human inborn errors of immunity (Fig. 6A). OAS1 GoF variants in human causes a polymorphic autoinflammatory immunodeficiency (Magg et al, 2021). Induction of OAS1 GoF A76V mutant, but not wild-type OAS1, resulted in 2–5A production and rRNA cleavage in the absence of dsRNA (Figs. 6B,C and EV3A). To test whether SKIV2L regulates pathogenic activation of OAS-RNase L pathway, we induced the expression of OAS1 GoF A76V mutant in WT and SKIV2LKO cells (Fig. 6D). We found that SKIV2LKO cells were more sensitive to OAS1 GoF mutant-induced cell death (Fig. 6D). SKIV2LKO cells also exhibited enhanced IFN response after induction of OAS1 GoF mutant, compared to WT cells (Figs. 6E and EV3B). Deletion of IFNAR1 did not ablate the increase in IFN response in SKIV2LKO cells after induction of OAS1 GoF mutant (Fig. EV3C,D), consistent with the findings of dsRNA-stimulated SKIV2LKO cells (Fig. EV1G,H). When RNASEL, MAVS or DDX58 was deleted, elevated IFN response was reduced in SKIV2LKO cells expressing OAS1 GoF mutant (Figs. 6F,G and EV3E). Induction of OAS1 GoF mutant in cells serves as an ideal model to study OAS-RNase L pathway without exogenous dsRNA that can activate other RNA sensing pathway. We found that after induction of OAS1 A76V mutant, cellular RNA isolated from SKIV2LKO A549 cells was more immunostimulatory when being transfected to MEFs (Fig. EV3F). ADAR1 LoF mutations lead to accumulation of endogenous dsRNA that has been reported to activate RLR-IFN and PKR-eIF2a pathways (Chung et al, 2018; Hartner et al, 2009; Liddicoat et al, 2015; Maurano et al, 2021; Rice et al, 2012; Tang et al, 2021). We further generated SKIV2LKOADAR1KO cells and found that deletion of SKIV2L had no effect on either PKR-eIF2a (Fig. 6H) or IFN response (Fig. 6I) of ADAR1KO cells after IFN-β treatment. These results of genetic mutations associated with human autoinflammatory diseases suggest that SKIV2L specifically regulates the OAS-RNase L pathway (Fig. 6J).Figure 6 SKIV2L restricts OAS1-mediated autoinflammation.

(A) A schematic diagram showing inborn errors of immunity associated with innate immune RNA sensing pathways. (B) Quantification of 2–5 A production in A549 cells after induction of OAS1 A76V mutant with doxycycline (0.25, 0.5, 0.75, 1.0 μg/ml) for 24 h. Data are mean ± SEM of four independent experiments. (C) rRNA cleavage analysis of total RNA in A549 cells after induction of OAS1 A76V mutant with doxycycline (0.25, 0.5, 0.75, 1.0 μg/ml) for 24 h. Data are representative of at least three independent experiments. (D) Western blot analysis of apoptosis markers in WT, SKIV2LKO cells after induction of OAS1 A76V mutant with increasing doses of doxycycline (0.25, 0.5, 1.0 μg/ml) for 24 h. Data are representative of at least three independent experiments. (E) RT-qPCR analysis of IFNB1 mRNA in WT, SKIV2LKO after induction of OAS1 A76V mutant with doxycycline (0.25, 0.5, 1.0 μg/ml) for 24 h. Data are mean ± SEM of four independent experiments. (F) RT-qPCR analysis of IFNB1 mRNA in WT, SKIV2LKO, RNASELKO, SKIV2LKORNASELKO cells after induction of OAS1 A76V mutant with doxycycline (1.0 μg/ml) for 24 h. Data are mean ± SEM of three independent experiments. Two-sided Student’s t test; **P < 0.01. P values = 0.009762 (WT vs SKIV2LKO), 0.004240 (SKIV2LKO vs SKIV2LKORNASELKO). (G) RT-qPCR analysis of IFNB1 mRNA in WT, SKIV2LKO, MAVSKO, SKIV2LKOMAVSKO cells after induction of OAS1 A76V mutant with doxycycline (1.0 μg/ml) for 24 h. Data are mean ± SEM of four independent experiments. Two-sided Student’s t test; ***P < 0.001. P values = 0.000005 (WT vs SKIV2LKO), 0.000002 (SKIV2LKO vs SKIV2LKOMAVSKO). (H) Western blot analysis of phosphor-eIF2α in WT, SKIV2LKO, ADAR1KO, SKIV2LKOADAR1KO cells after IFN-β treatment (20 ng/ml) for 24 h. (I) RT-qPCR analysis of ISGs in WT, SKIV2LKO, ADAR1KO, SKIV2LKOADAR1KO cells after IFN-β treatment (20 ng/ml) for 24 h. Data are mean ± SEM of three independent experiments. (J) A schematic diagram summarizing SKIV2L regulation of the OAS-RNase L pathway. Source data are available online for this figure.

Discussion

A long-standing conundrum of the OAS-RNase L pathway is its powerful enzymatic amplification of RIG-I-compatible 3’RNA ends (through RNase L cleavage of cellular and viral RNA) and yet its modest contribution to the overall IFN production during infection (Malathi et al, 2007). We show here that the human SKI complex, a cofactor complex that recruits RNA substrates for degradation by the cytoplasmic RNA exosome, plays a crucial role in limiting the antiviral activities of the OAS-RNase L pathway. This evolutionarily conserved RNA degradation machinery, normally involved in nonsense mediated decay of cellular RNA, now also acts as an unfortunate barrier to antiviral immunity. When this barrier is removed (e.g. SKIV2LKO cells), the OAS-RNase L pathway becomes fully unleashed and dominates the IFN response to exogenous RNA challenge (via RIG-I sensing of RNase L-processed RNA) and far exceeds that from RIG-I direct sensing of incoming RNA. On the other hand, the SKI complex may serve as a gatekeeper to prevent overactivation of innate immunity and inadvertent immune pathologies (e.g. cell death) in both viral infections and autoinflammation.

The mechanism described here has important parallels to a previous study (Eckard et al, 2014). In cells under endoplasmic reticulum (ER) stress, SKIV2L knockdown enhances RIG-I-mediated IFN response due to the accumulation of ribonuclease-IRE1-processed cellular RNA (Eckard et al, 2014). IRE1 and RNase L are two highly homologous ribonucleases, both activated by an upstream signal (unfolded proteins activate IRE1, 2–5A activate RNase L), both dimerize and then cleave substrate RNAs, and both are metal-ion-independent endoribonucleases that produces 3’ cyclic phosphate RNA ends that activate RIG-I (Jung et al, 2020; Shigematsu et al, 2018). The nuclease domain of RNase L also shares structural similarity to IRE1 (Dong et al, 2001). The one difference is that IRE1 is membrane anchored in the ER and RNase L is ubiquitously present in the cytosol, which makes sense for the upstream stimuli they each detect. Therefore, we propose that, during ER stress, the cytoplasmic SKIV2L RNA exosome degrades IRE1-processed RNA to prevent innate immune activation (as a protective mechanism for the host); during RNA virus infection, it similarly degrades RNase L-processed RNA thus (unfortunately) limiting antiviral immunity.

