NUDT21 links mitochondrial IPS 1 to RLR containing stress … · 2020. 4. 9. · 86 IPS-1 abrogated both its mitochondrial localization and the induction of type I IFN (8, 87 32)

1

1

NUDT21 links mitochondrial IPS-1 to RLR-containing stress granules and 2

activates host antiviral defense 3

4

5

Saeko Aoyama-Ishiwatari1, Tomohiko Okazaki1*, Shun-ichiro Iemura2†, Tohru 6

Natsume2, Yasushi Okada3,4,5, Yukiko Gotoh1,5 7

8

9 1Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-10

0033, Japan. 11 2Molecular Profiling Research Center for Drug Discovery (molprof), National Institute 12

of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan. 13 3Laboratory for Cell Dynamics Observation, Center for Biosystems Dynamics Research 14

(BDR), RIKEN, Osaka 565-0874, Japan. 15 4Department of Physics, Universal Biology Institute (UBI) 16

5International Research Center for Neurointelligence (WPI-IRCN), The University of 17

Tokyo, Tokyo 113-0033, Japan. 18

19

*Corresponding author: [email protected] 20

21

†Present address: Translational Research Center, Fukushima Medical University, 22

Fukushima 960-1295, Japan. 23

24

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 10, 2020. ; https://doi.org/10.1101/2020.04.09.033597doi: bioRxiv preprint

https://doi.org/10.1101/2020.04.09.033597

2

Summary 25

Viral RNA in the cytoplasm of mammalian host cells is recognized by retinoic acid–26

inducible protein–I (RIG-I)–like receptors (RLRs), which localize to cytoplasmic stress 27

granules (SGs). Activated RLRs associate with the mitochondrial adaptor protein IPS-1, 28

which activates antiviral host defense mechanisms including type I interferon (IFN) 29

induction. It has remained unclear, however, how RLRs in SGs and IPS-1 in the 30

mitochondrial outer membrane associate physically and engage in information transfer. 31

Here we show that NUDT21, an RNA binding protein that regulates alternative 32

transcript polyadenylation, physically associates with IPS-1 and mediates its 33

localization to SGs in response to transfection with poly(I:C), a mimic of viral double-34

stranded RNA. We found that, despite its well-established function in the nucleus, a 35

fraction of NUDT21 localizes to mitochondria in resting cells and becomes localized to 36

SGs in response to poly(I:C) transfection. NUDT21 was also found to be required for 37

efficient type I IFN induction in response to viral infection. Our results together indicate 38

that NUDT21 links RLRs in SGs to mitochondrial IPS-1 and thereby activates host 39

defense responses to viral infection. 40

41


https://doi.org/10.1101/2020.04.09.033597

3

Introduction 42

The innate immune system provides the first line of defense against viral infection. The 43

initial step of this defense is detection of “non-self” cues known as pathogen-associated 44

molecular patterns (PAMPs) by specialized sensors, known as pattern recognition 45

receptors (PRRs), in host cells. Recognition of viral PAMPs by PRRs results in the 46

activation of a series of mechanisms to combat viral propagation. In vertebrates, 47

activation of PRRs induces the production of type I interferons (IFNs) such as IFN-α 48

and IFN-β and the subsequent expression of hundreds of IFN-stimulated genes (ISGs) 49

that play a major role in restriction of viral replication within infected cells (1–3). 50

Retinoic acid–inducible protein–I (RIG-I)–like receptors (RLRs) are a family 51

of PRRs consisting of DEAD box–containing RNA helicases that recognize viral RNA 52

in the cytoplasm. Among RLRs, RIG-I recognizes RNA molecules containing a 5'-53

triphosphate group as well as relatively short (2 kb) dsRNAs, with both types of dsRNA being derived from a wide 56

range of RNA viruses (4–6). The binding of RLRs to such viral RNAs triggers their 57

interaction with a key antiviral hub protein, IFN-β promoter stimulator–1 (IPS-1, also 58

known as MAVS, CARDIF, and VISA) (7–10). IPS-1 is anchored to the mitochondrial 59

outer membrane, and activated IPS-1 forms large prionlike aggregates on these 60

organelles (11) that in turn activate transcription factors such as interferon regulatory 61

factor 3 (IRF3), nuclear factor–κB, and activator protein–1, resulting in the expression 62

of type I IFNs (8,12–16). The pivotal role of IPS-1 in antiviral responses is exemplified 63

by the finding that IPS-1–deficient mice are more vulnerable to viral infection than are 64

wild-type (WT) mice (17, 18). The NH2-terminal caspase activation and recruitment 65

domain (CARD) of RLRs is required for the interaction with IPS-1 and was indeed 66

found to be essential for IFN induction (19). 67

Viral RNAs, RLRs, and RLR-associated proteins have been found to be 68

localized to stress granules (SGs) in virus-infected cells. SGs are membraneless 69

structures that are composed of translation-stalled mRNAs and proteins and in which 70

translation is generally inhibited (20). They are formed in response to various cellular 71

stresses including viral infection. Viral RNA–mediated activation of dsRNA-activated 72

protein kinase (PKR) results in the phosphorylation of eukaryotic initiation factor 2a, 73

translational arrest, and SG nucleation (21). The RNA binding proteins Ras-GAP SH3 74

domain–binding protein 1 (G3BP1), T cell–restricted intracellular antigen 1 (TIA1), and 75

TIA1-related protein (TIAR) participate in the formation of SGs (22, 23), which is 76

thought to involve liquid-liquid phase separation (24). SGs have been proposed to serve 77


https://doi.org/10.1101/2020.04.09.033597

4

as a platform for RLR recognition of viral RNA and consequent activation of antiviral 78

responses, with such SGs also having been termed antiviral SGs (avSGs) (25). 79

Inhibition of SG formation by depletion of G3BP1 indeed suppressed type I IFN 80

expression in response to infection with influenza A virus or Newcastle disease virus 81

(NDV) (25, 26). The fact that many viral factors interfere with SG formation (27–31) 82

also supports an antiviral function of SGs. 83

The mitochondrial localization of IPS-1 has been thought to be essential for its 84

function. For instance, deletion of the COOH-terminal transmembrane (TM) domain of 85

IPS-1 abrogated both its mitochondrial localization and the induction of type I IFN (8, 86

32). Caspase-mediated cleavage of IPS-1 that results in detachment of the TM domain 87

also inactivates IPS-1 function (33, 34). Given the mitochondrial localization of IPS-1, 88

it has remained unclear how RLRs within SGs encounter and activate IPS-1. Of interest 89

in this regard, a fraction of IPS-1 appears to colocalize with TIAR, a marker of SGs, in 90

cells infected with viruses or transfected with polyinosinic-polycytidylic acid 91

[poly(I:C)], a synthetic analog of viral dsRNA (26, 35), suggesting that viral RNA may 92

induce the localization of IPS-1 to SGs and its association with RLRs. The mechanism 93

that might underlie such an effect has remained unknown, however. 94

We here identify nucleoside diphosphate–linked moiety X (Nudix)–type motif 95

21 (NUDT21) as an IPS-1 interactor. NUDT21 is an RNA binding protein that 96

constitutes the CFIm complex in nucleus and regulates the choice of polyadenylation 97

site (PAS) by binding to the UGUA element in the 3' untranslated region (UTR) of 98

transcripts (36–39). NUDT21-mediated alternative polyadenylation has been shown to 99

influence various cell fate decision processes as well as tumorigenesis (40, 41). Virus 100

infection was previously shown to induce genome-wide changes in PAS selection in 101

host transcripts and was also reported to limit a host gene expression through 102

NUDT21(42, 43). Unexpectedly, we found that, whereas NUDT21 is localized mostly 103

to the nucleus, where alternative polyadenylation takes place, a fraction of NUDT21 is 104

associated with mitochondria in resting cells but also localizes to SGs in response to 105

poly(I:C) transfection. Moreover, NUDT21 was found to associate with IPS-1 and to 106

play an important role in its localization to SGs as well as in the efficient induction of 107

IFN expression in response to poly(I:C) transfection or to infection with 108

encephalomyocarditis virus (EMCV). Our results thus suggest an unexpected role for 109

NUDT21 in the recruitment of mitochondrial IPS-1 to SGs and in the consequent 110

promotion of RLR-mediated activation of IPS-1 and IFN induction in virus-infected 111

cells. 112

113


https://doi.org/10.1101/2020.04.09.033597

5

Materials and Methods 114

Cell culture and transfection 115

HeLa S3 and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium 116

supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. HEK293T 117

cells were transfected with the use of the GeneJuice Transfection Reagent (Merck 118

Millipore), whereas transfection of HeLa S3 cells with expression vectors was 119

performed with Lipofectamine 2000 (Thermo Fisher Scientific). 120

121

Plasmids and reagents 122

The plasmid pEF-BOS-FLAG-IPS-1 encoding WT human IPS-1 was kindly provided 123

by M. Yoneyama (Division of Molecular Immunology, Medical Mycology Research 124

Center, Chiba University, Japan). A full-length cDNA for NUDT21 was amplified by 125

