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Human Nuclear RNAi-Defective 2 (NRDE2) is an essential RNA splicing factor
Alan L. Jiao,1,2 Roberto Perales,3 Neil T. Umbreit,4 Jeffrey R. Haswell,1,5 Mary E. Piper,6 Brian D. Adams,1,7 David Pellman,4,8,9 Scott Kennedy,3 Frank J. Slack1 *
1: HMS Initiative for RNA Medicine, Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
2: Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT USA.
3: Department of Genetics, Harvard Medical School, Boston, MA, USA.
4: Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
5: Department of Biological and Biomedical Sciences, Harvard University, Boston, MA, USA.
6: Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA.
7: Present address: Department of RNA Science, The Brain Institute of America, Groton, CT 06340, USA
8: Department of Pediatric Oncology, Dana-Farber Cancer Institute and Pediatric Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA.
9: Howard Hughes Medical Institute, Chevy Chase, MD, USA.
* Correspondence: [email protected]
Running title: NRDE2 suppresses intron retention Keywords: Splicing, nuclear RNAi, intron retention, centrosome, mitosis, genome stability
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1 Abstract
2 The accurate inheritance of genetic material is a basic necessity in all domains of life and an
3 unexpectedly large number of RNA processing factors are required for mitotic progression and
4 genome stability. NRDE2 (nuclear RNAi defective-2) is an evolutionarily conserved protein
5 originally discovered for its role in nuclear RNA interference (RNAi) and heritable gene silencing
6 in Caenorhabditis elegans (C. elegans). The function of the human NRDE2 gene remains poorly
7 understood. Here we show that human NRDE2 is an essential protein required for suppressing
8 intron retention in a subset of pre-mRNAs containing short, GC-rich introns with relatively weak
9 5’ and 3’ splice sites. NRDE2 preferentially interacts with components of the U5 small nuclear
10 ribonucleoprotein (snRNP), the exon junction complex, and the RNA exosome. Interestingly,
11 NRDE2-depleted cells exhibit greatly increased levels of genomic instability and DNA damage,
12 as well as defects in centrosome maturation and mitotic progression. We identify the essential
13 centriolar satellite protein, CEP131, as a direct NRDE2-regulated target. NRDE2 specifically
14 binds to and promotes the efficient splicing of CEP131 pre-mRNA, and depleting NRDE2
15 dramatically reduces CEP131 protein expression, contributing to impaired recruitment of critical
16 centrosomal proteins (e.g. γ-tubulin and Aurora Kinase A) to the spindle poles during mitosis.
17 Our work establishes a conserved role for human NRDE2 in RNA splicing, characterizes the
18 severe genomic instability phenotypes observed upon loss of NRDE2, and highlights the direct
19 regulation of CEP131 splicing as one of multiple mechanisms through which such phenotypes
20 might be explained.
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27 Introduction
28 The NRDE2 gene was initially discovered in C. elegans for its role in nuclear RNAi, i.e.
29 the small RNA-directed silencing of nuclear transcripts (Guang et al. 2010; Burton et al. 2011).
30 Consistent with a co-transcriptional mechanism of nuclear RNAi (Guang et al. 2010), recent
31 studies have implicated intriguing physical and functional links between nuclear RNAi factors
32 and components of the splicing machinery (Dumesic et al. 2013; Aronica et al. 2015; Akay et al.
33 2017). Indeed, the NRDE2 homolog in S. pombe, Nrl1, was found to associate with RNA splicing
34 and decay factors to regulate cryptic intronic sequences (Lee et al. 2013; Zhou et al. 2015;
35 Aronica et al. 2015). Nrl1 has also been linked to genomic instability, possibly through the
36 regulation of R-loop accumulation (Aronica et al. 2015). The function of human NRDE2 remains
37 poorly understood.