The nature of RNA substrates cleavage by RNase L and then rapidly degraded by SKIV2L RNA exosome remain unclear. We showed here that the RNA substrates accumulate in SKIV2L-deficient cells are immunogenic and activates RLR. One previous study in Drosophila cells has shown that the nuclear RNA exosome can also target viral RNA for degradation (Molleston et al, 2016). However, it remains unclear whether the mammalian cytoplasmic SKIV2L RNA exosome has a similar capacity to degrade viral RNA directly without prior RNase L cleavage. Extensive structural studies have elucidated the mechanism of RNA recognition by RLRs (Cadena and Hur, 2019; Chen and Hur, 2022; Rehwinkel and Gack, 2020). RIG-I filament assembly and oligomerization require dsRNA with a 5′-triphosphate (5’ppp) or 5′-diphosphate group, which is structurally characterized in 5’pppRNA-bound RIG-I multimer (Goubau et al, 2014; Myong et al, 2009; Peisley et al, 2013). 3’ cyclic phosphate RNA ends produced by metal-ion-independent endoribonucleases can also activate RIG-I (Jung et al, 2020; Shigematsu et al, 2018). Further, rearrangement of the actin cytoskeleton can facilitate RLR activation by promoting the dephosphorylation of RIG-I and MDA5 (Acharya et al, 2022). Therefore, a major possibility is that RNA virus activates OAS-RNase L, which cleavages both cellular and viral RNA and generates 3’ cyclic phosphate RNA ends that are either degraded by SKIV2L RNA exosome or sensed by RLR. A minor possibility is that SKIV2L RNA exosome directly degrades viral RNA and prevents them from triggering RLR.

We believe that SKIV2L-mediated regulation of RLR pathway is related to its function within SKI complex of RNA exosome, although our data does not exclue the possibility that SKIV2L may also have activity outside of the RNA exosome. We showed that the SKIV2LKO and TTC37KO cells phenocopied each other in all assays tested in our study. In both human and mouse cells, knockout of either SKIV2L or TTC37 of SKI complex destablized the whole complex and resulted in decrease protein level of the other component. While cryo-EM structural studies have elegantly elucidated the mechanism of RNA degradation by SKI complex and cytoplasmic RNA exosome (Halbach et al, 2013; Schmidt et al, 2016), it is intriguing to explore the function and potential RNA substrates of “free” SKIV2L outside of SKI complex.

The discovery of this cell-intrinsic barrier to antiviral immunity immediately opens up opportunities for therapy. Interestingly, a recent yeast suppressor screen identified the human SKI complex as a promising broad-spectrum antiviral drug target (Weston et al, 2020). Inhibition of SKIV2L RNA helicase activity by chemical compounds impaired the replication of influenza, filoviruses as well as several human coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) (Weston et al, 2020). The underlying mechanism is not yet defined, although based on our data presented here, we predict that the OAS-RNase L pathway may underlie the antiviral activity of SKIV2L inhibitors. Together, our results not only reveal the enormous hidden power of the OAS-RNase L signaling pathway, but also a targetable host barrier protein SKIV2L for antiviral therapy against many RNA viruses that cause severe diseases in humans.

Methods

Reagents and tools table

Reagent/resource	Reference or source	Identifier or catalog number	
Experimental models	
A549 cells (H. sapiens)	ATCC	CCL-185	
293T (H. sapiens)	ATCC	CRL-3216	
BHK-21 (Mesocricetus auratus)	ATCC	CCL-10	
Vero cells (Chlorocebus sabaeus)	ATCC	CCL-81	
SKIV2L-deficient THES2 patient-derived fibroblasts (H. sapiens)	(Yang et al, 2022a)	N/A	
Skiv2lfl/fl (M. musculus)	(Yang et al, 2022a)	N/A	
Skiv2lfl/flUBC-Cre/ERT2 (M. musculus)	(Yang et al, 2022a)	N/A	
Sindbis virus	(Orvedahl et al, 2010)	N/A	
Sendai virus	(Hasan et al, 2013)	N/A	
Encephalomyocarditis virus	(Aguilera et al, 2019)	N/A	
Recombinant DNA	
LentiCRISPRv2	Addgene	52961	
pMRX-SKIV2L	This study	N/A	
pMRX-SKIV2L-E424Q	This study	N/A	
pEasiLV-OAS1	This study	N/A	
pEasiLV-OAS1-A76V	This study	N/A	
Antibodies	
SKIV2L Rabbit Polyclonal Antibody	Proteintech	11462-1-AP	
TTC37 Rabbit Polyclonal Antibody	Proteintech	24594-1-AP	
Monoclonal Anti-α-Tubulin antibody (clone B-5-1-2)	Sigma	T5168	
Anti-HMGB1 antibody	Abcam	ab18256	
GAPDH (14C10) Rabbit mAb	Cell Signaling	2118	
Vinculin Antibody	Cell Signaling	4650	
OAS3 Rabbit Polyclonal Antibody	Proteintech	21915-1-AP	
OAS1 Recombinant antibody	Proteintech	82883-1-RR	
RNase L (D4B4J) Rabbit mAb	Cell Signaling	27281	
RIG-I (D14G6) Rabbit mAb	Cell Signaling	3743	
MDA-5 (D74E4) Rabbit mAb	Cell Signaling	5321	
MAVS (D5A9E) Rabbit mAb	Cell Signaling	24930	
TBK1/NAK (D1B4) Rabbit mAb	Cell Signaling	3504	
IRF-3 (D6I4C) XP® Rabbit mAb	Cell Signaling	11904	
Viperin (D5T2X) Rabbit mAb mAb	Cell Signaling	13996	
Recombinant Anti-Interferon alpha/beta receptor 1 antibody [EPR6244]	Abcam	ab124764	
Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb	Cell Signaling	9664	
Cleaved PARP (Asp214) (D64E10) Rabbit mAb	Cell Signaling	5625	
PKR (phospho T446) antibody [E120]	Abcam	ab32036	
PKR (D7F7) Rabbit mAb	Cell Signaling	12297	
Phospho-eIF2α (Ser51) (119A11) Rabbit mAb	Cell Signaling	3597	
eIF2α (D7D3) XP® Rabbit mAb	Cell Signaling	5324	
ADAR1 mouse monoclonal antibody (15.8.6)	Santa Cruz	sc-73408	
Goat anti-rabbit IgG (H/L):HRP	Bio-Rad	5196-2504	
Goat anti-mouse IgG (H/L):HRP	Bio-Rad	5178-2504	
Oligonucleotides and other sequence-based reagents	
Human SKIV2L gRNA1	This study	5’-GACGGATCCCTGGTCTCTTT-3’	
Human SKIV2L gRNA2	This study	5’-CTTTGGGCCTGTAGGTCGGA-3’	
Human TTC37 gRNA1	This study	5’-TGGTGTTTACCAAAAGCTCC-3'	
Human TTC37 gRNA2	This study	5’-TGATGTCTGCAAGAAACTTG-3’.	
Human MAVS gRNA	This study	5’-CTGTGAGCTAGTTGATCTCG-3’	
Human ADAR1 gRNA	This study	5’- TCTGTCAAATGCCATATGGG-3’	
OAS1-crRNA1	This study	/AlTR1/rArGrUrArCrGrArArGrCrUrGrArGrCrGrCrArCrGrGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AlTR2/	
OAS1-crRNA2	This study	/AlTR1/rGrCrUrCrCrCrArArGrCrArUrArGrArCrCrGrUrCrGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AlTR2/	
IFNAR1-crRNA1	This study	/AlTR1/rGrCrGrGrCrUrGrCrGrGrArCrArArCrArCrCrCrArGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AlTR2	
IFNAR1-crRNA2	This study	/AlTR1/rArArGrCrArGrCrArCrUrArCrUrUrArCrGrUrCrArGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AlTR2/	
IFIH1-crRNA1	This study	/AlTR1/rUrCrArUrGrArGrCrGrUrUrCrUrCrArArArCrGrArGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AlTR2/	
IFIH1-crRNA2	This study	/AlTR1/rUrUrGrGrArCrUrCrGrGrGrArArUrUrCrGrUrGrGrGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AlTR2/;	
DDX58-crRNA1	This study	/AlTR1/rGrGrArUrUrArUrArUrCrCrGrGrArArGrArCrCrCrGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AlTR2/	
DDX58-crRNA2	This study	/AlTR1/rGrArUrCrArGrArArArUrGrArUrArUrCrGrGrUrUrGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AlTR2/	
Alt-R® CRISPR-Cas9 tracrRNA	Integrated DNA Technologies	1072532	
PCR primers	(Tu et al, 2022; Wu et al, 2020; Yang et al, 2018)		
Chemicals, enzymes and other reagents	
TRI reagent	Sigma	T9424	
iScript™ cDNA Synthesis Kit	Bio-Rad	1708890	
iTaq™ Universal SYBR® Green Supermix	Bio-Rad	1725122	
Dulbecco′s Modified Eagle′s Medium - high glucose	Sigma	D5796	
Fetal bovine serum	Sigma	F2442	
Dulbecco′s Phosphate Buffered Saline	Sigma	D8537	
Gibco™ Puromycin Dihydrochloride	Thermo Fisher	A1113803	
Blasticidin S, Hydrochloride	Sigma	15205	
Poly(I:C) (LMW)	Invivogen	tlrl-picw	
Poly(I:C) (HMW)	Invivogen	tlrl-pic	
2’-5’p3A3	This study	N/A	
Doxycycline Hyclate	Sigma	D5207	
Recombinant Human IFN-β	PeproTech	300-02BC	
Alt-R S.p. Cas9 Nuclease V3	Integrated DNA Technologies	1081058	
Lipofectamine™ 2000 Transfection Reagent	Thermo Fisher	11668500	
Lipofectamine RNAiMAX reagent	Thermo Fisher	13778100	
FITC-Annexin V Apoptosis Detection Kit	BioLegend	640914	
SuperSignal™ West Pico PLUS Chemiluminescent Substrate	Thermo Fisher	34580	
miRNeasy Tissue/Cells Advanced Kits	QIAGEN	217684	
Software	
FlowJo 10.6	FlowJo, LLC		
GraphPad Prism 10.1.1	GraphPad Software		
BioTek Gen5 Software	BioTek		
Other	
BD FACSCalibur™ Flow Cytometer	BD Biosciences		
ChemiDoc Imaging Systems	Bio-Rad		
CFX96™ Real-Time PCR Detection System	Bio-Rad		
2100 Bioanalyzer system	Agilent		