PCR from a mouse cDNA library with the primers 5′-126

GGCAGATCTATGTCTGTGGTGCCGCCCAA-3′ and 5′-127

GGCGAATTCTCAGTTGTATATAAAATTGA-3′ (sense and antisense, respectively), 128

and was subcloned into either the BglII and EcoRI sites of the pCS4 vector or the 129

BamHI and EcoRI sites of pcDNA3.1. Human G3BP1 cDNAs with or without stop 130

codon were amplified by PCR from pN1/G3BP1-iRFP (Okada lab plasmids, 131

Addgene #129339) with the sense primer 5′-132

GCCAGATCTATGGTGATGGAGAAGCCTAG-3′ and the antisense primers 5′-133

GCCGAATTCGGATCCTTACTGCCGTGGCGC-3′ or 5′-134

GCCGAATTCGGATCCCTGCCGTGGCGCAAG-3′, respectively, and were 135

subcloned into the BamHI and EcoRI sites of pcDNA3. Myc epitope–tagged full-length 136

human IPS-1 and Myc–IPS-1(ΔTM) cDNAs were amplified by PCR from a Myc–IPS-1 137

expression vector described previously (44) with the sense primer 5′-138

ACTGCGGCCGCATGAGCAAAAGCTCATTT-3′ and the antisense primers 5′-139

GCCTCTAGACTAGTGCAGACGCCGCCG-3′ or 5′-140

GCCTCTAGACTAGTCTACCTGGGATGCCA-3, respectively, and were subcloned 141

into the NotI and XbaI sites of either pcDNA3 or the G3BP1 expression vector. Myc–142


https://doi.org/10.1101/2020.04.09.033597

6

IPS-1 (amino acids 1–98), Myc–IPS-1 (amino acids 99–200) and Myc– IPS-1 (amino 143

acids 201–540) cDNAs were amplified by PCR from a Myc–IPS-1 expression vector 144

with the sense primer 5′-GGCAGATCTATGCCGTTTGCTGAAGACAAGACC-3′ 145

and the antisense primer 5′-GGCAGATCTCTACCGAGGCTGGTAGCTCTGGT-3′, 146

the sense primer 5′-GGCAGATCTACCTCGGACCGTCCCCCAGAC-3′ and the 147

antisense primer 5′-GGCAGATCTCTATGTGTCCTGCTCCTGATGCCCGCT-3′, 148

or the sense primer 5′-GGCAGATCTATGGAACTGGGCAGTACCCACACAGCA-149

3′ and the antisense primer 5′-GGCAGATCTCTAGTGCAGACGCCGCCGGT-150

3′, respectively, and were subcloned into the BglII and EcoRI sites of the pCS4 vector. 151

Poly(I:C) was obtained from GE Healthcare and was introduced into cells by 152

transfection with Lipofectamine 2000 (Thermo Fisher Scientific). 153

154

Antibodies 155

Antibodies to NUDT21 were obtained from Proteintech; those to Myc (9E10), to p38, to 156

TIAR, and to TOMM20 were from Santa Cruz Biotechnology; those to IPS-1 for 157

immunostaining as well as those to phospho-p38, to phospho-JNK, to cleaved caspase-158

3, to cleaved PARP, and to phospho-IRF3 were from Cell Signaling Technology; those 159

to FLAG (M2) were from Sigma; those to HA were from Roche; those to IPS-1 for 160

immunoblot analysis were from Abcam; those to cytochrome c were from BD 161

Pharmingen; and those to CFIm68 were from Bethyl Laboratories. Antibodies to RIG-I 162

were kindly provided by M. Yoneyama (Chiba University). 163

164

RNA interference 165

Knockdown of NUDT21 was achieved with the use of Stealth RNA interference 166

(Thermo Fisher Scientific). HeLa S3 cells were transfected with siRNA 167

oligonucleotides with the use of the Lipofectamine RNAiMAX reagent (Thermo Fisher 168

Scientific) and were used for experiments after incubation for 72 h. The siRNA 169

sequences were 5′-UGAACCUCCU-CAGUAUCCAU-AUAUU-3′ and 5′-170

AAUAUAUGGA-UACUGAGGAG-GUUCA-3′ for NUDT21, and a negative control 171


https://doi.org/10.1101/2020.04.09.033597

7

siRNA (Thermo Fisher Scientific, catalog no. 12935300) was also used. 172

173

Mass spectrometry 174

Liquid chromatography and tandem mass spectrometry were performed as previously 175

described (45). 176

177

Immunoblot analysis 178

Immunoblot analysis was performed as described previously (44). In brief, cells were 179

lysed with a solution containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM -180

glycerophosphate, 5 mM EGTA, 1 mM Na4P2O7, 5 mM NaF, 0.5% Triton X-100, 1 mM 181

Na3VO4, 1 mM dithiothreitol, and aprotinin (1 mg/ml), cell lysates were fractionated by 182

SDS-polyacrylamide gel electrophoresis on a 10% gel, and the separated proteins were 183

transferred to a polyvinylidene difluoride membrane. The membrane was incubated first 184

with primary antibodies for 24 h at 4°C and then with horseradish peroxidase–185

conjugated secondary antibodies (GE Healthcare) for 1 h at room temperature. After a 186

wash with a solution containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% 187

Tween 20, the membrane was processed for detection of peroxidase activity with 188

chemiluminescence reagents and an Image Quant LAS4000 instrument (GE 189

Healthcare). 190

191

Co-immunoprecipitation analysis 192

HEK293T cells harvested 20 h after transfection with plasmids encoding FLAG–IPS-1 193

and Myc-NUDT21 were lysed with a solution containing 20 mM Tris-HCl (pH 7.5), 194

150 mM NaCl, 10 mM -glycerophosphate, 5 mM EGTA, 1 mM Na4P2O7, 5 mM NaF, 195

0.2% Triton X-100, 1 mM Na3VO4, 1 mM dithiothreitol, and aprotinin (1 mg/ml). The 196

cell lysates were incubated with rotation at 4°C first for 45 min with antibodies to 197

FLAG and then for 45 min with protein A–Sepharose (GE Healthcare). The resulting 198

immunoprecipitates were subjected to immunoblot analysis. 199

200

Immunofluorescence microscopy 201

Cells were fixed with 4% formaldehyde for 10 min at 37°C, permeabilized with 0.2% 202

Triton X-100 in phosphate-buffered saline (PBS) for 5 min, and incubated for 24 h in 203

PBS containing 2% fetal bovine serum and 2 % bovine serum albumin (blocking 204

buffer). They were then exposed at room temperature first for 1 h to primary antibodies 205

in blocking buffer and then for 1 h to Alexa Fluor–conjugated secondary antibodies 206

(Thermo Fisher Scientific) and Hoechst 33342 in blocking buffer. Moviol were used as 207


https://doi.org/10.1101/2020.04.09.033597

8

mounting medium. Images were acquired with a TCS SP5 confocal microscope (Leica) 208

and were processed with Image J (NIH). 209

210

Colocalization analysis and quantification of SG volume 211

Manders M1 and M2 coefficients for colocalization were calculated with Coloc 2 of 212

Fiji. A statistical significance test was derived by Costes (46). For the experiment shown 213

in Figure 2C, samples were prepared in the same way as for immunofluorescence 214

analysis described above, with the exceptions that ProLong Diamond (Thermo Fisher 215

Scientific) was used as mounting medium and that images were acquired with a TCS 216

SP8 confocal microscope (Leica). Three-dimensional images were acquired in order to 217

meet the Nyquist condition (pixel size of 40.6 nm for x and y, and of 100 nm for z) and 218

were deconvoluted with Huygens software (Scientific Volume Imaging). Manders M1 219

and M2 for the colocalization of IPS-1 and TOMM20 were then calculated. For the 220

experiment shown in Figure 2E, samples were again prepared in the same way as for 221

immunofluorescence analysis with the exception that ProLong Diamond (Thermo 222

Fisher Scientific) was used as mounting medium. Three-dimensional images were 223

acquired in order to match the Nyquist condition (pixel size of 50 nm for x and y, and of 224

100 nm for z) and were deconvoluted with Huygens software (Scientific Volume 225

Imaging). Manders M1 coefficient for the colocalization of IPS-1 and TIAR was then 226

calculated. The SG volume per cell was measured with the 3D object counter 227

(threshold, 25; size filter, 1) of Huygens software in images for which the background 228

intensity of the cytosol had been subtracted. 229

230

RT and real-time PCR analysis 231

Total RNA was obtained from cells with the use of RNAiso (TaKaRa). RT was 232

performed with 1 μg of total RNA and ReverTra Ace qPCR RT Master Mix with gDNA 233