38 Results and Discussion
39 Here we report the functional and biochemical characterization of the human NRDE2
40 (C14ORF102) gene. Unlike its homologs in nematodes and fission yeast (Guang et al. 2010;
41 Aronica et al. 2015), we demonstrate that NRDE2 is an essential gene in human cells. Depletion
42 of NRDE2 resulted in a complete arrest in cell growth and proliferation in all cell lines tested
43 (Fig. 1A). si-NRDE2 specificity was confirmed by the efficient knockdown of NRDE2 mRNA
44 and protein, and by the rescue of proliferation in cells carrying a NRDE2 overexpression
45 construct (Supplemental Fig. 1A-C). Following NRDE2 depletion, FACS analysis revealed an
46 accumulation of cells with 4N DNA content, indicative of an increase in the number of cells in
47 G2 or mitosis (Fig. 1B). Cyclin B1 and phosphorylated histone H3(Ser10) – markers upregulated
48 in late G2 and early mitosis – were also increased (Fig. 1C), further indicating defective cell cycle
49 progression. To investigate the nature and extent of the mitotic delay in individual cells, we
50 performed live cell imaging using RPE-1 (retinal pigment epithelial) cells expressing H2B-GFP
51 (for chromatin visualization) and α-tubulin-mCherry (for mitotic spindle visualization); while
52 50/51 NRDE2-depleted cells examined did complete mitosis, we observed a ~3-fold increase in
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53 mitotic time, with the majority of the delay occurring during prometaphase (Fig. 1D and
54 Supplemental Movies).
55 Prometaphase delays are often associated with chromosome missegregation, which can
56 result in micronuclei formation. Micronuclei are clinically relevant markers of genomic
57 instability, and have also been implicated as major sources of DNA damage (Zhang et al. 2015).
58 Indeed, NRDE2-depleted HEK293T cells displayed an increased mitotic index, followed by an
59 increase of cells containing micronuclei and γH2AX foci (a marker of double-stranded DNA
60 breaks), as well as activation of p53 (Supplemental Fig. 1D-F). In MDA-MB-231 breast cancer
61 cells, which are mutant for p53, NRDE2 depletion resulted in a similar, gradual accumulation of
62 DNA damage along with a broad range of aberrant nuclear morphologies characteristic of mitotic
63 defects (Fig. 1E-F). Taken together with recent reports identifying NRDE2 as one of ~1,600 core
64 fitness genes in the human genome (Blomen et al. 2015; Hart et al. 2015), we conclude that
65 NRDE2 plays an essential role in ensuring genomic stability and mitotic progression in most, if
66 not all, human cells.
67 NRDE2 features a conserved stretch of ~350 amino acids defined as the NRDE-2
68 domain, a nuclear localization sequence, and five HAT (half-a-TPR) repeats, short helical motifs
69 found in a variety of RNA processing factors (Hammani et al. 2012) (Supplemental Fig. 2A).
70 While multiple studies have found that RNA processing factors are the most enriched functional
71 category of genes required for mitosis and genome stability (Goshima et al. 2007; Paulsen et al.
72 2009; Neumann et al. 2010), to our knowledge NRDE2 has eluded the “hits” list of all such
73 screens, possibly because of the relatively lengthy time to phenotype seen here (Supplemental
74 Fig. 1E). To begin characterizing the molecular function of NRDE2, we examined the NRDE2
75 protein interactome by immunoprecipitation-mass spectrometry (IP-MS) in HEK293T cells stably
76 expressing NRDE2-GFP. Interestingly, NRDE2 interacted almost exclusively with other RNA
77 processing factors (Fig. 2A, Supplemental Fig. 2B,C Supplemental Table 1). Proteins co-
78 purifying with NRDE2 included known components of the RNA exosome (e.g. EXOSC10 and
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79 SKIV2L2) (Lubas et al. 2011), the U5 small nuclear ribonucleoprotein (snRNP) (e.g. EFTUD2)
80 (Fabrizio et al. 1997), and the exon junction complex (EJC) (e.g. eIF4A3) (Singh et al. 2012)
81 (Fig. 2A). SKIV2L2, the most abundant NRDE2-interacting protein we detected, is a conserved
82 DExH/D box RNA helicase component of the human nuclear exosome targeting (NEXT)
83 complex (Lubas et al. 2011); notably, previous proteomic analyses of human SKIV2L2, and its
84 homolog in S. pombe, have both revealed specific associations with NRDE2 (Ogami et al. 2017;