Methods and protocols

Cell culture

A549 cells, Vero, BHK-21 and HEK293T cells were maintained in DMED supplemented with 10% FBS. RNase L knockout and OAS3 knockout A549 cells were generated as described previously and kindly provided by Dr. Susan R. Weiss (University of Pennsylvania) (Li et al, 2016). SKIV2L-deficient THES2 patient-derived fibroblasts were described previously (Yang et al, 2022a). Mouse primary skin-derived fibroblasts (MSFb) were generated from Skiv2lfl/fl and Skiv2lfl/flUBC-Cre/ERT2 mice (Yang et al, 2022a) following the protocol described previously (Khan and Gasser, 2016). All mice were housed in pathogen-free barrier facilities at UT Southwestern Medical Center. The animal protocol was approved by the Institutional Animal Care and Use Committee at UT Southwestern Medical Center (APN 2017-101968). Ex vivo deletion of Skiv2l in culture cells was described previously (Yang et al, 2022a). All cells used in the study were tested negative for mycoplasma contamination.

CRISPR/Cas9-mediated gene editing

Knockout cell lines were generated using CRISPR/Cas9-mediated gene editing either through lentivrial transduction or transient cationic lipid delivery of CRISPR/Cas9 ribonucleoprotein (RNP) complex. For CRISPR/Cas9 lentivrial transduction, sgRNAs were designed and cloned into LentiCRISPRv2 vector. The packaging of pseudo lentiviruses carrying sgRNA and Cas9 was described previously (Yang et al, 2018). A549 cells were transduced with lentiviruses, followed by puromycin (2 μg/ml) selection for several days.

For transient CRIPSR/Cas9 RNP transfection, the following crRNAs were purchased from. Each crRNA was annealed with Alt-R® CRISPR-Cas9 tracrRNA to form crRNA: tracrRNA duplex, then incubate with to assemble RNP complex. RNP complex was transfected into cells using Lipofectamine RNAiMAX reagent. Single cell-derived clones were confirmed with western blot for knockout of gene of interest.

Retrovirusl and lentivirus preparation and transduction

SKIV2L were cloned into retroviral pMRX-ires-bsr vector (a kind gift from S. Akira) using EcoR I and Not I sites. SKIV2L E424Q mutant was generated by site-directed mutagenesis (Agilent Technologies). Synonymous mutations were introduced in SKIV2L sgRNA targeting sequences and protospacer adjacent motif (PAM). Human OAS1 A76V mutant was cloned into pEsiLV lentiviral vector as described previously (Wu et al, 2019). Retroviruses and lentiviruses were packaged in HEK293T cells following standard protocol. Retroviruses were used for transduction followed by selection with blasticidine (15 μg/ml) for 7 days. pEsiLV lentiviruses were used for transduction followed by single-cell clone selection. Cells transduced with human OAS1 A76V were verified with western blot and flow cytometry after doxycycline induction.

Transfections with poly(I:C) or 2–5A

Transfections of poly(I:C) and 2-5A were performed with Lipofectamine 2000 per manufacturer’s instruction. Briefly, poly(I:C) or 2–5A diluted in Opti-MEM was mixed with diluted Lipofectamine 2000 reagent to form complex, then added to sub-confluent cells. Lipofectamine alone was used as mock control. Cells were collected at indicated time points for RT-qPCR or western blot analysis.

RNA isolation and RT-qPCR

Total RNA was isolated from cultured cells using TRI reagent (Sigma) per manufacturer’s instruction, and cDNA were synthesized with iScript cDNA Synthesis Kit (Bio-Rad). iTaq Universal SYBR Green Supermix (Bio-Rad) was used to quantify mRNA expression with CFX96™ Real-Time PCR Detection System.

Small RNA isolation and transfection

The isolation of small RNA was performed as described previously (Malathi et al, 2007). Briefly, OAS1 A76V mutant was induced in WT and SKIV2LKO A549 for 24 h. Small RNAs were isolated using miRNeasy Tissue/Cells Advanced Kits. Small RNAs were transfected into MEFs using Lipofectamine 2000 and induction of mouse Ifnb was analyzed using RT-qPCR.