Remover (Toyobo). The resulting cDNA was subjected to real-time PCR analysis in a 234

Roche LightCycler with the use of a KAPA SYBR Fast qPCR Kit (Nippon Genetics). 235

The abundance of each target mRNA was normalized by that glyceraldehyde-3-236

phosphate dehydrogenase (GAPDH) mRNA. The PCR primers (sense and antisense, 237

respectively) were 5′-ACTCCTCCACCTTTGACGCT-3′ and 5′-238

TCCTCTTGTGCTCTTGCTGG-3′ for human GAPDH, 5′-239

CTGGCTGGAATGAGACTATTGTT- 3′ and 5′-CTTCAGTTTCGGAGGTAACCTG-3′ 240

for human IFN-β, 5′-AACCTGAACCTTCCAAAGATGG-3′ and 5′-241

TCTGGCTTGTTCCTCACTACT-3′ for human IL-6, and 5′-242

ATGAGCACTGAAAGCATGATCC-3′ and 5′-GAGGGCTGATTAGAGAGAGGTC-3′ 243


https://doi.org/10.1101/2020.04.09.033597

9

for human TNF-α. 244

245

Reporter gene analysis 246

HEK293T cells seeded in 24-well plates (7.5 × 104 cells/well) were transiently 247

transfected for 20 h with 8 ng of a reporter plasmid encoding Renilla luciferase under 248

the control of the human IFN-β gene promoter (p-125-RLuc) together with 5 ng of an 249

expression plasmid encoding FLAG-tagged full-length IPS-1 and either 50 or 500 ng of 250

a plasmid for Myc-tagged IPS-1(ΔTM). The activity of Renilla luciferase in total cell 251

lysates was measured with the use of a Dual-Luciferase Reporter Assay System 252

(Promega) and was normalized by that of firefly luciferase derived from 80 ng of a 253

control plasmid. 254

255

Virus infection 256

EMCV were kindly provided by M. Yoneyama (Chiba University). The cells were 257

incubated in culture medium with 0.1 (+) or 0.2 (++) PFU of EMCV for 2 h, and then 258

replaced in EMCV-free culture medium for 10 h. 259

260

Statistical analysis 261

Quantitative data are presented as means ± s.e.m. or ± s.d. and were compared with 262

Student’s t test. A P value of

10

Results 265

NUDT21 forms a complex with IPS-1 266

In a search for regulators of IPS-1, we performed co-immunoprecipitation analysis to 267

identify IPS-1–associated molecules. FLAG epitope–tagged IPS-1 expressed in 268

HEK293T cells was thus immunoprecipitated with antibodies to FLAG, and the 269

resultant precipitates were analyzed by a highly sensitive direct nanoflow liquid 270

chromatography–tandem mass spectrometry system. We identified NUDT21 among the 271

proteins that coprecipitated with FLAG–IPS-1. To confirm this result, we performed co-272

immunoprecipitation analysis with HEK293T cells expressing FLAG–IPS-1 and Myc 273

epitope–tagged NUDT21. We found that Myc-NUDT21 coprecipitated with FLAG–274

IPS-1 (Figure 1A), suggesting that NUDT21 indeed forms a complex with IPS-1. 275

To examine which domains of IPS-1 interact with NUDT21, we generated 276

Myc-tagged fragments of human IPS-1 that contain either CARD (amino acids 1–98), 277

the proline-rich domain (amino acids 99–200), or the COOH-terminal domain including 278

the TM domain (amino acids 201–540). We found that hemagglutinin epitope (HA)–279

tagged NUDT21 coprecipitated with Myc–IPS-1 fragments containing CARD or the 280

proline-rich domain, but not with that containing the COOH-terminal domain (Figure 281

S1). These results suggested that IPS-1 associates with NUDT21 through its NH2-282

terminal domains including CARD and the proline-rich domain. 283

284

A fraction of NUDT21 associates with mitochondria in resting cells and localizes to 285

SGs on exposure to cytoplasmic dsRNA 286

These results suggestive of a physical interaction between IPS-1 and NUDT21 were 287

unexpected, given the previously described localization of NUDT21 in the nucleus and 288

that of IPS-1 to the mitochondrial outer membrane (8). Immunofluorescence analysis of 289

HeLa S3 cells indeed revealed that most NUDT21 was localized to the nucleus (Figure 290

1B). However, we detected a fraction of the NUDT21 signals in the cytoplasm (Figure 291

1B). Knockdown of NUDT21 by transfection of the cells with a small interfering RNA 292

(siRNA) resulted in a reduction in these cytoplasmic and nuclear signals, supporting 293

their specificity for NUDT21 (Figure 1B). The NUDT21 signals in the cytoplasm 294

overlapped with those for the mitochondrial marker cytochrome c (Figure 1B). By 295

contrast, CFIm68, a cofactor of NUDT21 in its nuclear function, did not appear to 296

localize to mitochondria (Figure S2). Together, these results suggested that a fraction of 297

NUDT21 localizes to mitochondria, where it may associate with IPS-1. 298

We next transfected HeLa S3 cells with the viral dsRNA mimic poly(I:C) to 299

examine its possible effect on the intracellular distribution of NUDT21. NUDT21 300


https://doi.org/10.1101/2020.04.09.033597

11

immunoreactivity was detected as granulelike aggregates in the cytoplasm at 6 h after 301

poly(I:C) transfection, and these granulelike signals were again attenuated by NUDT21 302

knockdown (Figure 1C). Moreover, we found that these cytoplasmic signals of 303

NUDT21 colocalized with the SG marker TIAR (Figure 1C). Indeed, 26.89 ± 3.65% of 304

the area of TIAR-positive foci overlapped with NUDT21 immunoreactivity (Figure 305

1D), suggesting that NUDT21 becomes localized to SGs in the presence of cytoplasmic 306

dsRNA and consequent activation of the RLR pathway. 307

308

NUDT21 plays an important role in IPS-1 localization to SGs 309

The interaction of NUDT21 with IPS-1 as well as its mitochondrial localization and its 310

appearance at SGs in response to dsRNA exposure prompted us to examine whether 311

NUDT21 regulates the localization of IPS-1. Consistent with previous studies (35, 26), 312

we found that a fraction of IPS-1 colocalized with TIAR in cells transfected with 313

poly(I:C), whereas most IPS-1 appeared to colocalize with the mitochondrial import 314

receptor subunit TOM20 homolog (TOMM20) in both control and poly(I:C)-transfected 315

HeLa S3 cells (Figure 2A). We calculated Manders coefficients for the colocalization of 316

IPS-1 and TIAR (47). Manders M1 (sum of TIAR signal intensity overlapping with IPS-317

1 versus total TIAR signal intensity) was significantly increased by poly(I:C) 318

transfection (Figure 2B), whereas Manders M2 (sum of IPS-1 signal intensity 319

overlapping with TIAR versus total IPS-1 signal intensity) did not differ significantly 320

between cells with or without poly(I:C) exposure (Figure 2B). These results suggested 321

that a fraction of IPS-1 becomes localized to SGs in response to the presence of 322

cytoplasmic dsRNA. We also found the 34.21 ± 5.88% of the area of TIAR-positive foci 323

overlapped with IPS-1 at 6 h after poly(I:C) transfection, indicating that IPS-1 is not 324

distributed to all SGs. To examine IPS-1 localization at a higher resolution, we stained 325

endogenous IPS-1 and TOMM20 at 6 h after poly(I:C) transfection in HeLa S3 cells 326

expressing a fusion protein of G3BP1 and near-infrared fluorescent protein (iRFP) and 327

obtained z-stack images deconvoluted with Huygens software. A fraction of IPS-1 328

signals was found to localize to G3BP1-iRFP foci in poly(I:C)-transfected cells, 329

whereas TOMM20 was detected exclusively outside of G3BP1-iRFP foci (Figure 2C). 330

We then examined whether poly(I:C) transfection affects the colocalization of IPS-1 and 331

TOMM20. Although Manders M1 (sum of IPS-1 signal intensity overlapping with 332

TOMM20 versus total IPS-1 signal intensity) was not significantly altered by poly(I:C) 333

transfection, Manders M2 (sum of TOMM20 signal intensity overlapping with IPS-1 334

versus total TOMM20 signal intensity) was slightly but significantly reduced in cells 335

exposed to poly(I:C) (Figure 2D), suggesting that a fraction of IPS-1 separates from 336


https://doi.org/10.1101/2020.04.09.033597

12

TOMM20 in response to poly(I:C) stimulation. Together, these results indicated that a 337

fraction of IPS-1, but not of another mitochondrial protein (TOMM20), becomes 338

localized to SGs in response to the presence of cytoplasmic dsRNA. 339

We next examined the effect of NUDT21 on the distribution of IPS-1 in cells 340

transfected with poly(I:C). We thus calculated Manders coefficients for IPS-1 and TIAR 341

in z-stack images deconvoluted with Huygens software and found that Manders M1 342

