Lncrna Malat1 Inhibition of TDP43 Cleavage Suppresses IRF3-Initiated Antiviral Innate Immunity

LncRNA Malat1 inhibition of TDP43 cleavage suppresses IRF3-initiated antiviral innate immunity

Wei Liua, Ziqiao Wanga, Lun Liua, Zongheng Yanga, Shuo Liua, Zhongfei Maa, Yin Liua, Yuanwu Mab, Lianfeng Zhangb, Xuan Zhangc, Minghong Jianga,1, and Xuetao Caoa,d,e,1

aDepartment of Immunology, Center for Immunotherapy, Institute of Basic Medical Sciences, Peking Union Medical College, Chinese Academy of Medical Sciences, 100005 Beijing, China; bInstitute of Laboratory Animal Science, Chinese Academy of Medical Sciences, 100021 Beijing, China; cDepartment of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, 100730 Beijing, China; dNational Key Laboratory of Medical Immunology and Institute of Immunology, Navy Medical University, 200433 Shanghai, China; and eCollege of Life Science, Nankai University, 300071 Tianjin, China

Edited by Akinori Takaoka, Hokkaido University, Sapporo, Japan, and accepted by Editorial Board Member Tadatsugu Taniguchi August 3, 2020 (receivedfor review March 1, 2020) Long noncoding RNAs (lncRNAs) involved in the regulation of degradation of host mRNAs (8–11). Many viruses induce wide- antiviral innate immune responses need to be further identified. spread host RNA decay through virally encoded endonucleases to By functionally screening the lncRNAs in macrophages, here we reduce activation of immune response and provide access to the identified lncRNA Malat1, abundant in the nucleus but signifi- host’s resources for viral replication. It is estimated that up to two- cantly down-regulated after viral infection, as a negative regulator thirds of total mRNAs are degraded upon expression by these of antiviral type I IFN (IFN-I) production. Malat1 directly bound to viral endonucleases (12). A significant fraction of lncRNAs are the transactive response DNA-binding protein (TDP43) in the nu- located in the nucleus and nuclear lncRNAs play important roles cleus and prevented activation of TDP43 by blocking the activated in physiological, biological, and pathological processes via differ- caspase-3-mediated TDP43 cleavage to TDP35. The cleaved TDP35 ent mechanisms (13, 14). However, the role of nuclear abundant increased the nuclear IRF3 protein level by binding and degrading lncRNAs in regulating RNA metabolism and RBP activity in the Rbck1 pre-mRNA to prevent IRF3 proteasomal degradation upon antiviral innate immune response remains to be further investigated. viral infection, thus selectively promoting antiviral IFN-I production. In this study, we functionally screen the differently expressed Deficiency of Malat1 enhanced antiviral innate responses in vivo, lncRNAs in macrophages and identify the nuclear abundant IMMUNOLOGY AND INFLAMMATION accompanying the increased IFN-I production and reduced viral bur- lncRNA Malat1 as a negative regulator of antiviral type I IFN den. Importantly, the reduced MALAT1, augmented IRF3, and in- IFNA production. We find that Malat1 can bind TDP43, inhibit the creased mRNA were found in peripheral blood mononuclear cleavage of TDP43 to its active form TDP35, then reduce nuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients. IRF3 level in resting cells, maintaining immune homeostasis. Therefore, the down-regulation of MALAT1 in virus-infected cells Upon viral infection, Malat1 expression is down-regulated and or in human cells from autoimmune diseases will increase host resis- releases TDP43 for its activation via cleavage to its active form tance against viral infection or lead to autoinflammatory interferonopathies via the increased type I IFN production. Our results demonstrate TDP35, then TDP35 prevents IRF3 proteasomal degradation and that the nuclear Malat1 suppresses antiviral innate responses by thus promotes IRF3-initiated antiviral type I IFNs production, targeting TDP43 activation via RNA-RBP interactive network, adding in- feedback benefitting the host against viral infection. Interestingly, sight to the molecular regulation of innate responses and autoimmune pathogenesis. Significance

long noncoding RNA | Malat1 | TDP43 | type I interferon | innate immunity LncRNAs have been identified as regulating antiviral innate responses via different targets through various ways. Whether s the first line of defense against viral infection, the pro- there is an abundant and highly conserved lncRNA in the nu- Aduction of type I interferons (IFN-I) (IFN-α and IFN-β), cleus which may regulate antiviral innate signaling through a plays a central role by activating the expression of hundreds of new mechanism remains to be investigated. Here, we identify Malat1 IFN-stimulated genes (ISGs) for establishing an “antiviral state” that viral infection-reduced expression of feedback to restrict viral replication within infected cells (1). Insufficient promotes IRF3-initiated type I IFN production in the innate re- Malat1 production of IFNs causes chronic viral infections, while exces- sponse against viral infection. binds to TDP43 and sive amounts of IFNs are also harmful to the host, inducing prevents its cleavage mediated by activated caspase-3. The autoimmune inflammation and type I interferonopathies (2–4). cleaved TDP35 inhibits IRF3 degradation through promoting Therefore, it is of vital importance to precisely regulate the the degradation of pre-mRNA of Rbck1, an E3 ubiquitin ligase MALAT1 production of IFNs both in temporal and spatial dimensions to targeting IRF3. Aberrant reduction and IRF3 activation ensure the induction of potent antiviral innate response against are found in SLE patients, providing one mechanistic explanation viral infection but also avoid the occurrence of autoimmune for why SLE patients always have type I interferonopathies. diseases. The transcription factor IFN regulatory factor 3 (IRF3) Author contributions: M.J. and X.C. designed research; W.L., Z.W., L.L., Z.Y., S.L., Z.M., Y.L., is critical for IFNs production and directs expression of diverse Y.M., L.Z., and X.Z. performed research; M.J. and X.C. analyzed data; M.J. and X.C. wrote genes in the antiviral immune response (5). Activation of IRF3 the paper; and X.Z. provided the clinical samples. involves virus infection-induced phosphorylation at several sites The authors declare no competing interest. in the C terminal, IRF3 dimerization, and translocation to the This article is a PNAS Direct Submission. A.T. is a guest editor invited by the nucleus. However, how the IRF3 activity for initiating innate gene Editorial Board. expression is tightly regulated remains to be fully understood. Published under the PNAS license. Long noncoding RNAs (lncRNAs) are involved in many biolog- 1To whom correspondence may be addressed. Email: [email protected] or caoxt@ ical processes, including immunity and inflammation (6, 7). They immunol.org. participate in the battle between host and virus via different mech- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ anisms, including regulating the activity of RNA-binding proteins doi:10.1073/pnas.2003932117/-/DCSupplemental. (RBPs) as well as RNA metabolism, especially the translation and