Western blotting

Western blots were performed as described previously (Yang et al, 2018). Briefly, cell lysate was quantified using BCA and equal amounts of proteins were separated on SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Membranes were blocked with 5% non-fat milk in 1X TBS-T and incubated with diluted primary antibodies at 4 °C overnight per manufacturers’ instructions. Membranes were incubated with HRP-conjugated secondary antibody (Bio-Rad) diluted for 1 h at room temperature. SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) was used to develop the blots on X-ray film or using ChemiDoc™ Imaging System (Bio-Rad).

Annexin V apoptosis assay

Apoptosis was measured using FITC-Annexin V Apoptosis Detection Kit. Briefly, cells were transfected with poly(I:C) at indicated concentration for 4 h, and then collected and washed twice with cold PBS. Cells were resuspended in Annexin V Binding Buffer and stained with FITC-Annexin V for 15 min at room temperature in the dark. Stained cells were analyzed by flow cytometry (BD FACSCalibur) and data were analyzed using FlowJo software.

Quantification of 2-5A

Intracellular 2-5A was quantified by an indirect RNase L-based FRET assays as described before (Thakur et al, 2005). Briefly, poly(I:C)-treated cells were washed with PBS, and lysed in preheated (95 °C) Nonidet P-40 lysis buffer (50 mM Tris-HCl, pH 7.2, 0.15 M NaCl, 1% Nonidet P-40, 200 mM sodium orthovanadate, 2 mM EDTA, 5 mM MgCl2, 5 mM DTT) and heated to 95 °C for another 7 min. The cleared supernatants collected after centrifugation at 14,000 × g for 10 min. Levels of 2-5A were determined by RNase L-based FRET assays with recombinant human RNase L and synthetic fluorophore-labeled oligoribonucleotide as substrate.

rRNA cleavage assay

Total RNA was isolated using TRI reagent (Sigma) per manufacturer’s instruction. Equal amount of total RNA was then resolved on RNA nanochips using an Agilent 2100 BioAnalyzer.

Plaque assays

SINV was diluted serially in DMEM and 250 μL of diluted viruses were added to confluent Vero cell monolayers in six-well plates. The plates were incubated for 1 h at 37 °C with rocking at 15-min intervals. Then the cells were overlaid with 3 mL warm DMEM containing 2% FBS and 1% Agar. After 36-48 h incubation at 37 °C with 5% CO2, cells were fixed with 4% formaldehyde and plaques were visualized using 0.1% Crystal violet staining.

Statistical analysis

For statistical analyses, most of the experiments were repeated three or more times as indicated in each figure legend. The sample size was not pre-determined in this study. No data were excluded from the analyses. Investigators were not blinded during data collection. Graphpad Prism was used for statistical analysis. Statistical tests performed were indicated in figure legend. Numerical data were shown as mean ± SEM. P values of less than 0.05 were considered statistically significant.

Supplementary information

Peer Review File

Source data Fig. 1

Source data Fig. 2

Source data Fig. 3

Source data Fig. 4

Source data Fig. 5

Source data Fig. 6

EV Figure Source Data

Expanded View Figures

Expanded view

Figure EV1 SKIV2L deficiency enhances dsRNA-induced type I IFN response.

(A) RT-qPCR analysis of IFNB1 mRNA in WT and two independent lines of SKIV2LKO cells treated with high molecular weight (HMW) or low molecular weight (LMW) poly(I:C) (1.0 μg/ml) for 4 h. Data are shown as mean ± SEM of three independent experiments. Two-sided Student’s t test; ***P < 0.001. P values = 0.000008 (HMW, WT vs SKIV2LKO 1), 0.000123 (LMW, WT vs SKIV2LKO 1), 0.000965 (HMW, WT vs SKIV2LKO 2), 0.000293 (LMW, WT vs SKIV2LKO 2). (B) RT-qPCR analysis of IFNB1 mRNA in WT and SKIV2LKO cells treated with 3p-hpRNA at indicated concentration for 4 h. Data are shown as mean ± SEM of four independent experiments. Two-sided Student’s t test; **P < 0.01, ***P < 0.001. P values = 0.000007 (150 ng/ml), 0.000038 (500 ng/ml). (C) Western blot analysis of RSAD2 proteins in WT and two independent lines of SKIV2LKO A549 cells after poly(I:C) (0.1 μg/ml) treatment for 8 h. (D) Western blot analysis of proteins of RNA sensing pathway in WT and two independent lines of SKIV2LKO A549 cells. (E) RT-qPCR analysis of genes of RNA sensing pathway in WT and two independent lines of SKIV2LKO A549 cells. Data are shown as mean ± SEM of three independent experiments. (F) RT-qPCR analysis of ISG expression in WT and two independent lines of SKIV2LKO A549 cells. Data are shown as mean ± SEM of three independent experiments. (G) Western blot analysis of IFNAR1 and SKIV2L proteins in WT and indicated knockout cells. (H) RT-qPCR analysis of IFNB1 mRNA in WT and indicated knockout cells after poly(I:C) (1.0 μg/ml) treatment for 4 h or SINV infection (MOI 0.3) for 24 h. Data are shown as mean ± SEM of four independent experiments. Two-sided Student’s t test; ***P < 0.001. P values = 0.000003 (poly(I:C), IFNAR1KO vs IFNAR1KOSKIV2LKO 1), 0.000283 (poly(I:C), IFNAR1KO vs IFNAR1KOSKIV2LKO 2), 0.000366 (SINV, IFNAR1KO vs IFNAR1KOSKIV2LKO 1), 0.000002 (SINV, IFNAR1KO vs IFNAR1KOSKIV2LKO 2). (I) RT-qPCR analysis of IFNB1 and ISGs mRNA in WT and two independent lines of SKIV2LKO A549 cells after recombinant IFN-b treatment (20 ng/ml for 24 h). Data are shown as mean ± SEM of three independent experiments. Source data are available online for this figure.

Figure EV2 SKIV2L negatively regulates the OAS-RNase L pathway.

(A) Western blot analysis of apoptosis in WT, SKIV2LKO, SKIV2LKOOAS3KO, SKIV2LKOOAS3KOOAS1KO A549 cells after poly(I:C) (1.0 μg/ml) treatment for 4 h. (B) Western blot analysis of RIG-I and MDA5 in single or double gene knockout cells (as indicated on top) treated with IFN-β (20 ng/ml) for 24 h. (C) RT-qPCR analysis of IFNB1 mRNA in WT, DDX58KO and IFIH1KO after SeV infection for 24 h or EMCV infection for 6 h. Data are shown as mean ± SEM of three or four independent experiments. Two-sided Student’s t test; **P < 0.01, ***P < 0.001; ns, not significant. P values = 0.007922 (Sev, WT vs DDX58KO), 0.182148 (Sev, WT vs IFIH1KO), 0.853967 (EMCV, WT vs DDX58KO), 0.000002 (EMCV, WT vs IFIH1KO). (D) RT-qPCR analysis of IFNB1 mRNA in WT and indicated gene knockout cells after poly(I:C) treatment (0.3 μg/ml) for 4 h. Fold change of IFNB1 mRNA compared to mock-treated WT cells is shown. Data are shown as mean ± SEM of four independent experiments. Two-sided Student’s t test; ***P < 0.001. P values = 0.000007 (WT vs DDX58KO), <0.000001 (WT vs SKIV2LKO), <0.000001 (SKIV2LKO vs SKIV2LKODDX58KO). (E, F) DDX58KO cells were treated with HMW poly(I:C) or IFN-β. Expression of ISG and IFNB1 were analyzed using RT-qPCR. Data are shown as mean ± SEM of three independent experiments. Source data are available online for this figure.