(sum of TIAR signal intensity overlapping with IPS-1 versus total TIAR signal 343

intensity) was markedly reduced by NUDT21 knockdown (Figure 2E, F). Importantly, 344

NUDT21 knockdown did not affect the overall (mitochondrial) distribution of IPS-1 in 345

control (nontransfected) cells (Figure S3), indicating that the effect of NUDT21 on the 346

localization of IPS-1 was stimulus dependent. By contrast, NUDT21 knockdown did not 347

significantly alter the volume of TIAR-positive foci (SGs) per cell (Figure 2G) or the 348

abundance of IPS-1 (Figure S4). We also found that NUDT21 knockdown did not 349

significantly affect the ratio of SG-containing cells (Figure 2H) or RIG-I accumulation 350

at SGs in response to poly(I:C) transfection (Figure 2I). Collectively, these results 351

suggested that NUDT21 plays a role in IPS-1 localization to SGs in response to the 352

presence of cytoplasmic dsRNA, whereas it does not overtly affect SG formation or 353

RIG-I accumulation at SGs. 354

355

NUDT21 is required for antiviral responses induced by cytoplasmic dsRNA 356

The interaction between RLRs and IPS-1 triggers activation of downstream signaling 357

pathways in response to virus infection (7–10). Given that our data implicated NUDT21 358

in localization of IPS-1 to SGs, which contain RLRs, we next asked whether NUDT21 359

contributes to antiviral responses mediated by RLRs and IPS-1 such as the induction of 360

IFN-β and the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis 361

factor–α (TNF-α) in HeLa S3 cells transfected with poly(I:C). We found that NUDT21 362

knockdown markedly suppressed the increase in the amount of IFN-β mRNA induced 363

by poly(I:C) stimulation (Figure 3A). The up-regulation of IL-6 and TNF-α mRNAs in 364

response to poly(I:C) transfection was also significantly attenuated by depletion of 365

NUDT21 (Figure 3B, C). These results thus suggested that NUDT21 is necessary for 366

the efficient induction of IFN-β and proinflammatory cytokines in response to the 367

presence of cytoplasmic dsRNA. 368

We next examined whether NUDT21 mediates activation of the transcription 369

factor IRF3 or the mitogen-activated protein kinases (MAPKs) p38 and c-Jun NH2-370

terminal kinase (JNK), all of which are essential factors for activation of the promoters 371

of IFN-β and inflammatory cytokine genes in response to cytoplasmic dsRNA (15, 48). 372


https://doi.org/10.1101/2020.04.09.033597

13

Knockdown of NUDT21 suppressed the increase in the abundance of phosphorylated 373

(activated) forms of IRF3 as well as of p38 and JNK induced by poly(I:C) transfection 374

(Figure 3D), suggesting that NUDT21 promotes the activation of these signaling 375

molecules that is essential for the transcription of IFN-β and inflammatory cytokine 376

genes. In addition, NUDT21 knockdown suppressed the cleavage of caspase-3 and 377

poly(ADP-ribose) polymerase (PARP) induced by poly(I:C) transfection (Figure 3D), 378

suggesting that NUDT21 also plays a key role in the induction of caspase activation, 379

and perhaps cell death, in response to the presence of cytoplasmic dsRNA. Together, 380

these results indicated that NUDT21 mediates antiviral cellular responses induced by 381

the presence of cytoplasmic dsRNA. 382

383

NUDT21 mediates type I IFN induction in response to virus infection 384

To investigate further the role of NUDT21 in antiviral responses triggered by RLRs and 385

IPS-1, we asked whether NUDT21 knockdown affects type I IFN induction in cells 386

infected with viruses. To this end, we studied EMCV, whose RNAs released into the 387

cytoplasm are recognized predominantly by the RLRs MDA5 (6). We found that 388

NUDT21 knockdown significantly suppressed the EMCV-induced phosphorylation of 389

IRF3 and increase in IFN-β mRNA abundance (Figure 4A, B). Together, these results 390

implicated NUDT21 in the optimal induction of IFN-β in response to viral infection 391

392

Forced localization of IPS-1 to SGs promotes type I IFN induction in response to 393

cytoplasmic dsRNA 394

Disruption of SGs has been shown to attenuate IFN induction by dsRNA (25, 30), 395

although SGs appear to be dispensable for antiviral responses in some instances (49). 396

We therefore investigated whether forced targeting of IPS-1 to SGs might enhance IFN 397

induction in poly(I:C)-transfected cells. We first constructed an expression vector for a 398

fusion protein containing the SG protein G3BP1 and Myc-tagged IPS-1. However, 399

expression of G3BP1-Myc-IPS-1 resulted in formation of abnormal aggregates within 400

HeLa S3 cells that appeared to include mitochondria as shown by TOMM20 401

immunostaining (Figure S5). To avoid such mitochondrial aggregation, we generated a 402

fusion protein containing G3BP1 and a Myc-tagged form of IPS-1 that lacks the 403

COOH-terminal TM domain (Figure 5A). We found that this G3BP1-Myc-IPS-1(ΔTM) 404

fusion protein was present mostly in the cytosol of HeLa S3 cells in the absence of 405

poly(I:C) transfection, but preferentially localized to SGs in the presence of poly(I:C) 406

(Figure 5B). We then measured the level of IFN-β mRNA in cells expressing G3BP1, 407

Myc-tagged IPS-1(ΔTM), or G3BP1-Myc-IPS-1(ΔTM) at 9 h after poly(I:C) 408


https://doi.org/10.1101/2020.04.09.033597

14

transfection (Figure 5C, D). Forced expression of G3BP1 resulted in slight enhancement 409

of the increase in the amount of IFN-β mRNA induced by poly(I:C) transfection, 410

consistent with a previous observation (50). We also found that expression of Myc-411

tagged IPS-1(ΔTM) slightly enhanced the increase in IFN-β mRNA abundance induced 412

by poly(I:C). Expression of G3BP1-Myc-IPS-1(ΔTM) resulted in a markedly greater 413

increase in the level of IFN-β mRNA in poly(I:C)-transfected cells compared with that 414

induced by G3BP1 or Myc-tagged IPS-1(ΔTM) (Figure 5D). Importantly, the amount of 415

G3BP1-Myc-IPS-1(ΔTM) in the cells was markedly lower than that of Myc–416

IPS(1ΔTM). These results together suggested that the localization of IPS-1 to SGs 417

enhances IFN induction in the presence of cytoplasmic dsRNA. 418

419


https://doi.org/10.1101/2020.04.09.033597

15

Discussion 420

In the innate immune system, the site of receptor-ligand interaction is often dissociated 421

from that of signal transduction, likely in order to prevent aberrant activation of 422

antipathogen programs in the absence of infection (51). However, these distinct 423

compartments must encounter each other soon after ligand detection. We have now 424

identified NUDT21 as a link between RLR-containing stress granules and 425

mitochondrial IPS-1 as well as an essential mediator of antiviral responses. Our study 426

has therefore unveiled the existence of a cellular machinery that links the site of 427

pathogen recognition and that of antiviral signal transduction. 428

What is the mechanism by which NUDT21 mediates type I IFN production? 429

Although we cannot exclude the possibility that NUDT21 regulates type I IFN 430

expression through alternative polyadenylation in the nucleus, we propose a model 431

whereby NUDT21 promotes antiviral responses by recruiting IPS-1 to antiviral SGs on 432

the basis of the following observations: (1) Both mass spectrometry and co-433

immunoprecipitation analyses indicated that NUDT21 forms a complex with 434

mitochondrial IPS-1. (2) A fraction of NUDT21 was found to be localized to 435

mitochondria in resting cells and became localized to SGs in response to poly(I:C) 436

transfection. (3) Another component of the CFIm complex, CFIm68, appeared to be 437

localized only in the nucleus, not being detected in the cytoplasm, suggesting that 438

NUDT21 has a cytoplasmic function independent of the CFIm complex. (4) 439

Knockdown of NUDT21 suppressed the change in the localization of IPS-1 from 440

mitochondria to SGs, but not the formation of SGs, in response to poly(I:C) stimulation. 441

Further studies are required to understand in more detail how NUDT21 regulates IPS-1 442

localization in response to poly(I:C) transfection. 443

Caspase-mediated IPS-1 cleavage at a juxtamembrane site has been proposed 444

to inactivate the function of IPS-1 (33, 34). However, an IPS-1 mutant lacking the TM 445

domain was found to form prionlike fibers (“seeds”) that could convert native IPS-1 446

into functional aggregates (11). The role of IPS-1 cleavage therefore remains 447

controversial. We found that expression of an IPS-1(ΔTM) mutant significantly 448

enhanced the activity of the IFN-β gene promoter only in the presence of full-length 449