www.pnas.org/cgi/doi/10.1073/pnas.2003932117 PNAS Latest Articles | 1of12 Downloaded by guest on September 29, 2021 the reduced MALAT1 and increased IRF3 in peripheral blood transcript 1 (Malat1) robustly decreased virus replication in mac- mononuclear cells (PBMCs) from systemic lupus erythematosus rophages upon GFP-VSV infection for 12 h (SI Appendix, Fig. (SLE) patients with type I interferonopathies were confirmed. S1C). Notably, Malat1 was the fourth most abundant lncRNA in Thus, our findings provide a potential target for controlling viral the uninfected cells and its expression was significantly reduced infection and IFN-I-related inflammatory autoimmune diseases. upon VSV infection (Fig. 1A). Malat1 was universally and highly expressed in immune organs and immune cells including macro- Results phages (SI Appendix,Fig.S1D and E), suggesting that Malat1 The Expression of Nuclear lncRNA Malat1 Decreases upon Viral Infection. might be involved in the regulation of antiviral innate immune Through RNA sequencing (RNA-Seq), we analyzed nuclear response. lncRNA profiling in RAW264.7 cells with or without vesicular Northern blot and fluorescence in situ hybridization (FISH) stomatitis virus (VSV) infection after isolation of nuclear RNAs assay further showed that the expression of Malat1 significantly (SI Appendix,Fig.S1A). The differentially expressed lncRNAs decreased in macrophages upon VSV infection (Fig. 1 B and C). (more than twofold change) are ranked according to their expres- Fluorescence probes to Malat1 exhibited punctuate staining only sion abundance in cells without VSV infection (Fig. 1A). We iso- in the nuclear compartment of cells (Fig. 1C). In addition, in- lated primary peritoneal macrophages from 8-wk-old C57BL/6 male fections with encephalomyocarditis virus (EMCV) (recognized mice and transfected the mixture composition of small interfering by melanoma differentiation-associated protein 5 [MDA5]) and RNAs (siRNAs) and antisense oligonucleotide (ASO) targeting the DNA virus herpes simplex virus type 1 (HSV-1) could also re- top 20 candidate RNAs, respectively (SI Appendix,Fig.S1B). Then duce Malat1 expression (Fig. 1D). In contrast, Toll-like receptor we detected the intracellular GFP intensity with high-content (TLR) ligands, such as lipopolysaccharide (LPS) and poly (I:C), screening (HCS) in cells upon GFP-VSV infection. We found could not significantly reduce Malat1 expression (SI Appendix, that knockdown of metastasis-associated lung adenocarcinoma Fig. S1 F and G), consistent with the findings of the previous

A B VSV (h) 0 8 VSV (h) 0 46 8 12 (nt) ENSMUST00000192833 8000 ENSMUST00000182010 Malat1 ENSMUST00000210878 4000 Malat1/28s 1.00 0.53 0.32 0.24 0.16 ENSMUST00000172812 ENSMUST00000151517 4000 28S ENSMUST00000137766 2000 ENSMUST00000129917 ENSMUST00000166047 D ENSMUST00000190033 P < 0.0001 P < 0.0001 P < 0.0001 ENSMUST00000156374 P = 0.0001 P < 0.0001 P < 0.0001 ENSMUST00000144777 ENSMUST00000132080 P < 0.0001 P = 0.0001 P = 0.0019 ENSMUST00000200021 1.0 ENSMUST00000179894 1.0 1.0 ENSMUST00000207776 ENSMUST00000125290 ENSMUST00000146976 0.5 ENSMUST00000144964 0.5 0.5 relative level relative level ENSMUST00000170644 relative level ENSMUST00000135243 Malat1 Malat1 Low High Malat1 0.0 -3log (FPKM+1) 3 0.0 0.0 10 VSV (h) 0 4 8 12 EMCV (h) 0 4 8 12 HSV-1 (h) 04812 C P < 0.0001 P < 0.0001 VSV (h) 0 4 8 12 P = 0.0008 1.0 Malat1

0.5 relative intensity Merge Malat1 0.0 VSV (h) 0 4 8 12

Fig. 1. The expression of nuclear lncRNA Malat1 decreases upon viral infection. (A) Heat map of top 20 abundant nuclear lncRNAs in RAW264.7 cells at resting state which are changed more than twofold after VSV infection for 8 h. ENSMUST00000172812 (Malat1) is pointed out by black arrow. (B) Northern blot assay of Malat1 level in RAW264.7 cells along with VSV infection for indicated hours. Relative intensity of Malat1 is calculated by the ImageJ program. (C) Confocal microscope images from FISH assay of Malat1 level in RAW264.7 cells along with VSV infection for indicated hours. Relative fluorescence intensity of Malat1 is calculated by the ImageJ program (Right). Red, Malat1; blue, DAPI. (Scale bar, 5 μm.) (D) qRT-PCR analysis of relative Malat1 level in RAW264.7 cells along with VSV, EMCV, and HSV-1 infection for indicated hours, respectively. Data are representative of three independent experiments (B and C) or shown as mean ± SD of n = 3 biological replicates (C and D), two-tailed unpaired Student’s t test (C and D).