Figure EV3 SKIV2L restricts OAS1-mediated autoinflammation.

(A) rRNA cleavage analysis of total RNA in WT or SKIV2LKO cells after induction of WT or A76V mutant OAS1 with doxycycline (1.0 μg/ml) for 24 h. Data are representative of at least three independent experiments. (B) RT-qPCR analysis of IFNB1 mRNA in WT or SKIV2LKO cells after induction of WT or A76V mutant OAS1 with doxycycline (1.0 μg/ml) for 24 h. Data are shown as mean ± SEM of three independent experiments. (C) Western blot analysis of IFNAR1 and SKIV2L proteins in WT and indicated knockout cells stably expressing inducible A76V mutant OAS1. (D) RT-qPCR analysis of IFNB1 mRNA in in WT and indicated knockout cells after induction of A76V mutant OAS1 with doxycycline (0.5, 1.0 μg/ml) for 24 h. Data are shown as mean ± SEM of three independent experiments. Two-sided Student’s t test; ***P < 0.001. P values = 0.000086 (Dox 0.5), 0.000036 (Dox 1.0). (E) RT-qPCR analysis of IFNB1 mRNA in WT, SKIV2LKO, DDX58KO, SKIV2LKO DDX58KO cells after induction of OAS1 A76V mutant with doxycycline (1.0 μg/ml) for 24 h. Data are mean ± SEM of three independent experiments. Two-sided Student’s t test; **P < 0.01. P values = 0.002764 (WT vs SKIV2LKO), 0.002985 (DDX58KO vs SKIV2LKO DDX58KO). (F) MEFs were transfected with RNA (1.0 μg/ml) isolated from WT and SKIV2LKO A549 cells after induction of OAS1 A76V mutant (dox 1.0 μg/ml for 24 h) with or without RNase A treatment. Expression of Ifnb was measured by RT-qPCR. Data are mean ± SEM of four independent experiments. Source data are available online for this figure.

Supplementary information

Expanded view data, supplementary information, appendices are available for this paper at 10.1038/s44318-024-00187-1.

Acknowledgements

We thank Dr. Susan Weiss (U Penn) for providing RNASELKO and OAS3KO A549 cells, members of the Yan lab for helpful discussion. We thank Drs. Julie Pfeiffer and Carolyn Sturge (UT Southwestern Medical Center) for assistance with EMCV infection. This work was supported by the NIH (AI153576 to NY, AI135922 to RHS).

Author contributions

Kun Yang: Conceptualization; Data curation; Formal analysis; Validation; Investigation; Visualization; Methodology; Writing—original draft; Project administration; Writing—review and editing. Beihua Dong: Formal analysis; Investigation. Abhishek Asthana: Formal analysis; Investigation. Robert H Silverman: Conceptualization; Resources; Formal analysis; Methodology; Writing—original draft. Nan Yan: Conceptualization; Resources; Data curation; Formal analysis; Supervision; Funding acquisition; Investigation; Visualization; Writing—original draft; Project administration; Writing—review and editing.

Source data underlying figure panels in this paper may have individual authorship assigned. Where available, figure panel/source data authorship is listed in the following database record: biostudies:S-SCDT-10_1038-S44318-024-00187-1.

Data availability

This study includes no data deposited in external repositories.

The source data of this paper are collected in the following database record: biostudies:S-SCDT-10_1038-S44318-024-00187-1.