IPS-1 (Figure S6). Moreover, our results showed that forced localization of IPS-1(ΔTM) 450

to SGs enhanced type I IFN induction by poly(I:C) transfection. On the basis of these 451

observations, we propose a two-step model: In the early stage of virus infection, the 452

caspase-cleaved form of IPS-1 cooperates with full-length IPS-1 to form large 453

aggregates on the mitochondrial surface that associate with RLRs in SGs and thereby 454

elicit antiviral responses. In the late stage, caspases cleave the remaining intact IPS-1 455


https://doi.org/10.1101/2020.04.09.033597

16

molecules and thereby terminate IPS-1–mediated immune responses. This model may 456

reconcile the apparent discrepancy in the effects of caspase-mediated cleavage on IPS-1 457

function mentioned above. 458

The precise nature of the physical interaction between SGs and IPS-1 459

aggregates remains unclear. Antiviral SGs are membraneless organelles that are formed 460

by liquid-liquid phase separation within the cytoplasm. We observed that a fraction of 461

IPS-1 appeared to form fiberlike structures rather than being evenly distributed within 462

SGs (Figure 2A). This observation and the previous findings on the prionlike 463

aggregation of IPS-1 (11) suggest that IPS-1 aggregates may form a subcompartment 464

within SGs, perhaps by undergoing a liquid-to-solid phase transition (which typically 465

underlies the formation of solid prionlike aggregates or crystals), or that they may lie 466

adjacent to SGs and associate with RIG-I in SGs at the interface. In these two cases, the 467

presence of NUDT21 may facilitate functional solidification of IPS-1 in SGs or 468

functional association between RIG-I in SGs and IPS-1 aggregates, respectively. 469

Lysine-63–linked polyubiquitination by the ubiquitin ligase TRIM25 has been 470

implicated in the activation of RIG-I and subsequent formation of large IPS-1 471

aggregates (52, 53, 11). It would thus be of interest to examine the relation between 472

such TRIM25-mediated polyubiquitination and NUDT21 in the formation of IPS-1 473

aggregates and their association with SGs. Of note, RIG-I has been shown to associate 474

exclusively with either TRIM25 or IPS-1, with the two complexes being localized to 475

distinct compartments (54). Together with our observation that IPS-1 is not distributed 476

among all SGs in a cell, this finding suggests that RIG-I might shift from the TRIM25 477

compartment to the IPS-1 compartment in association with IPS-1 activation (54) and 478

that NUDT21 may facilitate this transition. 479

In closing, we have here demonstrated the cytoplasmic localization of 480

NUDT21 and its unexpected role in regulation of antiviral proteins in the cytoplasm in 481

addition to its well-established localization to nucleus and function in alternative 482

polyadenylation. We therefore propose that NUDT21 may function in broader biological 483

contexts, at least at mitochondria and SGs, than anticipated, and our results provide a 484

basis for the development of a new target for clinical intervention in viral propagation. 485

486


https://doi.org/10.1101/2020.04.09.033597

17

Acknowledgments 487

We thank M. Okajima and K. Takechi (Graduate School of Pharmaceutical Sciences, 488

The University of Tokyo) for technical assistance; M. Yoneyama (Chiba University) for 489

providing the pEF-BOS-FLAG-IPS-1 plasmid, antibodies to RIG-I, and ECMV; and 490

laboratory members for discussion. 491

492

Author Contributions 493

S.A.I. performed experiments and analyzed data. T.N. and S.I. conducted the mass 494

spectrometric analysis. Y.O. provided the pN1/G3BP1-iRFP plasmid and technical 495

assistance for imaging analysis. S.A.I., Y.G., and T.O. conceived the study and wrote the 496

manuscript. 497

498

Funding 499

This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, 500

Sports, Science, and Technology (MEXT) of Japan; by Core Research for Evolutionary 501

Science and Technology of the Japan Science and Technology Agency; by research 502

fellowships from the Japan Society for the Promotion of Science (JSPS) and the Global 503

Centers of Excellence Program (Integrative Life Science Based on the Study of 504

Biosignaling Mechanisms) of MEXT; by the Graduate Program for Leaders in Life 505

Innovation, The University of Tokyo Life Innovation Leading Graduate School, of 506

MEXT; and by JSPS KAKENHI grants, JP18gm0610013, JP15H05773, JP16H06481, 507

JP16H06279, and JP16H06479 to Y.G., JP16K19149 and JP18K07168 to T.O., and 508

JP16H06280 to Y.O., and JP15J10794 to S.A.I.. 509

510

Declaration of Interests 511

The authors declare no competing interests. 512

513


https://doi.org/10.1101/2020.04.09.033597

18

References 514

1. Stetson, D.B., and R. Medzhitov. 2006. Type I interferons in host defense. 515

Immunity. 25: 373–381. 516

2. Schneider, W.M., M.D. Chevillotte, and C.M. Rice. 2014. Interferon-stimulated 517

genes: a complex web of host defenses. Annu. Rev. Immunol. 32: 513–545. 518

3. Akira, S., S. Uematsu, and O. Takeuchi. 2006. Pathogen recognition and innate 519

immunity. Cell. 124: 783–801. 520

4. Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi, M. Miyagishi, 521

K. Taira, S. Akira, and T. Fujita. 2004. The RNA helicase RIG-I has an essential 522

function in double-stranded RNA-induced innate antiviral responses. Nat Immunol. 523

5: 730–737. 524

5. Gitlin, L., W. Barchet, S. Gilfillan, M. Cella, B. Beutler, R.A. Flavell, M.S. 525

Diamond, and M. Colonna. 2006. Essential role of mda-5 in type I IFN responses to 526

polyriboinosinic: polyribocytidylic acid and encephalomyocarditis picornavirus. 527

Proc Natl Acad Sci USA. 103: 8459–8464. 528

6. Kato, H., O. Takeuchi, S. Sato, M. Yoneyama, M. Yamamoto, K. Matsui, S. 529

Uematsu, A. Jung, T. Kawai, K.J. Ishii, O. Yamaguchi, K. Otsu, T. Tsujimura, C.S. 530

Koh, C. Reis e Sousa, Y. Matsuura, T. Fujita, and S. Akira. 2006. Differential roles 531

of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature. 441: 101–532

105. 533

7. Kawai, T., K. Takahashi, S. Sato, C. Coban, H. Kumar, H. Kato, K.J. Ishii, O. 534

Takeuchi, and S. Akira. 2005. IPS-1, an adaptor triggering RIG-I- and Mda5-535

mediated type I interferon induction. Nat Immunol. 6: 981–988. 536

8. Seth, R.B., L. Sun, C.K. Ea, and Z.J. Chen. 2005. Identification and 537

characterization of MAVS, a mitochondrial antiviral signaling protein that activates 538

NF-κB and IRF3. Cell. 122: 669–682. 539

9. Meylan, E., J. Curran, K. Hofmann, D. Moradpour, M. Binder, R. Bartenschlager, 540

and J. Tschopp. 2005. Cardif is an adaptor protein in the RIG-I antiviral pathway 541

and is targeted by hepatitis C virus. Nature. 437: 1167–1172. 542

10. Xu, L.G., Y.Y. Wang, K.J. Han, L.Y. Li, Z. Zhai, and H.B. Shu. 2005. VISA is an 543

adapter protein required for virus-triggered IFN-β signaling. Mol Cell. 19: 727–544

740. 545

11. Hou, F., L. Sun, H. Zheng, B. Skaug, Q.X. Jiang, and Z.J. Chen. 2011. MAVS 546

forms functional prion-like aggregates to activate and propagate antiviral innate 547

immune response. Cell. 146: 448–461. 548

12. Fitzgerald, K.A., S.M. Mcwhirter, K.L. Faia, D.C. Rowe, E. Latz, D.T. Golenbock, 549


https://doi.org/10.1101/2020.04.09.033597

19

A.J. Coyle, S.M. Liao, and T. Maniatis. 2003. IKKε and TBK1 are essential 550

components of the IRF3 signaling pathway. Nat Immunol. 4: 491–496. 551

13. Chu, W.M., D. Ostertag, Z.W. Li, L. Chang, Y. Chen, Y. Hu, B. Williams, J. 552

Perrault, and M. Karin. 1999. JNK2 and IKKβ are required for activating the innate 553

response to viral infection. Immunity. 11: 721–731. 554

14. Du, W., and T. Maniatis. 1992. An ATF / CREB binding site is required for virus 555

induction of the human interferon beta gene. Proc Natl Acad Sci USA. 89: 2150–556

2154. 557

15. Honda, K., A. Takaoka, and T. Taniguchi. 2006. Type I inteferon gene induction by 558

the interferon regulatory factor family of transcription factors. Immunity. 25: 349–559

360. 560

16. Okazaki, T., M. Higuchi, K. Takeda, K. Iwatsuki-Horimoto, M. Kiso, M. Miyagishi, 561

H. Yanai, A. Kato, M. Yoneyama, T. Fujita, T. Taniguchi, Y. Kawaoka, H. Ichijo, 562

and Y. Gotoh. 2015. The ASK family kinases differentially mediate induction of 563

type I interferon and apoptosis during the antiviral response. Sci Signal. 8: ra78. 564