85 Lee et al. 2013).
86 NRDE2 interactions with SKIV2L2, EXOSC10, EFTUD2 and EIF4A3 were confirmed
87 by co-immunoprecipitation (Fig. 2B), and further supported by indirect immunofluorescence
88 (Fig. 2C). Like many splicing factors (Spector and Lamond 2011), the subcellular localization of
89 NRDE2 was nucleoplasmic, reminiscent of nuclear speckles, largely excluded from nucleoli, and
90 diffuse throughout mitotic cells (Fig. 2C). Endogenously tagged zsGreen-NRDE2 exhibited a
91 similar expression pattern (Supplemental Fig. 3A), and endogenously tagged 3XFLAG-NRDE2
92 also co-precipitated with SKIV2L2 (Supplemental Fig. 3B). RNA immunoprecipitation (RIP) of
93 NRDE2 followed by qPCR revealed an enrichment of NRDE2-bound spliceosomal RNAs,
94 particularly the U5 small nuclear RNA (snRNA), with no detectable binding of abundant small
95 nucleolar RNAs (snoRNAs) (Fig. 2D). To our knowledge, NRDE2 has not been previously
96 detected in human spliceosome purifications. Our data demonstrating the association of NRDE2
97 with components of the RNA exosome and the U5 snRNP indicate that NRDE2 may play
98 conserved roles in regulating RNA splicing and/or stability in human cells.
99 A number of reports have suggested that the mechanisms linking various splicing factor
100 knockdowns to mitotic defects converged on the inefficient splicing of a single intron in the
101 CDCA5 gene, which regulates sister chromatid cohesion (Oka et al. 2014; Watrin et al. 2014;
102 Lelij et al. 2014). We thus performed RNA sequencing (RNA-seq) in NRDE2-depleted cells 48h
103 post-transfection, a time point prior to the appearance of mitotic defects and DNA damage. We
104 examined differential gene expression as well as intron retention (IR) events; we identified 84
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105 genes deregulated by greater than two-fold in NRDE2-depleted cells (Supplemental Table 2).
106 This set was not enriched for any biological process or molecular function by GO term analysis.
107 A panel of these genes were validated by qPCR using an additional NRDE2 siRNA, at a different
108 time point (96h), and in a different (RPE-1) cell line (Supplemental Fig. 4).
109 To investigate intron retention (IR) events, we used the recently refined IRFinder
110 algorithm (Middleton et al. 2017) to assess IR levels in 234,396 introns in the human genome.
111 Our analysis identified 2,172 IR events in MDA-MB-231 cells, of which 178 (8%) were
112 differentially regulated upon NRDE2-depletion. 163 (92%) of these introns showed increased
113 retention (Fig. 3A, Supplemental Fig. 5A, and Supplemental Table 3), indicating that NRDE2 is
114 generally required to suppress IR, i.e. promote splicing. We next surveyed the sequence
115 properties of these retained introns. The 2,172 retained introns in MDA-MB-231 cells were
116 significantly shorter, more GC-rich, and contained weaker 5’ and 3’ splice sites (ss) compared to
117 global intron averages, properties consistent with previous reports examining IR-prone introns
118 (Braunschweig et al. 2014) (Fig. 3B). Intriguingly, these properties were even more pronounced
119 in NRDE2-regulated introns: NRDE2-suppressed IR events (purple boxes) occurred in introns that
120 were shorter, more GC-rich, and contained weaker 5’ and 3’ ss even when compared to IR-prone
121 introns (Fig. 3B).
122 We do note that our ability to detect IR events from the RNA-seq data is not saturated.
123 For instance, an alternative IR analysis in SeqMonk identified 47 affected IR events following
124 NRDE2-depletion, many of which were not detected by IRFinder (Supplemental Table 4 and
125 Supplemental Fig. 5A). While part of this discrepancy is due to different intron annotations in
126 different programs, it is almost certain that increasing RNA-seq read depths will reveal additional
127 NRDE2-dependent IR events not detected in this study. Nonetheless, properties of NRDE2-
128 regulated introns remained consistent: 43 of 47 (91%) introns exhibited increased retention
129 following NRDE2 depletion, again indicating that NRDE2 generally suppresses IR. Moreover,
130 these introns were also significantly more GC-rich and contained weaker 5’ ss compared to global
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131 intron averages (Supplemental Fig. 5B). Global intron levels were not measurably affected by
132 NRDE2 depletion (Supplemental Fig. 5C). Thus, unlike many core splicing factors, together these
133 data suggest that NRDE2 is only required for the efficient splicing of a small subset of
134 particularly weak introns.