Disclosure and competing interests statement

The authors declare no competing interests.
==== Refs
References

Acharya D Reis R Volcic M Liu G Wang MK Chia BS Nchioua R Gross R Munch J Kirchhoff F Actin cytoskeleton remodeling primes RIG-I-like receptor activation Cell 2022 185 3588 3602.e3521 10.1016/j.cell.2022.08.011 36113429
Acharya D, Reis R, Volcic M, Liu G, Wang MK, Chia BS, Nchioua R, Gross R, Munch J, Kirchhoff F et al (2022) Actin cytoskeleton remodeling primes RIG-I-like receptor activation. Cell 185:3588–3602.e352136113429 10.1016/j.cell.2022.08.011
Aguilera ER Nguyen Y Sasaki J Pfeiffer JK Bacterial stabilization of a panel of picornaviruses mSphere 2019 4 e00183 19 10.1128/mSphere.00183-19 30944213
Aguilera ER, Nguyen Y, Sasaki J, Pfeiffer JK (2019) Bacterial stabilization of a panel of picornaviruses. mSphere 4:e00183–1930944213 10.1128/mSphere.00183-19
Banday AR Stanifer ML Florez-Vargas O Onabajo OO Papenberg BW Zahoor MA Mirabello L Ring TJ Lee CH Albert PS Genetic regulation of OAS1 nonsense-mediated decay underlies association with COVID-19 hospitalization in patients of European and African ancestries Nat Genet 2022 54 1103 1116 10.1038/s41588-022-01113-z 35835913
Banday AR, Stanifer ML, Florez-Vargas O, Onabajo OO, Papenberg BW, Zahoor MA, Mirabello L, Ring TJ, Lee CH, Albert PS et al (2022) Genetic regulation of OAS1 nonsense-mediated decay underlies association with COVID-19 hospitalization in patients of European and African ancestries. Nat Genet 54:1103–111635835913 10.1038/s41588-022-01113-z
Cadena C Hur S Filament-like assemblies of intracellular nucleic acid sensors: commonalities and differences Mol Cell 2019 76 243 254 10.1016/j.molcel.2019.09.023 31626748
Cadena C, Hur S (2019) Filament-like assemblies of intracellular nucleic acid sensors: commonalities and differences. Mol Cell 76:243–25431626748 10.1016/j.molcel.2019.09.023
Chakrabarti A Banerjee S Franchi L Loo YM Gale M Jr. Nunez G Silverman RH RNase L activates the NLRP3 inflammasome during viral infections Cell Host Microbe 2015 17 466 477 10.1016/j.chom.2015.02.010 25816776
Chakrabarti A, Banerjee S, Franchi L, Loo YM, Gale M Jr., Nunez G, Silverman RH (2015) RNase L activates the NLRP3 inflammasome during viral infections. Cell Host Microbe 17:466–47725816776 10.1016/j.chom.2015.02.010
Chen YG Hur S Cellular origins of dsRNA, their recognition and consequences Nat Rev Mol Cell Biol 2022 23 286 301 10.1038/s41580-021-00430-1 34815573
Chen YG, Hur S (2022) Cellular origins of dsRNA, their recognition and consequences. Nat Rev Mol Cell Biol 23:286–30134815573 10.1038/s41580-021-00430-1
Chung H Calis JJA Wu X Sun T Yu Y Sarbanes SL Dao Thi VL Shilvock AR Hoffmann HH Rosenberg BR Human ADAR1 prevents endogenous RNA from triggering translational shutdown Cell 2018 172 811 824.e814 10.1016/j.cell.2017.12.038 29395325
Chung H, Calis JJA, Wu X, Sun T, Yu Y, Sarbanes SL, Dao Thi VL, Shilvock AR, Hoffmann HH, Rosenberg BR et al (2018) Human ADAR1 prevents endogenous RNA from triggering translational shutdown. Cell 172:811–824.e81429395325 10.1016/j.cell.2017.12.038
Dong B Niwa M Walter P Silverman RH Basis for regulated RNA cleavage by functional analysis of RNase L and Ire1p RNA 2001 7 361 373 10.1017/S1355838201002230 11333017
Dong B, Niwa M, Walter P, Silverman RH (2001) Basis for regulated RNA cleavage by functional analysis of RNase L and Ire1p. RNA 7:361–37311333017 10.1017/S1355838201002230
Drappier M Michiels T Inhibition of the OAS/RNase L pathway by viruses Curr Opin Virol 2015 15 19 26 10.1016/j.coviro.2015.07.002 26231767
Drappier M, Michiels T (2015) Inhibition of the OAS/RNase L pathway by viruses. Curr Opin Virol 15:19–2626231767 10.1016/j.coviro.2015.07.002
Eckard SC Rice GI Fabre A Badens C Gray EE Hartley JL Crow YJ Stetson DB The SKIV2L RNA exosome limits activation of the RIG-I-like receptors Nat Immunol 2014 15 839 845 10.1038/ni.2948 25064072
Eckard SC, Rice GI, Fabre A, Badens C, Gray EE, Hartley JL, Crow YJ, Stetson DB (2014) The SKIV2L RNA exosome limits activation of the RIG-I-like receptors. Nat Immunol 15:839–84525064072 10.1038/ni.2948
Goubau D Schlee M Deddouche S Pruijssers AJ Zillinger T Goldeck M Schuberth C Van der Veen AG Fujimura T Rehwinkel J Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5’-diphosphates Nature 2014 514 372 375 10.1038/nature13590 25119032
Goubau D, Schlee M, Deddouche S, Pruijssers AJ, Zillinger T, Goldeck M, Schuberth C, Van der Veen AG, Fujimura T, Rehwinkel J et al (2014) Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5’-diphosphates. Nature 514:372–37525119032 10.1038/nature13590
Halbach F Reichelt P Rode M Conti E The yeast ski complex: crystal structure and RNA channeling to the exosome complex Cell 2013 154 814 826 10.1016/j.cell.2013.07.017 23953113
Halbach F, Reichelt P, Rode M, Conti E (2013) The yeast ski complex: crystal structure and RNA channeling to the exosome complex. Cell 154:814–82623953113 10.1016/j.cell.2013.07.017
Hartner JC Walkley CR Lu J Orkin SH ADAR1 is essential for the maintenance of hematopoiesis and suppression of interferon signaling Nat Immunol 2009 10 109 115 10.1038/ni.1680 19060901
Hartner JC, Walkley CR, Lu J, Orkin SH (2009) ADAR1 is essential for the maintenance of hematopoiesis and suppression of interferon signaling. Nat Immunol 10:109–11519060901 10.1038/ni.1680
Hasan M Koch J Rakheja D Pattnaik AK Brugarolas J Dozmorov I Levine B Wakeland EK Lee-Kirsch MA Yan N Trex1 regulates lysosomal biogenesis and interferon-independent activation of antiviral genes Nat Immunol 2013 14 61 71 10.1038/ni.2475 23160154
Hasan M, Koch J, Rakheja D, Pattnaik AK, Brugarolas J, Dozmorov I, Levine B, Wakeland EK, Lee-Kirsch MA, Yan N (2013) Trex1 regulates lysosomal biogenesis and interferon-independent activation of antiviral genes. Nat Immunol 14:61–7123160154 10.1038/ni.2475
Houseley J LaCava J Tollervey D RNA-quality control by the exosome Nat Rev Mol Cell Biol 2006 7 529 539 10.1038/nrm1964 16829983
Houseley J, LaCava J, Tollervey D (2006) RNA-quality control by the exosome. Nat Rev Mol Cell Biol 7:529–53916829983 10.1038/nrm1964
Jung S von Thulen T Yang I Laukemper V Rupf B Janga H Panagiotidis GD Schoen A Nicolai M Schulte LN A ribosomal RNA fragment with 2’,3’-cyclic phosphate and GTP-binding activity acts as RIG-I ligand Nucleic Acids Res 2020 48 10397 10412 10.1093/nar/gkaa739 32946572
Jung S, von Thulen T, Yang I, Laukemper V, Rupf B, Janga H, Panagiotidis GD, Schoen A, Nicolai M, Schulte LN et al (2020) A ribosomal RNA fragment with 2’,3’-cyclic phosphate and GTP-binding activity acts as RIG-I ligand. Nucleic Acids Res 48:10397–1041232946572 10.1093/nar/gkaa739
Kerr IM Brown RE pppA2’p5’A2’p5’A: an inhibitor of protein synthesis synthesized with an enzyme fraction from interferon-treated cells Proc Natl Acad Sci USA 1978 75 256 260 10.1073/pnas.75.1.256 272640