17. Kumar, H., T. Kawai, H. Kato, S. Sato, K. Takahashi, C. Coban, M. Yamamoto, S. 565

Uematsu, K.J. Ishii, O. Takeuchi, and S. Akira. 2006. Essential role of IPS-1 in 566

innate immune responses against RNA viruses. J Exp Med. 203: 1795–1803. 567

18. Sun, Q., L. Sun, H.H. Liu, X. Chen, R.B. Seth, J. Forman, and Z.J. Chen. 2006. The 568

specific and essential role of MAVS in antiviral innate immune responses. 569

Immunity. 24: 633–642. 570

19. Yoneyama, M., M. Kikuchi, K. Matsumoto, T. Imaizumi, M. Miyagishi, K. Taira, E. 571

Foy, Y.M. Loo, M. Gale Jr., S. Akira, S. Yonehara, A. Kato, and T. Fujita. 2005. 572

Shared and unique functions of the DExD/H-Box helicases RIG-I, MDA5, and 573

LGP2 in antiviral innate immunity. J Immunol. 175: 2851–2858. 574

20. Buchan, J.R., and R. Parker. 2009. Eukaryotic stress granules : the ins and outs of 575

translation. Mol Cell. 36: 932–941. 576

21. McCormick, C., and D.A. Khaperskyy. 2017. Translation inhibition and stress 577

granules in the antiviral immune response. Nat Rev Immunol. 17: 647-660. 578

22. Kedersha, N.L., M. Gupta, W. Li, I. Miller, and P. Anderson. 1999. RNA-binding 579

proteins TIA-1 and TIAR link the phosphorylation of eIF-2α to the assembly of 580

mammalian stress granules. J Cell Biol. 147: 1431–1442. 581

23. Tourrière, H., K. Chebli, L. Zekri, B. Courselaud, J.M. Blanchard, E. and Bertrand, 582

J. Tazi. 2003. The RasGAP-associated endoribonuclease G3BP assembles stress 583

granules. J Cell Biol. 160: 823–831. 584

24. Boeynaems, S., S. Alberti, N.L. Fawzi, T. Mittag, M. Polymenidou, F. Rousseau, J. 585


https://doi.org/10.1101/2020.04.09.033597

20

Schymkowitz, J. Shorter, B. Wolozin, L. Van Den Bosch, P. Tompa, and M. 586

Fuxreiter. 2018. Protein Phase Sparation: A New Phase in Cell Biology. Trends Cell 587

Biol. 28: 420–435. 588

25. Onomoto, K., M. Jogi, J.S. Yoo, R. Narita, S. Morimoto, A. Takemura, S. 589

Sambhara, A. Kawaguchi, S. Osari, K. Nagata, T. Matsumiya, H. Namiki, M. 590

Yoneyama, and T. Fujita. 2012. Critical role of an antiviral stress granule containing 591

RIG-I and PKR in viral detection and innate immunity. PLoS One. 7: e43031. 592

26. Oh, S.W., K. Onomoto, M. Wakimoto, K. Onoguchi, F. Ishidate, T. Fujiwara, M. 593

Yoneyama, H. Kato, and T. Fujita. 2016. Leader-Containing Uncapped Viral 594

Transcript Activates RIG-I in Antiviral Stress Granules. PLoS Pathog. 12: 595

e1005444. 596

27. White, J.P., A.M. Cardenas, W.E. Marissen, and R.E. Lloyd. 2007. Inhibition of 597

cytoplasmic mRNA stress granule formation by a viral proteinase. Cell Host 598

Microbe. 2: 295–305. 599

28. Khaperskyy, D.A., T.F. Hatchette, and C. McCormick. 2012. Influenza A virus 600

inhibits cytoplasmic stress granule formation. FASEB J. 26: 1629–1639. 601

29. Borghese, F., and T. Michiels. 2011. The leader protein of cardioviruses inhibits 602

stress granule assembly. J Virol. 85: 9614–9622. 603

30. Ng, C.S., M. Jogi, J.S. Yoo, K. Onomoto, S. Koike, T. Iwasaki, M. Yoneyama, H. 604

Kato, and T. Fujita. 2013. Encephalomyocarditis virus disrupts stress granules, the 605

critical platform for triggering antiviral innate immune responses. J Virol. 87: 606

9511–9522. 607

31. Finnen, R.L., M. Zhu, J. Li, D. Romo, and B.W. Banfield. 2016. Herpes Simplex 608

Virus 2 Virion Host Shutoff Endoribonuclease Activity Is Required To Disrupt 609

Stress Granule Formation. J Virol. 90: 7943–7955. 610

32. Li, X.D., L. Sun, R.B. Seth, G. Pineda, and Z.J. Chen. 2005. Hepatitis C virus 611

protease NS3/4A cleaves mitochondrial antiviral signaling protein off the 612

mitochondria to evade innate immunity. Proc Natl Acad Sci USA. 102: 17717–613

17722. 614

33. Rebsamen, M., E. Meylan, J. Curran, and J. Tschopp. 2008. The antiviral adaptor 615

proteins Cardif and Trif are processed and inactivated by caspases. Cell Death 616

Differ. 15: 1804–1811. 617

34. Ning, X., Y. Wang, M. Jing, M. Sha, M. Lv, P. Gao, R. Zhang, X. Huang, J.M. 618

Feng, and Z. Jiang. 2019. Apoptotic Caspases Suppress Type I Interferon 619

Production via the Cleavage of cGAS, MAVS, and IRF3. Mol Cell. 74: 19-31.e7. 620

35. Zhang, Peifen, Y. Li, J. Xia, J. He, J. Pu, J. Xie, S. Wu, L. Feng, X. Huang, and 621


https://doi.org/10.1101/2020.04.09.033597

21

Ping Zhang. 2014. IPS-1 plays an essential role in dsRNA-induced stress granule 622

formation by interacting with PKR and promoting its activation. J Cell Sci. 127: 623

2471–2482. 624

36. Brown, K.M., and G.M. Gilmartin. 2003. A mechanism for the regulation of pre-625

mRNA 3’ processing by human cleavage factor I m. Mol Cell. 12: 1467–1476. 626

37. Di Giammartino, D.C., K. Nishida, and J.L. Manley. 2011. Mechanisms and 627

consequences of alternative polyadenylation. Mol Cell. 43: 853–866. 628

38. Yang, Q., M. Coseno, G.M. Gilmartin, and S. Doublié. 2011. Crystal structure of a 629

human cleavage factor CFI(m)25 / CFI(m)68 / RNA complex provides an insight 630

into poly (A) site recognition and RNA looping. Structure. 19: 368–377. 631

39. Elkon, R., A.P. Ugalde, and R. Agami. 2013. Alternative cleavage and 632

polyadenylation: extent, regulation and function. Nat Rev Genet. 14: 496–506. 633

40. Masamha, C.P., Z. Xia, J. Yang, T.R. Albrecht, M. Li, A.B. Shyu, W. Li, and E.J. 634

Wagner. 2014. CFIm25 links alternative polyadenylation to glioblastoma tumour 635

suppression. Nature. 510: 412–416. 636

41. Brumbaugh, J., B. Di Stefano, X. Wang, M. Borkent, E. Forouzmand, K.J. Clowers, 637

F. Ji, B.A. Schwarz, M. Kalocsay, S.J. Elledge, Y. Chen, R.I. Sadreyev, S.P. Gygi, 638

G. Hu, Y. Shi, and K. Hochedlinger. 2018. Nudt21 Controls Cell Fate by 639

Connecting Alternative Polyadenylation to Chromatin Signaling. Cell. 172: 106-640

120.e21. 641

42. Jia, X., S. Yuan, Y. Wang, Y. Fu, Yong Ge, Yutong Ge, X. Lan, Y. Feng, F. Qiu, P. 642

Li, S. Chen, and A. Xu. 2017. The role of alternative polyadenylation in the 643

antiviral innate immune response. Nat Commun. 8: 14605. 644

43. Gaucherand, L., B.K. Porter, R.E. Levene, E.L. Price, S.K. Schmaling, C.H. 645

Rycroft, Y. Kevorkian, C. McCormick, D.A. Khaperskyy, and M.M. Gaglia. 2019. 646

The Influenza A Virus Endoribonuclease PA-X Usurps Host mRNA Processing 647

Machinery to Limit Host Gene Expression. Cell Rep. 27: 776-792.e7. 648

44. Okazaki, T., M. Higuchi, and Y. Gotoh. 2013. Mitochondrial localization of the 649

antiviral signaling adaptor IPS-1 is important for its induction of caspase activation. 650