2of12 | www.pnas.org/cgi/doi/10.1073/pnas.2003932117 Liu et al. Downloaded by guest on September 29, 2021 study (15). In addition, it has been reported that widespread function of these proteins in antiviral innate response with siRNAs RNAs are efficiently degraded during apoptosis (16), and then and found that knockdown of Tardbp (siTardbp, encoding TDP43) we investigated whether the reduction of Malat1 was also in- increased viral replication in macrophages upon GFP-VSV duced by apoptosis. We found the inducer of apoptosis ABT-737 infection compared with negative control (siNC)(SI Appendix, (inhibiting the inhibitors Bcl-xL, Bcl-2, and Bcl-w), could sig- Fig. S4B). nificantly decrease Malat1 level (SI Appendix, Fig. S1H). Fur- Then we confirmed the interaction of Malat1 with TDP43 by thermore, the reduction of Malat1 mediated by VSV or ABT-737 ChIRP assay followed by Western blot (SI Appendix, Fig. S4C). was reversed by pretreatment of pan caspase inhibitor Z-VAD- FISH assay further demonstrated that Malat1 colocalized with FMK or Q-VD-Oph (SI Appendix, Fig. S1H), indicating that host TDP43 in the nucleus with or without VSV infection (Fig. 4A). apoptosis might promote the destabilization of Malat1. Taken To determine the position of Malat1 responsible for interacting together, these data above suggest that the expression of Malat1 with TDP43, we performed individual-nucleotide resolution cross- decreases upon viral infection, indicting its possible role in an- linking and immunoprecipitation (iCLIP) in RAW264.7 cells. After tiviral innate immune response. RNA digestion, the TDP43-RNA complexes were immunoprecipitated and appeared above the molecular weight of TDP43 (SI Malat1 Selectively Inhibits Type I IFN Production in Macrophages Appendix,Fig.S4D). The signal of the TDP43-RNA complex was upon Viral Infection. We generated Malat1 knockout RAW264.7 sharply reduced upon RNase extensive digestion, and no signal was − − cells (Malat1 / cells) which lacked a full 7,691-bp genomic re- detected by using IgG (SI Appendix,Fig.S4D), demonstrating the gion of Malat1 with the CRISPR-Cas9 system (SI Appendix, Fig. high specificity of the immunoprecipitated RNA. Then we ana- − − S2A). Upon viral infection, Malat1 / cells produced more IFN-α lyzed the cross-link-induced truncation positions of Malat1 in the and IFN-β than Malat1+/+ cells and this effect was reversed by iCLIP library and found that Malat1 cross-linkedtoTDP43via − − rescue of Malat1 in Malat1 / cells (Fig. 2A and SI Appendix, Fig. 3,618 and 4,887 sites of Malat1 (Fig. 4B). We constructed 9 frag- S2B), while the production of proinflammatory cytokines (TNF- ments of Malat1 in a head-to-tail overlap manner, each truncation α and IL-6) had no difference among these three groups (SI about 1,000 nt, and found that both fragments of Malat1 (3,006 Appendix, Fig. S2C). Correspondingly, the relative mRNA levels to 4,020 nt) and Malat1 (4,569 to 5,609 nt), which contained the of Ifna, Ifnb, and ISG (Isg15) (Fig. 2 B and C), but not Tnfa, Il6, twocross-linksites,couldbindtoTDP43,respectively(Fig.4C). and Il1b (SI Appendix, Fig. S2 D and E), significantly increased in Through RNA pull-down assay, we found that both fragments could − − Malat1 / cells upon viral infection. Furthermore, Malat1 could bind to the RRM1 domain (Fig. 4D), which was the main RNA- inhibit IRF3-induced IFN-β promoter activity (Fig. 2D) but not binding domain of TDP43 (17). These data suggest that Malat1 can NF-κB-induced IL-6 promoter activity (SI Appendix, Fig. S2F) directly bind to the RRM1 domain of TDP43 with two sites. IMMUNOLOGY AND INFLAMMATION upon VSV infection. These data indicated that Malat1 selectively To further analyze the function of TDP43, we generated TDP43- − − inhibits IFN-I production. To investigate whether Malat1 could deficient RAW264.7 cells (Tardbp / cells) with the CRISPR-Cas9 affect IFN-mediated antiviral function, we treated the Malat1+/+ system. TDP43 deficiency did not affect the expression of Malat1 in − − and Malat1 / cells with IFN-α or IFN-β and then checked ISG RAW264.7 cells with or without VSV infection (SI Appendix,Fig. − − expression. There was no difference on IFN-I effector signaling S4E). Of note, Tardpb / cells produced much less IFN-α and (p-STAT1 and IFIT1) (SI Appendix, Fig. S2G) and ISG expres- IFN-β than Tardbp+/+ cells (Fig. 4E), but produced almost the same − − sion (Fig. 2 E and F) between Malat1+/+ and Malat1 / cells, level of TNF-α and IL-6 upon VSV or HSV-1 infection (SI Ap- suggesting that Malat1 does not affect IFN function. So, Malat1 pendix,Fig.S4F). Overexpression of TDP43 could enhance IFN-β selectively inhibits the production of IFN-I upon viral infection but not IL-6 promoter activity upon VSV infection in a dose- but does not affect IFN-I antiviral function. dependent manner (Fig. 4F and SI Appendix,Fig.S4G). These results suggest that Malat1-bound TDP43 selectively promotes Deficiency of Malat1 Enhances Antiviral Response In Vivo. To further the expression of type I IFNs in response to viral infection. evaluate the role of Malat1 in host antiviral innate immune re- − − sponse in vivo, we generated Malat1-deficient mice (Malat1 / Malat1 Inhibits IFN-I Production by Blocking TDP43 Cleavage to Its mice, deleting the full 7,691-bp genomic region of Malat1). After Active Form TDP35. We next investigated how Malat1 inhibited the − − i.v. infection with VSV or HSV-1 respectively, Malat1 / mice production of type I IFNs through binding to TDP43. TDP43 +/+ produced more IFN-α and IFN-β in serum than Malat1 mice was reported to be cleaved to TDP35 mainly by cleaved caspase- (Fig. 3A). Consistently, the relative mRNA level of Ifna, Ifnb, 3 during apoptosis (18). We found that deficiency of Malat1 did − − and Isg15 in organs (liver, spleen, and lung) of Malat1 / mice was not affect the protein level of TDP43 in RAW264.7 cells in re- significantly higher than that of their littermates (Fig. 3 B–D). sponse to VSV infection (Fig. 5A). Unexpectedly, we found that − − Consequently, the VSV RNA replication of organs above was much more TDP43 was cleaved to TDP35 in Malat1 / cells − − notably reduced in Malat1 / mice (Fig. 3E). However, there was compared with that in Malat1+/+ cells upon VSV infection − − no difference of TNF-α or IL-6 expression in serum and or- (Fig. 5A). Rescue of Malat1 expression in Malat1 / cells de- gans between two groups infected with VSV or HSV-1 (SI Ap- creased the cleavage of TDP43 to TDP35 (Fig. 5A). To detect the − − pendix,Fig.S3A–D). Consistent with the higher IFNs, Malat1 / TDP43 protein level without the influence of cleavage by caspase- mice showed improved survival compared with Malat1+/+ mice 3 upon viral infection, we pretreated RAW264.7 cells with pan after being challenged with a lethal dose of VSV or HSV-1 caspase inhibitor Z-VAD-FMK, and found that the activation of infection (Fig. 3F and SI Appendix,Fig.S3E). These data suggest caspase-3 and the cleavage of TDP43 were suppressed in both − − that Malat1 inhibits antiviral innate response in vivo by selec- Malat1+/+ and Malat1 / cells with Z-VAD-FMK treatment and tively suppressing IFN-I production. VSV infection (SI Appendix,Fig.S5A). As expected, the protein − − level of TDP43 was increased in Malat1 / cells with treatment of Identification of Malat1-Bound TDP43 in Selectively Promoting IFN-I Z-VAD-FMK (SI Appendix,Fig.S5A). To determine the function Production. To analyze the underlying mechanism how Malat1 of cleaved TDP35 in innate immunity, we overexpressed TDP35 − − inhibits the production of type I IFNs, we conducted the chro- into Tardbp / cells and found that rescue of TDP35 expression matin isolation by RNA purification (ChIRP) assay followed by could increase the relative mRNA levels of Ifna, Ifnb,andIsg15 mass spectrography to determine the Malat1-bound proteins in (Fig. 5B and SI Appendix,Fig.S5B) but not proinflammatory cy- the nucleus. The 10 Malat1-bound proteins with the highest score tokines (Tnfa, Il6,andIl1b)(SI Appendix,Fig.S5C), suggesting in nuclear fraction of macrophages without viral infection are that TDP35 is the key subunit of TDP43 that promoted the pro- shown in SI Appendix, Fig. S4A. Then we used HCS to screen the duction of type I IFNs.

Liu et al. PNAS Latest Articles | 3of12 Downloaded by guest on September 29, 2021 A D pGL4.17 vector IFN-β promoter SV40 poly(A) signal

P = 0.0892 +/+ P = 0.0006 luc2 P = 0.8694 Malat1 -/- P = 0.0003 Malat1 P = 0.0031 1500 P = 0.0002 5000 -/- 80000 Malat1 +Malat1 80 P = 0.0425 P = 0.2133 60000 4000 P = 0.0004 60 40000 1000 20000 3000 P = 0.8146 40 2000 100 500 P = 0.0004 relative level 80 activity IFN-α (pg/ml) IFN-β (pg/ml) 20 60 1000 40 Relative IFN-β luc

Malat1 20 0 0 0 0 MockVSV HSV-1 Mock VSV HSV-1 Malat1 --+ Malat1 --+ Mock VSV(8h) B P < 0.0001 150000 80000 P = 0.0004 6000 P < 0.0001 P = 0.0005 Malat1+/+ 100000 60000 P < 0.0001 Malat1-/- 4000 P < 0.0001 50000 40000

P = 0.0144 mRNA (fold) P < 0.0001 mRNA (fold) 6000 mRNA (fold) 2000 20000 P < 0.0001 b1 1000 P = 0.0002 P = 0.0001

Ifna4 P = 0.0008 Ifn

500 Isg15 0 0 0 VSV (h) 0 4 8 12 16 VSV (h) 0481216 VSV (h) 0481216

C P < 0.0001 P < 0.0001 25000 8000 2500 Malat1+/+ P < 0.0001 20000 2000 -/- 6000 Malat1 15000 P = 0.0001 1500 4000 P < 0.0001 10000 1000 P < 0.0001 mRNA (fold) mRNA (fold) P < 0.0001 P < 0.0001 2000 P = 0.0002 5000 P < 0.0001 b1 mRNA (fold) P < 0.0001 500 P < 0.0001 Ifna4 Isg15 Ifn 0 0 0 HSV-1 (h) 0 4 8 12 16 HSV-1 (h) 0481216 HSV-1 (h) 0 4 8 12 16 E P = 0.1719