Kerr IM, Brown RE (1978) pppA2’p5’A2’p5’A: an inhibitor of protein synthesis synthesized with an enzyme fraction from interferon-treated cells. Proc Natl Acad Sci USA 75:256–260272640 10.1073/pnas.75.1.256
Khan M, Gasser S (2016) Generating primary fibroblast cultures from mouse ear and tail tissues. J Vis Exp 107:e53565
Kilchert C Wittmann S Vasiljeva L The regulation and functions of the nuclear RNA exosome complex Nat Rev Mol Cell Biol 2016 17 227 239 10.1038/nrm.2015.15 26726035
Kilchert C, Wittmann S, Vasiljeva L (2016) The regulation and functions of the nuclear RNA exosome complex. Nat Rev Mol Cell Biol 17:227–23926726035 10.1038/nrm.2015.15
Kristiansen H Gad HH Eskildsen-Larsen S Despres P Hartmann R The oligoadenylate synthetase family: an ancient protein family with multiple antiviral activities J Interferon Cytokine Res 2011 31 41 47 10.1089/jir.2010.0107 21142819
Kristiansen H, Gad HH, Eskildsen-Larsen S, Despres P, Hartmann R (2011) The oligoadenylate synthetase family: an ancient protein family with multiple antiviral activities. J Interferon Cytokine Res 31:41–4721142819 10.1089/jir.2010.0107
Li Y Banerjee S Wang Y Goldstein SA Dong B Gaughan C Silverman RH Weiss SR Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses Proc Natl Acad Sci USA 2016 113 2241 2246 10.1073/pnas.1519657113 26858407
Li Y, Banerjee S, Wang Y, Goldstein SA, Dong B, Gaughan C, Silverman RH, Weiss SR (2016) Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses. Proc Natl Acad Sci USA 113:2241–224626858407 10.1073/pnas.1519657113
Liddicoat BJ Piskol R Chalk AM Ramaswami G Higuchi M Hartner JC Li JB Seeburg PH Walkley CR RNA editing by ADAR1 prevents MDA5 sensing of endogenous dsRNA as nonself Science 2015 349 1115 1120 10.1126/science.aac7049 26275108
Liddicoat BJ, Piskol R, Chalk AM, Ramaswami G, Higuchi M, Hartner JC, Li JB, Seeburg PH, Walkley CR (2015) RNA editing by ADAR1 prevents MDA5 sensing of endogenous dsRNA as nonself. Science 349:1115–112026275108 10.1126/science.aac7049
Magg T Okano T Koenig LM Boehmer DFR Schwartz SL Inoue K Heimall J Licciardi F Ley-Zaporozhan J Ferdman RM Heterozygous OAS1 gain-of-function variants cause an autoinflammatory immunodeficiency Sci Immunol 2021 6 eabf9564 10.1126/sciimmunol.abf9564 34145065
Magg T, Okano T, Koenig LM, Boehmer DFR, Schwartz SL, Inoue K, Heimall J, Licciardi F, Ley-Zaporozhan J, Ferdman RM et al (2021) Heterozygous OAS1 gain-of-function variants cause an autoinflammatory immunodeficiency. Sci Immunol 6:eabf956434145065 10.1126/sciimmunol.abf9564
Malathi K Dong B Gale M Jr. Silverman RH Small self-RNA generated by RNase L amplifies antiviral innate immunity Nature 2007 448 816 819 10.1038/nature06042 17653195
Malathi K, Dong B, Gale M Jr., Silverman RH (2007) Small self-RNA generated by RNase L amplifies antiviral innate immunity. Nature 448:816–81917653195 10.1038/nature06042
Maurano M Snyder JM Connelly C Henao-Mejia J Sidrauski C Stetson DB Protein kinase R and the integrated stress response drive immunopathology caused by mutations in the RNA deaminase ADAR1 Immunity 2021 54 1948 1960.e1945 10.1016/j.immuni.2021.07.001 34343497
Maurano M, Snyder JM, Connelly C, Henao-Mejia J, Sidrauski C, Stetson DB (2021) Protein kinase R and the integrated stress response drive immunopathology caused by mutations in the RNA deaminase ADAR1. Immunity 54:1948–1960.e194534343497 10.1016/j.immuni.2021.07.001
Molleston JM Sabin LR Moy RH Menghani SV Rausch K Gordesky-Gold B Hopkins KC Zhou R Jensen TH Wilusz JE A conserved virus-induced cytoplasmic TRAMP-like complex recruits the exosome to target viral RNA for degradation Genes Dev 2016 30 1658 1670 10.1101/gad.284604.116 27474443
Molleston JM, Sabin LR, Moy RH, Menghani SV, Rausch K, Gordesky-Gold B, Hopkins KC, Zhou R, Jensen TH, Wilusz JE et al (2016) A conserved virus-induced cytoplasmic TRAMP-like complex recruits the exosome to target viral RNA for degradation. Genes Dev 30:1658–167027474443 10.1101/gad.284604.116
Myong S Cui S Cornish PV Kirchhofer A Gack MU Jung JU Hopfner KP Ha T Cytosolic viral sensor RIG-I is a 5’-triphosphate-dependent translocase on double-stranded RNA Science 2009 323 1070 1074 10.1126/science.1168352 19119185
Myong S, Cui S, Cornish PV, Kirchhofer A, Gack MU, Jung JU, Hopfner KP, Ha T (2009) Cytosolic viral sensor RIG-I is a 5’-triphosphate-dependent translocase on double-stranded RNA. Science 323:1070–107419119185 10.1126/science.1168352
Orvedahl A MacPherson S Sumpter R Jr. Talloczy Z Zou Z Levine B Autophagy protects against Sindbis virus infection of the central nervous system Cell Host Microbe 2010 7 115 127 10.1016/j.chom.2010.01.007 20159618
Orvedahl A, MacPherson S, Sumpter R Jr. Talloczy Z, Zou Z, Levine B (2010) Autophagy protects against Sindbis virus infection of the central nervous system. Cell Host Microbe 7:115–12720159618 10.1016/j.chom.2010.01.007
Peisley A Wu B Yao H Walz T Hur S RIG-I forms signaling-competent filaments in an ATP-dependent, ubiquitin-independent manner Mol Cell 2013 51 573 583 10.1016/j.molcel.2013.07.024 23993742
Peisley A, Wu B, Yao H, Walz T, Hur S (2013) RIG-I forms signaling-competent filaments in an ATP-dependent, ubiquitin-independent manner. Mol Cell 51:573–58323993742 10.1016/j.molcel.2013.07.024
Rehwinkel J Gack MU RIG-I-like receptors: their regulation and roles in RNA sensing Nat Rev Immunol 2020 20 537 551 10.1038/s41577-020-0288-3 32203325
Rehwinkel J, Gack MU (2020) RIG-I-like receptors: their regulation and roles in RNA sensing. Nat Rev Immunol 20:537–55132203325 10.1038/s41577-020-0288-3
Rice GI Kasher PR Forte GM Mannion NM Greenwood SM Szynkiewicz M Dickerson JE Bhaskar SS Zampini M Briggs TA Mutations in ADAR1 cause Aicardi-Goutieres syndrome associated with a type I interferon signature Nat Genet 2012 44 1243 1248 10.1038/ng.2414 23001123
Rice GI, Kasher PR, Forte GM, Mannion NM, Greenwood SM, Szynkiewicz M, Dickerson JE, Bhaskar SS, Zampini M, Briggs TA et al (2012) Mutations in ADAR1 cause Aicardi-Goutieres syndrome associated with a type I interferon signature. Nat Genet 44:1243–124823001123 10.1038/ng.2414
Schmidt C Kowalinski E Shanmuganathan V Defenouillere Q Braunger K Heuer A Pech M Namane A Berninghausen O Fromont-Racine M The cryo-EM structure of a ribosome-Ski2-Ski3-Ski8 helicase complex Science 2016 354 1431 1433 10.1126/science.aaf7520 27980209
Schmidt C, Kowalinski E, Shanmuganathan V, Defenouillere Q, Braunger K, Heuer A, Pech M, Namane A, Berninghausen O, Fromont-Racine M et al (2016) The cryo-EM structure of a ribosome-Ski2-Ski3-Ski8 helicase complex. Science 354:1431–143327980209 10.1126/science.aaf7520