Genes Cells. 18: 493–501. 651

45. Natsume, T., Y. Yamauchi, H. Nakayama, T. Shinkawa, M. Yanagida, N. Takahashi, 652

and T. Isobe. 2002. A direct nanoflow liquid chromatography - tandem mass 653

spectrometry system for interaction proteomics. Anal Chem. 74: 4725–4733. 654

46. Costes, S.V., D. Daelemans, E.H. Cho, Z. Dobbin, G. Pavlakis, and S. Lockett. 655

2004. Automatic and quantitative measurement of protein-protein colocalization in 656

live cells. Biophys J. 86: 3993–4003. 657


https://doi.org/10.1101/2020.04.09.033597

22

47. Manders, E.M.M., F.J. Verbeek, and J.A. Aten. 1993. Measurement of co-658

localization of objects in dual-colour confocal images. J. Microsc. 169: 375–382. 659

48. Mikkelsen, S.S., S.B. Jensen, S. Chiliveru, J. Melchjorsen, I. Julkunen, M. Gaestel, 660

J.S. Arthur, R.A. Flavell, S. Ghosh, and S.R. Paludan. 2009. RIG-I-mediated 661

activation of p38 MAPK is essential for viral induction of interferon and activation 662

of dendritic cells: dependence on TRAF2 and TAK1. J Biol Chem. 284: 10774–663

10782. 664

49. Langereis, M.A., Q. Feng, and F.J. van Kuppeveld. 2013. MDA5 localizes to stress 665

granules, but this localization is not required for the induction of type I interferon. J 666

Virol. 87: 6314–6325. 667

50. Kim, S.S., L. Sze, C. Liu, and K.P. Lam. 2019. The stress granule protein G3BP1 668

binds viral dsRNA and RIG-I to enhance interferon-β response. J Biol Chem. 294: 669

6430–6438. 670

51. Kagan, J.C. 2012. Signaling organelles of the innate immune system. Cell. 151: 671

1168–1178.. 672

52. Gack, M.U., Y.C. Shin, C.H. Joo, T. Urano, C. Liang, L. Sun, O. Takeuchi, S. Akira, 673

Z. Chen, S. Inoue, and J.U. Jung. 2007. TRIM25 RING-finger E3 ubiquitin ligase is 674

essential for RIG-I-mediated antiviral activity. Nature. 446: 916–920. 675

53. Jiang, X., L.N. Kinch, C.A. Brautigam, X. Chen, F. Du, N.V. Grishin, and Z.J. 676

Chen. 2012. Ubiquitin-induced oligomerization of the RNA sensors RIG-I and 677

MDA5 activates antiviral innate immune response. Immunity. 36: 959–973. 678

54. Sánchez-Aparicio, M.T., J. Ayllón, A. Leo-Macias, T. Wolff, and A. García-Sastre. 679

2017. Subcellular Localizations of RIG-I, TRIM25, and MAVS Complexes. J Virol. 680

91: e01155-16. 681

682

683


https://doi.org/10.1101/2020.04.09.033597

23

Figure Legends 684

Figure 1. A fraction of NUDT21 localizes to mitochondria in resting cells and 685

contributes to SGs in the cytoplasm in response to poly(I:C) transfection. 686

(A) Extracts of HEK293T cells transiently transfected with expression vectors for 687

FLAG-tagged IPS1 and Myc-tagged NUDT21 were subjected to immunoprecipitation 688

(IP) with antibodies to FLAG, and the resulting precipitates as well as the original cell 689

extracts (Total) were subjected to immunoblot analysis with antibodies to FLAG and to 690

Myc. The asterisk indicates a nonspecific band. Data are representative of three 691

independent experiments. 692

(B) Immunofluorescence analysis of HeLa S3 cells transfected with control or NUDT21 693

siRNAs. The cells were stained with antibodies to NUDT21 and to cytochrome c as 694

well as with Hoechst 33342. The boxed regions in the upper panels are shown at higher 695

magnification in the corresponding middle panels. Scale bars, 5 μm or 2 μm (higher 696

magnification images). Data are representative of three independent experiments. 697

(C) Immunofluorescence analysis of control and NUDT21-depleted HeLa S3 cells at 6 698

h after transfection with poly(I:C) (0.25 μg/ml) or mock transfection. The cells were 699

stained with antibodies to NUDT21 and to TIAR as well as with Hoechst 33342. Scale 700

bars, 5 μm. Data are representative of three independent experiments. 701

(D) Quantification of the NUDT21-positive area of TIAR-positive foci per cell in 702

images similar to those in (C). Data are means ± s.e.m. from three independent 703

experiments (control, n = 16; NUDT21 knockdown, n = 13). ***P < 0.005 (Student’s t 704

test). 705

706

Figure 2. NUDT21 knockdown impairs the localization of IPS-1 to SGs. 707

(A) Immunofluorescence analysis of HeLa S3 cells at 6 h after transfection with 708

poly(I:C) (0.25 μg/ml) or mock transfection. The cells were stained with antibodies to 709

IPS-1, to TOMM20, and to TIAR as well as with Hoechst 33342. The boxed regions of 710

the middle panels are shown at higher magnification in the corresponding lower panels. 711

Arrowheads indicate SGs. Scale bars, 5 μm or 2 μm (higher magnification images). 712

Data are representative of three independent experiments. 713

(B) Colocalization (Manders M1 and M2) between IPS-1 and TIAR was determined for 714

images as in (A). Manders M1 is the sum of TIAR signal intensity overlapping with 715

IPS-1 versus total TIAR signal intensity, whereas Manders M2 is the sum of IPS-1 716

signal intensity overlapping with TIAR versus total IPS-1 signal intensity. Data are 717

means ± s.e.m. from three independent experiments (mock, n = 5; poly(I:C), n = 11). 718

**P < 0.01 (Student’s t test). 719


https://doi.org/10.1101/2020.04.09.033597

24

(C) HeLa S3 cells transiently expressing iRFP-fused G3BP1 were subjected to 720

immunofluorescence staining with antibodies to IPS-1 and to TOMM20 at 6 h after 721

transfection with poly(I:C) (0.25 μg/ml) or mock transfection. The fluorescence of iRFP 722

was monitored directly. The z-stack images were deconvoluted with Huygens software. 723

The boxed regions of the left images are shown at higher magnification in those on the 724

right. Scale bars, 5 μm or 2 μm (higher magnification images). Line-scan analysis of the 725

relative fluorescence intensity of IPS-1, TOMM20, and G3BP1-iRFP along the white 726

broken lines (6 μm) in the enlarged images is also shown. Data are representative of 727

three independent experiments. 728

(D) Colocalization (Manders M1 and M2) between IPS-1 and TOMM20 was 729

determined for images as in (C). Manders M1 is the sum of IPS-1 signal intensity 730

overlapping with TOMM20 versus total IPS-1 signal intensity, and Manders M2 is the 731

sum of TOMM20 signal intensity overlapping with IPS-1 versus total TOMM20 signal 732

intensity. Data are means ± s.e.m. from three independent experiments (mock, n = 12; 733

poly(I:C), n = 18). *P < 0.05 (Student’s t test). 734

(E) Immunofluorescence analysis of HeLa S3 cells expressing control or NUDT21 735

siRNAs at 6 h after transfection with poly(I:C) (0.25 μg/ml). The cells were stained with 736

antibodies to IPS-1, to TOMM20, and to TIAR. The z-stack images were deconvoluted 737

with Huygens software. The boxed region of the upper image of each pair is shown at 738

higher magnification in the lower image. Arrowheads indicate SGs. Scale bars, 5 μm or 739

2 μm (higher magnification images). Data are representative of three independent 740

experiments. 741

(F) Colocalization (Manders M1, sum of TIAR signal intensity overlapping with IPS-1 742

versus total TIAR signal intensity) between IPS-1 and TIAR was determined for images 743

as in (E). Data are means ± s.e.m. of three independent experiments (control, n = 10; 744

NUDT21 knockdown, n = 10). ****P < 0.001 (Student’s t test). 745

(G) Quantification of SG volume per cell for images as in (E). Data are means ± s.e.m. 746

of three independent experiments (control, n = 10; NUDT21 knockdown, n = 10). The P 747

value was determined with Student’s t test. 748

(H) Quantification of SG-containing cells. HeLa S3 cells expressing control or 749

NUDT21 siRNAs were subjected to immunofluorescence staining for TIAR in order to 750

determine the proportion of SG-containing cells at 6 h after transfection with poly(I:C) 751

(0.25 μg/ml) or mock transfection. Data are means ± s.e.m. of three independent 752

experiments. The P value was determined with Student’s t test. 753

(I) Immunofluorescence analysis of control and NUDT21-knockdown HeLa S3 cells at 754