135 Intron-retaining transcripts are susceptible to degradation via nonsense-mediated decay
136 (NMD) (Ge and Porse 2014), representing a potential mechanism of post-transcriptional gene
137 regulation. Strikingly, in NRDE2-depleted cells, we found that genes identified by IRFinder
138 exhibiting increased IR were predominantly downregulated, whereas genes exhibiting decreased
139 IR were predominantly upregulated (Fig. 3C). Furthermore, GO term analyses revealed that the
140 163 IR events suppressed by NRDE2 (which occurred in 138 unique genes) were enriched in
141 chromosome components (Bonferroni-corrected p=0.02), and included the histone
142 acetyltransferase KAT2A, and the condensin II complex subunit NCAPG2, whose mRNA
143 expression based on RNA-seq analysis were reduced by ~1.6 and ~1.7-fold, respectively
144 (Supplemental Tables 2 and 3). These data not only suggest that dysregulated intron retention
145 contributes to the gene expression changes in NRDE2-depleted cells, but also that these changes
146 may cumulatively be an important source of genomic instability.
147 We used qPCR to confirm IR events in a number of genes previously implicated in
148 directly promoting accurate chromosome segregation and mitotic progression, including CEP131
149 (Staples et al. 2012), TUBGCP2 (Neben et al. 2004), and KIF2C (Andrews et al. 2004) (Fig. 4A).
150 We sought to further investigate the role of NRDE2 in regulating CEP131 (Centrosomal Protein
151 131) expression, as both of our IR analyses identified CEP131 as a major NRDE2-regulated
152 transcript. CEP131 was also one of only four genes affected by an IR event identified by
153 IRFinder that increased >2-fold and resulted in an IR ratio > 0.5 in NRDE2-depleted cells (Fig.
154 4B). The IR ratio is defined as the ratio of transcripts retaining a particular intron to the sum of
155 transcripts of a given gene. Moreover, CEP131 had previously been identified as an essential
156 centriolar satellite factor and regulator of genome stability (Staples et al. 2012). The IR event
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157 suppressed by NRDE2 occurs within the 16th intron of CEP131, where loss of NRDE2 resulted in
158 intronic RNA levels to nearly those of flanking exons (Fig. 4C). Critically, RIP-qPCR revealed
159 that NRDE2 preferentially bound to unspliced CEP131 transcripts (Fig. 4D). As the retained
160 intron in CEP131 contained multiple in-frame stop codons (Supplemental Fig. 6), we expected
161 NRDE2 depletion to reduce CEP131 protein levels. Indeed, a striking loss of CEP131 protein was
162 observed in NRDE2-depleted HEK293T and MDA-MB-231 cells (Fig. 4E, Supplemental Fig.
163 7A,B). The expression of CEP68, another centrosome factor, was unaffected (Supplemental Fig.
164 7B). Taken together, we conclude that NRDE2 is a major regulator of CEP131 expression, likely
165 through its role in suppressing IR in CEP131 pre-mRNA.
166 Interestingly, unlike most other genes exhibiting increased IR, our RNA-seq data
167 indicated that CEP131 transcripts were upregulated ~1.5-fold in NRDE2-depleted cells
168 (Supplemental Table 2). We hypothesized that, given the interactions between NRDE2 and
169 nuclear RNA exosome components, NRDE2 may couple the regulation of RNA splicing and
170 decay of certain transcripts. We thus tested the effect of NRDE2 on CEP131 pre-mRNA stability.
171 Consistent with a role for NRDE2 in promoting pre-mRNA decay, unspliced CEP131 transcripts
172 were significantly stabilized relative to actin mRNA in NRDE2-depleted cells (Supplemental Fig.
173 7C). These data suggest that the increase in unspliced CEP131 transcripts may be due to
174 contributions from both reduced splicing and a reduced rate of RNA decay. Nonetheless, by four
175 days post-NRDE2 depletion, overall CEP131 RNA was reduced to ~60% that of control-treated
176 cells (Supplemental Fig. 7D), consistent with the eventual decay of aberrantly spliced transcripts
177 and the concomitant reduction in CEP131 protein.