2500 P = 0.1146 800 800 Malat1+/+ P = 0.2938 P = 0.6413 Malat1-/- 2000 P = 0.9096 600 600 1500 P = 0.1326 400 400 1000 P = 0.7586 mRNA (fold) P = 0.7045 P = 0.5443 mRNA (fold) mRNA (fold) 200 500 200 Ifit1 Isg15 Mx1 0 0 0 IFN-α (h) 0 2 4 8 IFN-α (h) 0 2 4 8 IFN-α (h) 0 2 4 8 F 4000 2500 +/+ P = 0.6083 20000 P = 0.5418 P = 0.3170 Malat1 P = 0.6667 -/- 2000 Malat1 P = 0.3657 15000 3000 P = 0.5691 1500 10000 2000 P = 0.5176 P = 0.1797 1000 P = 0.3519 mRNA (fold) 5000 mRNA (fold) 1000 500 Ifit1 Mx1 mRNA (fold) Isg15 0 0 0 IFN-β (h) 0 2 4 8 IFN-β (h) 0 2 4 8 IFN-β (h) 0 2 4 8

Fig. 2. Malat1 selectively inhibits the production of type I IFNs in macrophages upon viral infection. (A) ELISA analysis of IFN-α and IFN-β in culture − − − − supernatants of Malat1+/+, Malat1 / , and Malat1 / rescued by Malat1 RAW264.7 cells infected with VSV or HSV-1 for 12 h, respectively. (B) qRT-PCR analysis of relative Ifna4, Ifnb1, and Isg15 mRNA level in Malat1+/+ and Malat1−/− RAW264.7 cells along with VSV infection for indicated hours. (C) qRT-PCR analysis of relative Ifna4, Ifnb1, and Isg15 mRNA level in Malat1+/+ and Malat1−/− RAW264.7 cells along with HSV-1 infection for indicated hours. (D) Luciferase assay of IFN-β promoter activity influenced by gradually enhanced Malat1 in HEK293T cells transfected with RIG-I and Malat1 vectors upon VSV infection for indicated hours (Left) and qRT-PCR analysis of Malat1 relative level in HEK293T cells above transfected with 200, 400, and 800 ng Malat1 vector, respectively − − (Right). (E) qRT-PCR analysis of relative Mx1, Ifit1, and Isg15 mRNA level in Malat1+/+ and Malat1 / RAW264.7 cells treated with IFN-α for indicated hours. (F) − − qRT-PCR analysis of relative Mx1, Ifit1, and Isg15 mRNA level in Malat1+/+ and Malat1 / RAW264.7 cells treated with IFN-β for indicated hours. All data are shown as mean ± SD of n = 3 biological replicates, two-tailed unpaired Student’s t test.

4of12 | www.pnas.org/cgi/doi/10.1073/pnas.2003932117 Liu et al. Downloaded by guest on September 29, 2021 Malat1+/+ A Malat1-/- P = 0.0005 4000 1500 P = 0.0005 3000 1000 P = 0.0016 Serum 2000 P = 0.0020 500

1000 IFN-β (pg/ml) IFN-α (pg/ml)

0 0 Mock VSV HSV-1 Mock VSV HSV-1

1500 8000 1500 +/+ B P = 0.0002 P = 0.0040 Malat1

P = 0.0066 -/- 6000 Malat1 1000 1000 Liver P = 0.0014 4000 P = 0.0017

mRNA (fold) P = 0.0012 500 mRNA (fold) mRNA (fold) 500 2000 b1 Ifna4 Ifn Isg15 0 0 0 Mock VSV HSV-1 Mock VSV HSV-1 Mock VSV HSV-1

+/+ P = 0.0001 Malat1 C = 0.0011 800 P 2000 250 -/- P = 0.0005 Malat1 600 1500 200 P = 0.0005 150 P = 0.0001 Spleen 400 1000 P = 0.0111

100 IMMUNOLOGY AND INFLAMMATION mRNA (fold) mRNA (fold) mRNA (fold) 200 a4 500 50 Ifn Ifnb1 Isg15 0 0 0 Mock VSV HSV-1 Mock VSV HSV-1 Mock VSV HSV-1

D Malat1+/+ P = 0.0001 1000 3000 P = 0.0023 -/- 400 P < 0.0001 Malat1 800 2000 300 600 Lung 200 400

mRNA (fold) P = 0.0033 mRNA (fold) 1000 P = 0.0003 P = 0.0016 mRNA (fold)

200 b1 100 Ifna4 Ifn Isg15 0 0 0 Mock VSV HSV-1 Mock VSV HSV-1 Mock VSV HSV-1

EFMalat1+/+ +/+ Liver Spleen Lung Malat1-/- Malat1 100 Malat1-/- 8000 1500 4000 P < 0.0001 P = 0.0006 P < 0.0001 6000 3000 1000 50 4000 2000 P = 0.0113 500 (%) Survival 2000 1000 0 VSV RNA Replicates VSV RNA Replicates

0 20406080

0 VSV RNA Replicates 0 0

Mock VSV Mock VSV Mock VSV Time after VSV infection (h)

Fig. 3. Deficiency of Malat1 enhances antiviral response in vivo. (A) ELISA of IFN-α and IFN-β in serum from Malat1+/+ and Malat1−/− mice infected with VSV (5 × 107 pfu/g) for 18 h or HSV-1 (1 × 107 pfu/g) for 24 h, respectively, via tail i.v. injection. (B) qRT-PCR analysis of relative Ifna4, Ifnb1, and Isg15 mRNA level in liver from Malat1+/+ and Malat1−/− mice corresponding to A.(C) qRT-PCR analysis of relative Ifna4, Ifnb1, and Isg15 mRNA level in spleen from Malat1+/+ and − − − − Malat1 / mice corresponding to A.(D) qRT-PCR analysis of relative Ifna4, Ifnb1,andIsg15 mRNA level in lung from Malat1+/+ and Malat1 / mice corresponding to − − A.(E) qRT-PCR analysis of relative VSV RNA replication in liver, spleen, and lung from Malat1+/+ and Malat1 / mice corresponding to A.(F) Survival curves of 6- to − − 8-wk-old mice of Malat1+/+ and Malat1 / infected with VSV (1 × 108 pfu/g) via tail i.v. injection. Data are shown as mean ± SD of n = 3biologicalreplicates(A–E) or demonstrated as Kaplan–Meier survival curve (F, n = 7), two-tailed unpaired Student’s t test (A–E), or Log-rank (Mantel–Cox) test (F).

The caspase-3 cleavage in virus-infected cells has been shown or HSV-1 in macrophages could induce the activation of caspase-3 recently for HIV, adenovirus, hepatitis C virus, and many other (SI Appendix,Fig.S5D), which showed a similar cleavage pattern kinds of virus (19). We found that the infection of VSV, EMCV, to TDP43. Moreover, TDP43 mutant (TDP43D89E), in which the

Liu et al. PNAS Latest Articles | 5of12 Downloaded by guest on September 29, 2021 AD Malat1 TDP43 Merge Merge VSV TDP43 WT 1 82 104 191 200 262 274 413 NTD 0h CTD △RRM1 △RRM2

RRM1 RRM2 △

Mock Flag-WT Flag-NTDFlag-CTDFlag- Flag-△MW

(KDa)

45 8h 35 Flag

B Malat1 (3006-4020 nt) Chromosome 19 7.50kb 5,795,000bp 5,796,000bp 5,797,000bp 5,798,000bp 5,799,000bp 5,800,000bp 5,801,000bp 5,802,000bp VSV Pull Down 45 35 120 Flag 0h