6 h after transfection with poly(I:C) (0.25 μg/ml) or mock transfection. The cells were 755


https://doi.org/10.1101/2020.04.09.033597

25

stained with antibodies to RIG-I and to TIAR as well as with Hoechst 33342. Scale 756

bars, 5 μm. Data are representative of three independent experiments. 757

758

Figure 3. NUDT21 plays a key role in the induction of type I IFN and caspase 759

activation in response to poly(I:C) transfection. 760

(A–C) Quantitative reverse transcription and polymerase chain reaction (RT-PCR) 761

analysis of IFN-β, IL-6, and TNF-α mRNAs, respectively, in HeLa S3 cells transiently 762

expressing control or NUDT21 siRNAs at 9 h after transfection with poly(I:C) (0.25 763

μg/ml) or mock transfection. Data are expressed relative to the corresponding amount of 764

GAPDH mRNA and are means ± s.e.m. from four independent experiments. **P < 765

0.01, ****P < 0.001 (Student’s t test). 766

(D) Immunoblot analysis of phosphorylated (p-) forms of IRF3, p38 MAPK, and JNK 767

as well as of cleaved forms of caspase-3 and PARP in control or NUDT21-knockdwon 768

HeLa S3 cells at 9 h after transfection with poly(I:C) [0.25 (+) or 2.5 (++) μg/ml] or 769

mock transfection. The amounts of NUDT21 and total p38 were examined as controls 770

for knockdown efficiency and protein loading, respectively. Asterisks indicate 771

nonspecific bands. Data are representative of three independent experiments. 772

773

Figure 4. NUDT21 is required for the optimal induction of type I IFN in response to 774

EMCV or NDV infection. 775

(A) Quantitative RT-PCR analysis of IFN-β mRNA in HeLa S3 cells transiently 776

expressing control or NUDT21 siRNAs at 12 h after infection with 0.2 plaque-forming 777

units (PFU) of EMCV. Data are means ± s.e.m. from four independent experiments. *P 778

< 0.05 (Student’s t test). 779

(B) Immunoblot analysis of phosphorylated IRF3, NUDT21, and p38 in control or 780

NUDT21-knockdown HeLa S3 cells infected with 0.1 (+) or 0.2 (++) PFU of EMCV 781

for 12 h. The asterisk indicates nonspecific bands. Data are representative of three 782

independent experiments. 783

784

Figure 5. Forced localization of IPS-1 to SGs enhances type I IFN induction in 785

response to poly(I:C) transfection. 786

(A) Schematic representation of human IPS-1, Myc-tagged IPS-1(ΔTM), and G3BP1-787

Myc-IPS-1(ΔTM). 788

(B) Immunofluorescence analysis of HeLa S3 cells transiently expressing G3BP1-Myc-789

IPS-1(ΔTM) at 6 h after transfection with poly(I:C) (0.25 μg/ml) or mock transfection. 790

The cells were stained with antibodies to Myc, to TIAR, and to TOMM20. Scale bars, 5 791


https://doi.org/10.1101/2020.04.09.033597

26

μm. Data are representative of three independent experiments. 792

(C) Immunoblot analysis with antibodies to Myc of HeLa S3 cells transiently 793

expressing Myc–IPS-1(ΔTM) or G3BP1-Myc-IPS-1(ΔTM) at 9 h after transfection with 794

poly(I:C) (0.25 μg/ml) or mock transfection. Black and blue arrowheads indicate Myc–795

IPS-1(ΔTM) and G3BP1-Myc-IPS-1(ΔTM), respectively. Data are representative of 796

three independent experiments. 797

(D) Quantitative RT-PCR analysis of IFN-β mRNA in HeLa S3 cells transiently 798

expressing G3BP1, Myc–IPS-1(ΔTM), or G3BP1-Myc-IPS-1(ΔTM) at 9 h after 799

transfection with poly(I:C) (0.25 μg/ml) or mock transfection. Data are means ± s.e.m. 800

from four independent experiments. *P < 0.05, **P < 0.01, ****P < 0.001 (Student’s t 801

test). 802


https://doi.org/10.1101/2020.04.09.033597

Con

trol

siR

NA

NU

DT2

1si

RN

A

Poly(I:C)

-

-

+

+

Hoechst NUDT21 TIAR

HoechstNUDT21

TIARC

HoechstNUDT21

(low gain)NUDT21

(high gain) Cytochrome c

ControlsiRNA

NUDT21siRNA

HoechstNUDT21

Cytochrome cB

D

FLAG

Myc

FLAG

Myc

IP: FLAG

Total

FLAG-IPS-1Myc-NUDT21

+ +++

--

A

*

26.590

90 kDa

26.5

Figure 1

0

20

40

60

80

100

ControlsiRNA

NUDT21siRNA

NU

DT2

1+ a

rea/

TIA

R+

area

(%)

***


https://doi.org/10.1101/2020.04.09.033597

C

D

B

*Colocalization

TOMM20 and IPS-1

050

100150200

0 2 4 6

050

100150200

0 2 4 6Distanc

Rel

ativ

e In

tens

ityR

elat

ive

Inte

nsity

Distanc

TOMM20IPS-1

G3BP1-iRFP

-

+

Figure 2

F

Man

ders

M2

- +0

0.20.40.60.81.0

Con

trol

siR

NA

NU

DT2

1si

RN

A

-

-

+

+

Hoechst RIG-I TIAR

HoechstRIG-ITIAR

I

H

ControlsiRNA

NUDT21siRNA

P = 0.287

0

304050

2010

Volu

e of

TIA

R fo

ci (µ

³

0

10

20

30

ControlsiRNA

NUDT21siRNA

SG+

+ - +-

P = 0.522

P = 0.543

GColocalizationTIAR and IPS-1

A

IPS-1TOMM20

TIARIPS-1 TIAR

ControlsiRNA

NUDT21siRNA

TOMM20E

Man

ders

M1

- +0

0.20.40.60.81.0

Man

ders M

1

0

0.2

0.4

0.6

0.8

1.0 ****

ControlsiRNA

NUDT21siRNA

00.20.40.60.81.0

Man

ders

M1

+-0

0.20.40.60.81.0

+-

Man

ders

M2**

P = 0.993

ColocalizationTIAR and IPS-1

Hoechst IPS-1 TOMM20 TIAR

HoechstIPS-1

TOMM20

HoechstIPS-1TIAR

-

+Deconvoluted


https://doi.org/10.1101/2020.04.09.033597

D

p-IRF3

p-p38

p-JNK

Cleavedcaspase-3

CleavedPARP

NUDT21

p38

ControlsiRNA

NUDT21siRNA

- + - +Poly(I:C)

*++ ++

*

*

Rel

ativ

em

RN

A a

mou

nt

- + - +Poly(I:C)

ControlsiRNA

NUDT21siRNA

****

1.2

1.0

0.8

0.6

0.4

0.2

0

1.4

Rel

ativ

e IL

-6m

RN

A a

mou

nt- + - +Poly(I:C)

ControlsiRNA

NUDT21siRNA

**

1.2

1.0

0.8

0.6

0.4

0.2

0

1.4

Rel

ativ

em

RN

A a

mou

nt

- + - +Poly(I:C)

ControlsiRNA

NUDT21siRNA

**1.2

1.0

0.8

0.6

0.4

0.2

0

1.4

A B C

48.5 kDa

90

36.5

36.5

48.5

*26.5

26.5

Figure 3


https://doi.org/10.1101/2020.04.09.033597

p-IRF3

NUDT21

p38

EMCV

ControlsiRNA

NUDT21siRNA

- + - +++ ++

A B

*

Rel

ativ

em

RN

A a

mou

nt

EMCV - - ++

*

ControlsiRNA

NUDT21siRNA

3.0

2.5

2.0

1.5

1.0

0.5

0

36.5

26.5

48.5 kDa

Figure 4


https://doi.org/10.1101/2020.04.09.033597

Rela

tive

mRN

A am

ount

Control G3BP1 Myc-IPS-1(ΔTM)

G3BP1-Myc-IPS-1 (ΔTM)

- - ++

**3.0

2.5

2.0

1.5

1.0

0.5

0- - ++

P = 0.113

Poly(I:C)

****

*

A

B

C D

Myc

(long exposure)

p38

Myc-IPS-1(ΔTM)

G3BP1-Myc-

IPS-1 (ΔTM)- - ++Poly(I:C)

MycTIAR

TOMM20TOMM20Myc TIARPoly(I:C)

-

+

IPS-1 (full length)

Myc-IPS-1 (ΔTM)

G3BP1-Myc-IPS-1 (ΔTM) 4291

CARDG3BP1 Myc

TM5401

D429

CARD

4291CARDMyc

G3B

P1-M

yc-

IPS-

1 ( Δ

TM)

48.558

90110 kDa

48.558

90110

36.5

Myc

Figure 5

https://doi.org/10.1101/2020.04.09.033597

Documents

NUDT21 links mitochondrial IPS 1 to RLR containing stress … · 2020. 4. 9. · 86 IPS-1 abrogated both its mitochondrial localization and the induction of type I IFN (8, 87 32)