178 Centriolar satellites are thought to regulate protein trafficking to the centrosomes
179 (Tollenaere et al. 2015). Centrosomes are large proteinaceous complexes critical for spindle
180 microtubule organization, whose activity and duplication must be tightly regulated to maintain
181 genome stability (Nigg and Stearns 2011). We tested whether the reduction of CEP131 protein
182 following NRDE2 depletion was sufficient to cause defects in mitosis. Indeed, siRNA-mediated
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183 depletion of CEP131, which reduced CEP131 protein to levels comparable to those following si-
184 NRDE2 treatment (Supplemental Fig. 8A), significantly impaired the centrosomal recruitment of
185 Aurora Kinase A (AURKA), a major mitotic kinase required for centrosome maturation
186 (Hochegger et al. 2013) (Supplemental Fig. 8B). CEP131 depletion also resulted in the loss of
187 cell proliferation (data not shown) and a range of aberrant nuclear morphologies remarkably
188 similar to those induced by NRDE2 depletion (Supplemental Fig. 8C). These results support
189 previous findings that CEP131 is essential for centrosome maturation and genome stability
190 (Staples et al. 2012; Li et al. 2017), and indicate that impaired CEP131 expression was sufficient
191 to partially explain the genomic instability seen upon NRDE2 depletion. However, ectopic
192 CEP131 cDNA expression failed to rescue a range of NRDE2 knockdown phenotypes
193 (Supplemental Fig. 9), suggesting that dysregulation of additional genes, at least in part through
194 IR-dependent mechanisms, cumulatively contribute to the genomic instability following loss of
195 NRDE2.
196 Nonetheless, six additional lines of evidence suggest that the impaired function of
197 centrosomes, which organize and nucleate spindle microtubules to ensure accurate chromosome
198 segregation, underlie the requirement for NRDE2 in proper mitotic progression. First, we
199 confirmed that the centrosomal localization of CEP131 was also reduced in NRDE2-depleted
200 mitotic cells (Fig. 5A). Second, we confirmed that the centrosomal localization of both active
201 AURKA (Fig. 5B) and total AURKA (Supplemental Fig. 10A) was similarly reduced. This was a
202 specific centrosomal recruitment defect, as overall AURKA expression was in fact increased
203 (Supplemental Fig. 10B,C), consistent with AURKA levels peaking during mitosis (Hochegger et
204 al. 2013) and an increased fraction of cells in mitosis following NRDE2 depletion. Third, we
205 observed that centrosomal γ-tubulin, which directly mediates microtubule nucleation and is
206 recruited to centrosomes by AURKA (Oakley et al. 2015; Hochegger et al. 2013), was also
207 significantly reduced – most notably in mitotic cells (Fig. 5C) – further indicating impaired
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208 centrosome maturation. A moderate reduction in total γ-tubulin expression was also observed
209 (Supplemental Fig. 10D), possibly exacerbating centrosomal recruitment defects.
210 Fourth, we found that CEP192 (Centrosomal protein 192), which is required for both γ-
211 tubulin and AURKA recruitment, and therefore central to centrosome function (Gomez-Ferreria
212 et al. 2007; Joukov et al. 2014), was not only reduced at spindle poles but also formed additional,
213 non-microtubule-nucleating foci (Fig. 5D), suggesting impaired centrosome integrity and
214 mislocalization/aggregation of certain centrosome proteins. Interestingly, through mechanisms
215 that remain unclear, total CEP192 protein was also considerably reduced, without a major effect
216 on CEP192 mRNA levels (Supplemental Fig. 10E,F). Reduced CEP192 expression is known to
217 cause a complete loss of centrosome function (Gomez-Ferreria et al. 2007), and thus likely plays
218 a key role (in addition to the loss of CEP131) in contributing to mitotic defects in NRDE2-
219 depleted cells. Fifth, analysis of the centriole marker centrin-2 revealed that while centriole
220 duplication was largely unaffected by NRDE2 depletion, a significantly increased number of
221 mitotic cells displayed prematurely disengaged centrioles (Fig. 5E), providing further evidence
222 for impaired centrosome integrity. Finally, consistent with multiple centrosome defects,
223 frequencies of aberrant mitotic spindles were significantly increased following NRDE2 depletion
224 (Supplemental Fig. 10G). We conclude that NRDE2 is required for the function of centrosomes
225 during mitosis.