0 Malat1 (4569-5609 nt) 200 45 35 Flag 8h Input 0 15 Malat1 45 β-Actin C Pull Down Input

5’ Malat1 3’ P < 0.0001 1 6983 nt F 20 P < 0.0001

P = 0.0050 beads 15 MW (KDa) 1-1032 nt707-17481453-2491 nt 2216-3288 nt 3006-4020 nt 3777-4775 nt 4569-5609 nt 5355-6299 nt 6069-6983 nt nt 10 45 P = 0.0028 V5-TDP43 5 35

Relative IFN-β luc activity 0 TDP43 --+ +/+ E 500 2500 P < 0.0001 Tardbp Mock VSV(8h) P = 0.0007 MW Tardbp-/- 400 2000 (KDa) P = 0.0012 45 300 1500 P < 0.0001 Flag-TDP43 200 1000 35 IFN-β (pg/ml) IFN-α (pg/ml) 100 500 β-Actin 45 0 0 MockVSV HSV-1 MockVSV HSV-1

Fig. 4. Identification of Malat1-bound TDP43 in selectively promoting IFN-I production. (A) Confocal microscope images from sequential FISH assay of Malat1 and TDP43 in RAW264.7 cells along with VSV infection for indicated hours. Red, Malat1; green, TDP43; and blue, DAPI. (Scale bar, 5 μm.) (B) iCLIP assay of V5- TDP43 overexpressed in RAW264.7 cells and the likely interaction sites of Malat1 that bind with TDP43. (C) RNA pull-down assay of different truncations of Malat1 with overexpressed V5-TDP43 in RAW264.7 cells. (D) RNA pull-down assay of Malat1 (3,006 to 4,020 nt) and Malat1 (4,569 to 5,609 nt) fragments with HEK293T cell lysates overexpressed different truncations of TDP43, respectively. (E) ELISA analysis of IFN-α and IFN-β in culture supernatants of Tardbp+/+ and − − Tardbp / RAW264.7 cells infected with VSV or HSV-1 for 12 h, respectively. (F) Luciferase assay of IFN-β promoter activity influenced by gradually enhanced TDP43 in HEK293T cells transfected with RIG-I and TDP43 vectors upon VSV infection for indicated hours (Upper) and Western blot analysis of TDP43/TDP35 level in HEK293T cells above transfected with 200, 400, and 800 ng TDP43 vector, respectively (Lower). Data are representative of three independent experiments (A, C, D, and F) or shown as mean ± SD of n = 3 biological replicates (E and F), two-tailed unpaired Student’s t test (E and F).

6of12 | www.pnas.org/cgi/doi/10.1073/pnas.2003932117 Liu et al. Downloaded by guest on September 29, 2021 TDP43 Malat1+/+ Malat1-/- Malat1-/- +Malat1 A C 1 82 104 191 200 262 274 413 MW TDP35 VSV (h) 0 6 8 10 0 6 8100 6 810(KDa) TDP43D89E 45 D89E 20 TDP43

35 P = 0.0002 15 P < 0.0001 45 β-Actin

10 P = 0.0011

P = 0.6564 P = 0.9964 B Tardbp+/+ 5 Tardbp-/- -/- Tardbp +TDP35 Relative IFN-β luc activity 0 2500 P = 0.0022 Malat1 --+ --+-+-+-+

P = 0.0010 VSV - ++- + +++++++ 2000 P = 0.0002 TDP43 ------+- +-- - P = 0.0022 TDP35 ------+ -+- - 1500 TDP43D89E ------+ + Tardbp mRNA (fold) 1000 +/+ -/- P = 0.0004

Ifna4 500 P = 0.0027

0 D Malat1 ----++ VSV (h) 04812 Malat1 +/+ +/+ -/- -/- -/- -/- MW VSV (h) 080808(KDa) Cleaved- 15 IMMUNOLOGY AND INFLAMMATION P = 0.0001 Casp3 P = 0.0007 45 5000

TDP43 4000 TDP43 IP P = 0.0001 35 P = 0.0006 3000 P = 0.0021 45 P = 0.0015 mRNA (fold) 2000 TDP43 35

Ifnb1 1000

Input Cleaved- 15 0 Casp3 VSV (h) 04812 β-Actin 45

Fig. 5. Malat1 inhibits IFN-I production by blocking TDP43 cleavage to its active form TDP35. (A) Western blot analysis of Malat1’s effect on TDP43 along with − − − − VSV infection for the indicated hours in RAW264.7 cells. (B) qRT-PCR analysis of relative Ifna4 and Ifnb1 mRNA level in Tardbp+/+, Tardbp / , and Tardbp / − − rescued by TDP35 RAW264.7 cells upon VSV infection for indicated hours. (C) Luciferase analysis of IFN-β promoter activity in Tardbp+/+ and Tardbp / L929 cells transfected with Malat1, RIG-I, as well as wild-type, truncation, or mutant of TDP43 vectors, respectively. (D) Coimmunoprecipitation analysis of interaction between TDP43 and cleaved caspase-3 affected by Malat1 in RAW264.7 cells upon virus infection for indicated hours. Data are representative of three independent experiments (A and D) or shown as mean ± SD of n = 3 biological replicates (B and C), two-tailed unpaired Student’s t test (B and C).

consensus motif for caspase-3 cleavage was mutated, failed to of the activated caspase-3 and TDP43, and then reduces the cleavage promote the IRF3-induced IFN-β promoter activity and STAT1- of TDP43 to TDP35 during viral infection. Of note, Malat1 inhibited induced ISRE promoter activity upon VSV infection (Fig. 5C and the IFN-β promoter activity with rescue of TDP43 or TDP35 but not − − SI Appendix,Fig.S5E), confirming that TDP43 cleavage into TDP43D89E in Tardbp / L929 cells (Fig. 5C). Taken together, these TDP35 subunit by the activated caspase-3 plays an important role data demonstrate that Malat1 inhibits the production of type I IFNs in antiviral innate immune response. through inhibiting the activated caspase-3-mediated cleavage of Then we further investigated how Malat1 inhibits TDP43 from TDP43 to TDP35 upon viral infection. being cleaved to TDP35 by the activated caspase-3. We found that deficiency of Malat1 increased both the cleavage of TDP43 The Cleaved TDP35 Increases the Nuclear IRF3 Protein Level by and the interaction between TDP43/35 (TDP43 antibody targets Degrading Pre-mRNA of Rbck1. We next investigated how the TDP43 C-terminal domain and fails to distinguish TDP43 from cleaved TDP35 promotes the production of type I IFNs upon viral TDP35) and cleaved caspase-3 compared with Malat1+/+ cells, infection. It is demonstrated that the intracellular localization of while the opposite results were observed after rescue of Malat1 TDP43 is important for its function in the central nervous system − − expression in Malat1 / cells. However, the activated caspase-3 (20). We found that both TDP43 and TDP35 mainly located in the remained unchanged among three groups (Fig. 5D). Furthermore, nucleus after viral infection, and the cleavage pattern of TDP43 immunoprecipitation with cleaved caspase-3 antibody showed that was positively associated with the level of IRF3 in the nucleus only TDP43 but not TDP35 interacted with the cleaved caspase-3 upon VSV infection (SI Appendix,Fig.S6A). Furthermore, defi- (SI Appendix,Fig.S5F), indicating that Malat1 inhibits the interaction ciency of the Tardbp gene in macrophages significantly decreased