Shigematsu M Kawamura T Kirino Y Generation of 2’,3’-cyclic phosphate-containing RNAs as a hidden layer of the transcriptome Front Genet 2018 9 562 10.3389/fgene.2018.00562 30538719
Shigematsu M, Kawamura T, Kirino Y (2018) Generation of 2’,3’-cyclic phosphate-containing RNAs as a hidden layer of the transcriptome. Front Genet 9:56230538719 10.3389/fgene.2018.00562
Soveg FW Schwerk J Gokhale NS Cerosaletti K Smith JR Pairo-Castineira E Kell AM Forero A Zaver SA Esser-Nobis K Endomembrane targeting of human OAS1 p46 augments antiviral activity eLife 2021 10 e71047 10.7554/eLife.71047 34342578
Soveg FW, Schwerk J, Gokhale NS, Cerosaletti K, Smith JR, Pairo-Castineira E, Kell AM, Forero A, Zaver SA, Esser-Nobis K et al (2021) Endomembrane targeting of human OAS1 p46 augments antiviral activity. eLife 10:e7104734342578 10.7554/eLife.71047
Tang Q Rigby RE Young GR Hvidt AK Davis T Tan TK Bridgeman A Townsend AR Kassiotis G Rehwinkel J Adenosine-to-inosine editing of endogenous Z-form RNA by the deaminase ADAR1 prevents spontaneous MAVS-dependent type I interferon responses Immunity 2021 54 1961 1975.e1965 10.1016/j.immuni.2021.08.011 34525337
Tang Q, Rigby RE, Young GR, Hvidt AK, Davis T, Tan TK, Bridgeman A, Townsend AR, Kassiotis G, Rehwinkel J (2021) Adenosine-to-inosine editing of endogenous Z-form RNA by the deaminase ADAR1 prevents spontaneous MAVS-dependent type I interferon responses. Immunity 54:1961–1975.e196534525337 10.1016/j.immuni.2021.08.011
Thakur CS Xu Z Wang Z Novince Z Silverman RH A convenient and sensitive fluorescence resonance energy transfer assay for RNase L and 2’,5’ oligoadenylates Methods Mol Med 2005 116 103 113 16000857
Thakur CS, Xu Z, Wang Z, Novince Z, Silverman RH (2005) A convenient and sensitive fluorescence resonance energy transfer assay for RNase L and 2’,5’ oligoadenylates. Methods Mol Med 116:103–11316000857
Tu X Chu TT Jeltema D Abbott K Yang K Xing C Han J Dobbs N Yan N Interruption of post-Golgi STING trafficking activates tonic interferon signaling Nat Commun 2022 13 6977 10.1038/s41467-022-33765-0 36379959
Tu X, Chu TT, Jeltema D, Abbott K, Yang K, Xing C, Han J, Dobbs N, Yan N (2022) Interruption of post-Golgi STING trafficking activates tonic interferon signaling. Nat Commun 13:697736379959 10.1038/s41467-022-33765-0
Weston S Baracco L Keller C Matthews K McGrath ME Logue J Liang J Dyall J Holbrook MR Hensley LE The SKI complex is a broad-spectrum, host-directed antiviral drug target for coronaviruses, influenza, and filoviruses Proc Natl Acad Sci USA 2020 117 30687 30698 10.1073/pnas.2012939117 33184176
Weston S, Baracco L, Keller C, Matthews K, McGrath ME, Logue J, Liang J, Dyall J, Holbrook MR, Hensley LE et al (2020) The SKI complex is a broad-spectrum, host-directed antiviral drug target for coronaviruses, influenza, and filoviruses. Proc Natl Acad Sci USA 117:30687–3069833184176 10.1073/pnas.2012939117
Wickenhagen A Sugrue E Lytras S Kuchi S Noerenberg M Turnbull ML Loney C Herder V Allan J Jarmson I A prenylated dsRNA sensor protects against severe COVID-19 Science 2021 374 eabj3624 10.1126/science.abj3624 34581622
Wickenhagen A, Sugrue E, Lytras S, Kuchi S, Noerenberg M, Turnbull ML, Loney C, Herder V, Allan J, Jarmson I et al (2021) A prenylated dsRNA sensor protects against severe COVID-19. Science 374:eabj362434581622 10.1126/science.abj3624
Wu J Chen YJ Dobbs N Sakai T Liou J Miner JJ Yan N STING-mediated disruption of calcium homeostasis chronically activates ER stress and primes T cell death J Exp Med 2019 216 867 883 10.1084/jem.20182192 30886058
Wu J, Chen YJ, Dobbs N, Sakai T, Liou J, Miner JJ, Yan N (2019) STING-mediated disruption of calcium homeostasis chronically activates ER stress and primes T cell death. J Exp Med 216:867–88330886058 10.1084/jem.20182192
Wu J Dobbs N Yang K Yan N Interferon-independent activities of mammalian STING mediate antiviral response and tumor immune evasion Immunity 2020 53 115 126.e115 10.1016/j.immuni.2020.06.009 32640258
Wu J, Dobbs N, Yang K, Yan N (2020) Interferon-independent activities of mammalian STING mediate antiviral response and tumor immune evasion. Immunity 53:115–126.e11532640258 10.1016/j.immuni.2020.06.009
Yang K Han J Asada M Gill JG Park JY Sathe MN Gattineni J Wright T Wysocki CA de la Morena MT Cytoplasmic RNA quality control failure engages mTORC1-mediated autoinflammatory disease J Clin Investig 2022 132 e146176 10.1172/JCI146176 35040435
Yang K, Han J, Asada M, Gill JG, Park JY, Sathe MN, Gattineni J, Wright T, Wysocki CA, de la Morena MT et al (2022a) Cytoplasmic RNA quality control failure engages mTORC1-mediated autoinflammatory disease. J Clin Investig 132:e14617635040435 10.1172/JCI146176
Yang K Han J Gill JG Park JY Sathe MN Gattineni J Wright T Wysocki C de la Morena MT Yan N The mammalian SKIV2L RNA exosome is essential for early B cell development Sci Immunol 2022 7 eabn2888 10.1126/sciimmunol.abn2888 35658009
Yang K, Han J, Gill JG, Park JY, Sathe MN, Gattineni J, Wright T, Wysocki C, de la Morena MT, Yan N (2022b) The mammalian SKIV2L RNA exosome is essential for early B cell development. Sci Immunol 7:eabn288835658009 10.1126/sciimmunol.abn2888
Yang K Huang R Fujihira H Suzuki T Yan N N-glycanase NGLY1 regulates mitochondrial homeostasis and inflammation through NRF1 J Exp Med 2018 215 2600 2616 10.1084/jem.20180783 30135079
Yang K, Huang R, Fujihira H, Suzuki T, Yan N (2018) N-glycanase NGLY1 regulates mitochondrial homeostasis and inflammation through NRF1. J Exp Med 215:2600–261630135079 10.1084/jem.20180783
Zhou A Hassel BA Silverman RH Expression cloning of 2-5A-dependent RNAase: a uniquely regulated mediator of interferon action Cell 1993 72 753 765 10.1016/0092-8674(93)90403-D 7680958
Zhou A, Hassel BA, Silverman RH (1993) Expression cloning of 2-5A-dependent RNAase: a uniquely regulated mediator of interferon action. Cell 72:753–7657680958 10.1016/0092-8674(93)90403-D
Zhou A Paranjape J Brown TL Nie H Naik S Dong B Chang A Trapp B Fairchild R Colmenares C Interferon action and apoptosis are defective in mice devoid of 2’,5’-oligoadenylate-dependent RNase L EMBO J 1997 16 6355 6363 10.1093/emboj/16.21.6355 9351818
Zhou A, Paranjape J, Brown TL, Nie H, Naik S, Dong B, Chang A, Trapp B, Fairchild R, Colmenares C et al (1997) Interferon action and apoptosis are defective in mice devoid of 2’,5’-oligoadenylate-dependent RNase L. EMBO J 16:6355–63639351818 10.1093/emboj/16.21.6355
Zinder JC Wasmuth EV Lima CD Nuclear RNA exosome at 3.1 A reveals substrate specificities, RNA Paths, and allosteric inhibition of Rrp44/Dis3 Mol Cell 2016 64 734 745 10.1016/j.molcel.2016.09.038 27818140
Zinder JC, Wasmuth EV, Lima CD (2016) Nuclear RNA exosome at 3.1 A reveals substrate specificities, RNA Paths, and allosteric inhibition of Rrp44/Dis3. Mol Cell 64:734–74527818140 10.1016/j.molcel.2016.09.038