Liu et al. PNAS Latest Articles | 7of12 Downloaded by guest on September 29, 2021 the level of nuclear IRF3, and rescue of TDP35 expression sig- expression of its ubiquitin ligase Rbck1 in an antiviral innate nificantly increased the level (Fig. 6A). Since TDP43 as a RNA- immune response. binding protein participates in different RNA processes, in- Next we demonstrated that deficiency of Malat1 significantly cluding alternative splicing, mRNA stability, and transcriptional increased the cleavage of TDP43 to TDP35, decreased the ex- regulation, we wondered whether the cleaved TDP35 increases pression of Rbck1, and increased the nuclear IRF3 protein com- IRF3 protein level through regulating Irf3 mRNA. However, pared with that in control cells upon VSV infection, while − − TDP35 was not bound to Irf3 mRNA and there was no difference knockdown of Tardbp in Malat1 / cells increased Rbck1 expres- − − in the level of mRNA of Irf3 in Tardbp+/+ and Tardbp / cells sion and decreased the IRF3 level, suggesting that Malat1 regu- with or without TDP35 rescue upon VSV infection (SI Appendix, lates the expression of Rbck1 and IRF3 depending on TDP35 Fig. S6B). (Fig. 6E). Furthermore, deficiency of Malat1 reduced the K48- Then, we wondered whether TDP35 directly targets the mRNA linked polyubiquitination on IRF3 compared with that in control of genes associated with IRF3 protein degradation. We analyzed cells (Fig. 6F), indicating that Malat1 promotes IRF3 degradation the interaction of TDP35 with genes encoding the proteins which through the proteasome pathway upon VSV infection. can regulate IRF3 protein level, including Rbck1 (21), Pin1 (22), Cullin1 (23), and PP2A (24), and found that TDP43/35 robustly The Decreased Expression of MALAT1 Is Associated with Increased cross-linked to intron 7 of Rbck1 transcript upon VSV infection TDP43 Cleavage, IRF3 Protein, and IFNA Expression in PBMCs of SLE but not the other three genes in iCLIP libraries of TDP43/35 (Fig. Patients. Dysregulation of innate immune response plays a criti- 6B). RNA pull-down confirmed that intron 7 of Rbck1 pre-mRNA cal role in the development of many autoimmune diseases. SLE, could interact with TDP35 but not TDP43 or its mutants (Fig. 6C). a common and potentially fatal autoimmune disorder, has been We then constructed 20 fragments of Rbck1 pre-mRNA each of characterized by autoantibody production and higher type I IFN which was about 1,000 nt in a head-to-tail overlap manner. Through signature (25). Early studies indicated that higher expression of RNA pull-down assay, we found that only one fragment (10,801 to p-IRF3 is found in PBMCs from patients with SLE (26). Inspired 11,777 nt), which contained the cross-link site, was bound to TDP35 by these indications, we analyzed the expression of MALAT1 in (SI Appendix,Fig.S6C), suggesting that Rbck1 pre-mRNA can di- PBMCs isolated from SLE patients and control normal donors. rectly bind to TDP35 through its seventh intron. As expected, MALAT1 expression was drastically reduced and Furthermore, the level of Rbck1, both pre-mRNA and mRNA, IFNA mRNA level significantly increased in SLE PBMCs com- − − increased robustly in Tardbp / cells compared with that in pared with that in control cells (Fig. 7A). Furthermore, the key − − Tardbp+/+ cells, and rescue of TDP35 expression in Tardbp / molecules, including increased TDP43 cleavage, reduced Rbck1, cells decreased both levels (Fig. 6D). Correspondingly, the change and enhanced IRF3 and p-IRF3, were observed in PBMCs from of Rbck1 protein expression was similar to Rbck1 mRNA among SLE patients (Fig. 7B). these cells (SI Appendix,Fig.S6D). To investigate whether TDP43 The glucocorticoids and antimalarial drugs (hydroxychloroquine or Malat1 directly regulates Rbck1 pre-mRNA stability, we ex- and chloroquine) have been a standard and prominent treatment for amined the half-life time of Rbck1 pre-mRNA with actinomycin D SLE patients for a long time (27). The treatment can achieve rapid (ActD) which can inhibit RNA polymerase II activity. We dem- immune suppression and alleviated inflammation through inhib- onstrated that the level of Rbck1 pre-mRNA decreased much iting TLR signaling and cGAS stimulator of IFN genes (STING) − − more quickly with ActD treatment in Malat1 / cells upon VSV signaling and reducing the production of proinflammatory cyto- − − infection. Rescue of the Malat1 level in Malat1 / cells signifi- kines, including type I interferons (27–29). We demonstrated that cantly retarded the reduction of Rbck1 pre-mRNA (SI Appendix, effective treatment not only rescues the IFNA expression but also − − Fig. S6E). Additionally, overexpressing TDP35 in Malat1 / cells MALAT1 expression in PBMCs from SLE patients (Fig. 7C). robustly accelerated the decrease of Rbck1 pre-mRNA (SI Ap- Furthermore, a series of molecular events, from reduced TDP43 pendix,Fig.S6E). Consistently, the level of Rbck1 pre-mRNA cleavage to decreased IRF3, were also achieved in PBMCs from decreased much more slowly along with the treatment of ActD SLE patients after effective treatment (Fig. 7D) − − − − in Tardbp / cells. Rescue of TDP35 expression in Tardbp / cells significantly promoted the reduction of Rbck1 pre-mRNA, while Discussion − − rescue of Malat1 in Tardbp / cells had little effect on its level (SI Here, we identified Malat1 as a negative regulator of antiviral Appendix, Fig. S6F), indicating that TDP43/TDP35 directly regu- IFN-I production. The viral infection-induced reduction of Malat1 lates Rbck1 pre-mRNA stability, while Malat1 depends on TDP43/ promotes IRF3 activation and type I IFNs production. The results TDP35 to achieve its regulation on Rbck1 pre-mRNA stability. of this study demonstrated that, in resting cells, Malat1 binds to TDP43, inhibits IRF3-induced IFNs expression, and maintains The Cleaved TDP35 Degrades Pre-mRNA of E3 Ligase Rbck1 for immune homeostasis. Upon viral infection, Malat1 expression is Preventing IRF3 Degradation. Next we detected whether Rbck1 reduced, and without the binding of Malat1, TDP43 is cleaved into acts as the E3 ubiquitin ligase targeting IRF3 for degradation in TDP35 by activated caspase-3. Then cleaved TDP35 increases macrophages upon viral infection. We demonstrated that over- nuclear IRF3 level by inhibiting its ubiquitination through deg- expression of Rbck1 reduced the protein level of IRF3 in a dose- radation of Rbck1 pre-mRNA, and the increased production of dependent manner and this effect was significantly diminished in IFNs participate in antiviral effects. A series of molecular events, the presence of proteasome inhibitor MG132. And both TDP43 from MALAT1 reduction to IFN production, spontaneously exist and TDP35 but not TDP43D89E could decrease Rbck1 and in the PBMCs of SLE patients. In sum, we found a nuclear lncRNA increase the IRF3 level as well (SI Appendix, Fig. S7A). Exposure inhibits the production of type I IFNs, decreasing the antiviral ca- of cells under the same culture conditions resulted in the cor- pacity and inhibiting the progress of autoimmune diseases (Fig. 7E). responding changes of K48-linked polyubiquitination of IRF3 LncRNAs have been reported closely associated with a reper- (SI Appendix, Fig. S7B). Furthermore, deficiency of Tardbp toire of physiological and pathological processes, including anti- showed increased Rbck1 but decreased IRF3 levels in the nu- viral innate immune responses (30). Generally, the level of most cleus of cells upon VSV infection, whereas knockdown of Rbck1 lncRNAs is much lower than that of mRNAs. Consistently, the − − by siRNA in Tardbp / cells significantly increased the level of abundance of lncRNAs is much lower than that of the proteins nuclear IRF3 (SI Appendix, Fig. S7C). Taken together, these which results in the limitation of the interaction between lncRNAs data demonstrate that virus infection promotes the cleavage of and proteins. The great function of lncRNAs (link-EPS and lnc13) TDP43 to TDP35, which increases the nuclear IRF3 protein with few copy numbers is debatable (31). Most lncRNAs have a level through preventing IRF3 protein degradation by inhibiting 5′ cap and a poly(A) tail, yet some of the most abundant long

8of12 | www.pnas.org/cgi/doi/10.1073/pnas.2003932117 Liu et al. Downloaded by guest on September 29, 2021 A C Nucleus TDP43 1 82 104 191 200 262 274 413 Tardbp+/+ Tardbp-/- Tardbp-/- +TDP35 TDP35 MW TDP43D89E VSV (h) 048121604812160481216(KDa) D89E 45 TDP43 △RRM1 IRF3 TDP43 △RRM2 45 TDP43 RRM2 35 △RRM1 △ 75 Lamin A/C 65 MW Mock Flag-TDP43 Flag-TDP35Flag-TDP43D89EFlag-TDP43Flag-TDP43 (KDa) B 45 Chromosome 2 16kb VSV 152,316kb 152,318kb 152,320kb 152,322kb 152,324kb 152,326kb 152,328kb 152,330kb 152,332kb Flag 35 Pull Down 100

0h Rbck1-pre-mRNA 45

0 Flag 100 35 Input 8h 45 β-Actin 0 Rbck1 D F IP IgG IRF3 Tardbp+/+ Tardbp-/- Malat1 +/+ -/- +/+ -/- +/+ -/- IMMUNOLOGY AND INFLAMMATION Tardbp-/-+TDP35 VSV (h) 0 0 0 0 8 8 MW MG132 + ++++ + (KDa) P = 0.0005 P = 0.0002 P = 0.0003 P = 0.0013 P = 0.0013 180 2.5 P = 0.0002 2.0 P = 0.0042 P = 0.0075 P = 0.0004 P = 0.0003 135 P = 0.0001 P = 0.0029 P = 0.0042 P = 0.0009 P = 0.1037 P = 0.0561 100 2.0 1.5 75 1.5 1.0 65 1.0 K48 45 mRNA level (fold)

pre-mRNA level (fold) 0.5 0.5 35 Rbck1

Rbck1 0.0 0.0 VSV (h) 04812 VSV (h) 0 4 8 12 25

45 E siNC siTardbp IRF3 Malat1 +/+ +/+ +/+ +/+ -/- -/- -/- -/- -/- -/- -/- -/- Input MW 180 VSV (h) 068100681006810(KDa) 135 45 100 TDP43 75 35 65 65 K48 Rbck1 45 35 IRF3 45

p-IRF3 45 25 75 Lamin A/C IRF3 45 65 GAPDH 35

Fig. 6. The cleaved TDP35 increases nuclear IRF3 protein level by binding and degrading Rbck1 pre-mRNA to prevent IRF3 proteasomal degradation upon − − − − viral infection. (A) Western blot analysis of TDP35’s effect on protein level of IRF3 in the nucleus of Tardbp+/+, Tardbp / , and Tardbp / rescued by TDP35 RAW264.7 cells upon VSV infection for indicated hours. (B) iCLIP assay of V5-TDP43 overexpressed RAW264.7 cells and the likely interaction sites of Rbck1 pre- mRNA that binding with TDP43/TDP35. (C) RNA pull-down assay of Rbck1 pre-mRNA and the different truncations or mutant of TDP43 overexpressed in − − − − HEK293T cells, respectively. (D) qRT-PCR analysis of relative pre-mRNA and mRNA level of Rbck1 in Tardbp+/+, Tardbp / , and Tardbp / rescued by TDP35 RAW264.7 cells upon virus infection for indicated hours. (E) Western blot analysis of the necessity of TDP35 for Malat1 regulating the expression of Rbck1 and − − phosphorylation of IRF3 in nucleus of Malat1+/+ and Malat1 / RAW264.7 cells transfected with siRNA targeting negative control (siNC)orTardbp (siTardbp), respectively, upon VSV infection for indicated hours. (F) Coimmunoprecipitation analysis of the K48-linked polyubiquitination on IRF3 in Malat1+/+ and Malat1−/− RAW264.7 cells infected with VSV for indicated hours. Data are representative of three independent experiments (A, C, E, and F) or shown as mean ± SD of n = 3 biological replicates (D), two-tailed unpaired Student’s t test (D).

Liu et al. PNAS Latest Articles | 9of12 Downloaded by guest on September 29, 2021 P < 0.0001 P = 0.0065 A P = 0.0045 C 2.0

n 2.0 2.0 1.5 1.5 1.5 1.0 1.0 1.0 relative expression 0.5 relative expressio

0.5 relative expression 0.5

MALAT1 0.0

0.0 IFNA Before treatment After treatment

MALAT1 0.0 Healthy SLE Healthy SLE

P = 0.0082 B 2.5

Healthy SLE n MW 12345 12345(KDa) 2.0 45 1.5 TDP43 35 1.0

65 relative expressio Rbck1 0.5

IFNA 0.0 p-IRF3 45 Before treatment After treatment 65 IRF3 E Actin 45 Immunity homeostasis Innate immune responses Viral infection SLE patients Resting cells D Macrophages PBMCs SLE Cytoplasm Before Treatment After Treatment pro-Caspase3 MW pro-Caspase3spasse3e3 12345 12345(KDa) 45 Nuclear Cleaved-Caspase3 Malat1 TDP43 P TDP43TTDDDPPP43P44343 35 TDP43TD IRF3

65 Rbck1 Rbck1 Rbck1 pre-mRNA TDP35 Degradation Ub IRF3 Ub p-IRF3 Ub Ub 45 IRF3 IRF3 Rbck1 IFNs IRF3 45 45 Proteasomal Antiviral effect IFN signature Actin degradation in SLE

Fig. 7. The decreased expression of MALAT1 is associated with increased TDP43 cleavage, IRF3 protein, and IFNA expression in PBMCs of SLE patients. (A) qRT-PCR analysis of relative MALAT1 and IFNA in PBMCs from healthy donors and SLE patients. (B) Western blot analysis of key molecules in MALAT1- regulated antiviral response in PBMCs from healthy donors and SLE patients. (C) qRT-PCR analysis of relative MALAT1 and IFNA in PBMCs from SLE patients before and after effective treatment. (D) Western blot analysis of key molecules in MALAT1-regulated antiviral response in PBMCs from SLE patients before and after effective treatment. (E) Proposed working model for Malat1 as a negative regulator of antiviral innate response by targeting TDP43 activation via an RNA-RBP interactive network. Data are shown as mean ± SD of n = 12 (A)orn = 6(C) biological replicates, two-tailed unpaired Student’s t test (A and C).

noncoding RNAs are processed in unexpected ways and lack triple helical structures, which accumulates to high levels in the these canonical structures. It has been reported that widespread nucleus (32). Upon viral infection, however, Malat1 was reduced. RNA decay during apoptosis is initiated by the mitochondrial As Malat1 used noncanonical strategies to ensure its stability, exoribonuclease PNPT1 (16). RNAs with 3′ end structures, like Malat1 may be regulated by unique posttranscriptional control many nonpoly(A) ncRNAs, block PNPT1 degradation and likely mechanisms. The study by Bhattacharyya and Vrati detected an contribute to the relative resistance of nonpoly(A) ncRNAs to increased Malat1 level by flaviviral infection in Neuro2a but not accelerated decay during apoptosis. It is possible that structured in macrophages (BV2 cells) in the nervous system (33). The sequences near the 3′ end of imported RNAs protect them from study by Yao et al. showed Malat1 KO had no defects following being degraded during import. The Malat1 is not polyadenylated lymphocytic choriomeningitis virus (LCMV) infection in T cells as the tRNA biogenesis machinery generates its mature 3′ end. (34). These studies indicate that the function of Malat1 varied in In place of a poly(A) tail, Malat1 is stabilized by highly conserved different kinds of cells which respond diversely to viral infection.

10 of 12 | www.pnas.org/cgi/doi/10.1073/pnas.2003932117 Liu et al. Downloaded by guest on September 29, 2021 In addition, we found the reduction of Malat1 was induced by in SLE patients. Our findings uncover an unexpected patho- apoptosis caused by viral infection; therefore, we speculated that physiological connection between lncRNA and type I IFNs, which various degrees of apoptosis induced by different types of virus allows us to gain insight into therapeutic targets and disease lead to specific changes in Malat1 expression. Malat1 has been biomarkers to provide potential support for clinical treatment reported closely associated with many tumors, ranging from breast in autoimmune diseases. cancer to prostate cancer. It is involved in tumorigenesis, progression, and metastasis, which has been generally studied since Materials and Methods it was discovered as metastasis-associated lung adenocarcinoma Mice and Cells. Malat1-deficient mice were generated via the CRISPR-Cas9 transcript 1 (35–39). Our findings identify that Malat1 reduction genomic editing system based on C57BL/6J background. Primary peritoneal contributes to the production of type I IFNs upon viral infection, macrophages were acquired from the peritoneal lavage fluids of mice that which uncovered a mechanism that host cells can actively decrease were intraperitoneally injected with 3% thioglycollate 72 to 96 h in advance. the expression of certain types of suppressive lncRNAs in response All animal experiments were performed according to the National Institutes to viral infection and then promote the induction of type I IFNs to of Health Guide for the Care and Use of Laboratory Animals and with ap- resist viral infection. proval of the Scientific Investigation Board of the Chinese Academy of The most popular role of nuclear lncRNAs is regulating the Medical Sciences. Malat1-deficient RAW264.7, TDP43-deficient RAW264.7, genes’ expression through epigenetic regulation. They could and L929 cell lines were produced through the CRISPR-Cas9 system with pGL3-U6 plasmid containing guide RNA sequence. Sequences for sgRNAs promote or suppress the transcription of certain genes directly and genotype identification are listed in SI Appendix, Table S1. HEK293T, or by transcription factors (30). Our study provides a mecha- RAW264.7, L929, BHK-21, and Vero cell lines were purchased from American − − nism for nuclear lncRNA in reducing the cleavage of TDP43 to Type Culture Collection (ATCC) company. Malat1 / mice and cells were TDP35 by blocking the interaction of TDP43 and cleaved generated as described in SI Appendix, Materials and Methods. caspase-3. Malat1 wasdirectlyboundtoTDP43withtwofrag- ments around the cross-link sites of Malat1 transcript at 3,618 Control Normal Donors, SLE Patients, and Treated SLE Patients. Healthy normal and 4,887 nt in different sequences. It has been previously dem- donors had no history of autoimmune diseases. Patients with concurrent onstrated that Malat1 with a short interspersed nuclear element infection were excluded from the study. All SLE patients (ages ranging from (SINE) deletion form creates more available TDP43-binding 17 to 55, mainly female, SI Appendix, Table S2) fulfilled the American College sites, suggesting that Malat1 has a specific secondary structure of Rheumatology (ACR) classification criteria for SLE (46). The Systemic Lupus which lays the foundation for the binding to TDP43 (40). How- Erythematosus Disease Activity Index (SLEDAI) score was determined for ever, it needs more profound investigation of secondary struc- each patient at the time of the blood draw. Patients were categorized as > ≤ tures of RNAs to fully explain the properties of RNA-binding having active disease (scores 4) or inactive disease (scores 4) based on the IMMUNOLOGY AND INFLAMMATION proteins. SLEDAI results. The study was approved by the Research Ethics Board of The function of TDP43, especially as a RBP, in antiviral re- Peking Union Medical College Hospital (JS-1239). Written informed consent was signed before sample collection. Information of healthy donors, SLE sponse is rarely investigated. Here, we discovered that TDP35 patients, and treated patients are listed in SI Appendix, Table S2. was a positive regulator of type I IFN production by binding with pre-mRNA of Rbck1 and facilitating its degradation. In a previous High-Content Screening. Primary peritoneal macrophages from wild-type study, TDP43 binds GU-rich distal intronic sites (41). However, the mice were seeded into 96-well plates. Then the lncRNA Smart Silencer two sites where TDP43 binds to Malat1 are not classical GU-rich (a mixture of three ASOs and three siRNAs with equal proportion; RiboBio) sites which enlarge the TDP43-binding RNA sequences. Moreover, (47–49) were transfected into the macrophages at the optimal final con- TDP35 likely differs from TDP43 in secondary structure which centration of 50 nM to silence target genes, respectively. Forty-eight hours provides the basis that full-length TDP43 could bind to Malat1 but later, the transfected macrophages were infected with or without GFP-VSV not Rbck1 pre-mRNA, while cleaved TDP35 could interact with for 12 h. Cells were then fixed by 4% paraformaldehyde for 15 mins at room Rbck1 pre-mRNA. It’s possible that secondary structure changes temperature (RT) and stained with DAPI for 10 min at RT to dye the nucleus. of TDP35 caused by cleavage exposes the sites for binding Rbck1 GFP intensity which represented the titer of VSV was measure by the pre-mRNA. ArrayScan High-Content System (Thermo Scientific). The siRNA- and ASO- Notably, it has been widely accepted that persistent type I IFN targeted sequences for knockdown of 20 lncRNAs are outlined in SI Ap- exposure and subsequent type I IFN signaling leading to ISG pendix, Table S1. expression (referred to as the IFN-I signature) are characteristic features of SLE pathogenesis (42). In animal models, reduced ChIRP Assay. Cells infected with or without VSV were washed three times with PBS. The assay was performed with Magna ChIRP RNA Interactome Kits IFN-I expression or IFN-I signaling showed clear beneficial ef- (Merck Millipore) according to the manufacturer’s protocols and followed by fects for lupus treatment (43). Clinically, anti-IFN-I therapies Western blot analysis or mass spectrography. attracted much attention for treatment of SLE patients (44). However, the mechanisms governing negative regulation of IFNs Statistical Analysis. Two-tailed unpaired Student’s t test was applied to an- is incomplete and requires further investigation and clarification. alyze the statistical significance of data from two groups with GraphPad Malat1 is one of the most conserved lncRNAs between human Prism software. Mice survival curve data were demonstrated as Kaplan– and mouse (about 70% conserved between human and mouse) Meier curves and analyzed with Log-rank (Mantel–Cox) test. P values less (45). We found SLE patients displayed reduced MALAT1 ex- than 0.05 were regarded as statistically significant. pression, coupled with enhanced cleavage of TDP43, decreased Rbck1 expression, increased IRF3 activation, and finally in- Data Availability. The sequencing data for RNA-Seq and iCLIP have been creased IFNA production. Furthermore, posttreatment of SLE deposited in the Gene Expression Omnibus with accession no. GSE134032 patients could robustly increase MALAT1 expression and alle- (50). All study data are included in the article and SI Appendix. viate the aberrant IRF3 activation cascade in patient-derived PBMCs, consistent with the available data in the Gene Expres- ACKNOWLEDGMENTS. This work is supported by grants from the National Natural Science Foundation of China (81788101), National 135 Mega Program sion Omnibus that MALAT1 expression is decreased dramati- of China (2017ZX10102032-001, 2017ZX10202203-002, and 2017ZX10203206- cally in monocytes and lymphocytes (accession no. GSE51997) of 001), and Chinese Academy of Medical Sciences Innovation Fund for Medical SLE patients, further supporting the potential role of MALAT1 Sciences (2016-12M-1-003).

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