226 Lastly, we tested a possible non-mitotic mechanism, the impaired processing of R-loops, that
227 might also contribute to NRDE2-dependent genome maintenance. R-loops are RNA-DNA
228 hybrids known to threaten genomic stability, and have been shown to be regulated by the NRDE2
229 homolog in fission yeast (Lee et al. 2013; Aronica et al. 2015). However, we did not observe any
230 significant increases in R-loop accumulation upon NRDE2 depletion in human cells
231 (Supplemental Fig. 11). These data are in agreement with a recent study (Richard et al. 2018),
232 which also concluded that the role of human NRDE2 in the DNA damage response was likely
233 independent of R-loop processing.
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234 Taken together, our work identifies NRDE2 as a novel human U5 snRNP-associated splicing
235 factor required for centrosome function and mitotic progression. Further, NRDE2 is directly
236 required for the efficient splicing of a single intron in an essential centrosome gene (CEP131).
237 However, there is no reason to believe that NRDE2 is a specific regulator of mitosis. Our data
238 simply positions NRDE2 as a peripheral splicing factor required for suppressing IR in a subset of
239 pre-mRNAs, particularly those with short, weak, GC-rich introns. The fact that NRDE2-regulated
240 IR events happen to occur in mitotic genes including CEP131, TUBGCP2, and KIF2C should
241 nonetheless provide a partial mechanistic explanation for the mitotic defects observed following
242 reduced NRDE2 expression (Supplemental Fig. 12). How might NRDE2 selectively regulate
243 specific introns? It is interesting that our IP-MS data uncovered ACIN1 as a NRDE2-interacting
244 protein (Fig. 2A). ACIN1 is a peripheral EJC component that has been shown to preferentially
245 bind short, GC-rich introns with weak splice signals (Rodor et al. 2016) – precisely the types of
246 introns retained following NRDE2 depletion. It will be interesting to determine whether the intron
247 specificity of NRDE2 is intrinsic or mediated by its protein partners. Moreover, it was recently
248 reported that the preferential killing of spliceosome-mutant cancer cells could be mediated by a
249 small molecule that induced the retention of short, GC-rich introns (Seiler et al. 2018). Whether
250 certain populations of low NRDE2-expressing cells show specific sensitivities to spliceosome
251 and/or mitotic inhibitors may also be an interesting area of future investigation.
252
253 Materials and Methods
254 Cell culture and siRNA transfections
255 HEK293T, MDA-MB-231, H23, Hela, and RPE-1 cells were obtained from the American Type
o 256 Culture Collection (ATCC), grown at 37 C in a 5% CO2 atmosphere, and passaged according to
257 standard procedures, in 10% FBS and 1% Penicillin/Streptavidin. All siRNA transfections were
258 performed at 20nM concentration, with Lipofectamine RNAiMAX Transfection Reagent
259 (Thermo Fisher) according to the manufacturer’s protocol. siRNAs used were: si-Control
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260 (Ambion AM4611), si-NRDE2-1 (Ambion Silencer Select s30063, 5’
261 GGUGUUGUUUGAUGAUAUUTT 3’), si-NRDE2-2 (Ambion Silencer Select s30064, 5’
262 GUUUAGUACCUUUUCGAUATT 3’), si-CEP131 (AZI1) (Ambion Silencer AM16708 5’
263 GCUGAUUGCAAGGCACAAGTT 3’)
264
265 Cell proliferation
266 Cell proliferation was determined by MTT assays according to the manufacturer’s protocol
267 (Thermo Fisher). Transfected cells were seeded in 96-well plates 48h (or 24h for MDA-MB-231)
268 post-transfection at ~1,000 cells/well. The following day was considered Day 1 of the MTT
269 assay, to which all A560 readings were normalized.
270
271 RNA extraction, reverse transcription and quantitative PCR
272 Cells were collected in Trizol (Invitrogen) and total RNA was isolated with Direct-zol RNA
273 Miniprep Plus spin columns (Zymo Research) according to the manufacturer’s protocol,
274 including the on-column Dnase I treatment. Total cDNA synthesis was performed on 50ng-1ug
275 total RNA using Superscript IV Reverse Transcriptase (Thermo Fisher Scientific), with random
276 hexamers. cDNA was diluted a minimum of 1:2 before expression analysis by qPCR with SYBR
277 Green I Master Mix (Roche) in a Roche LightCycler 480 II (qPCR program: 95oC for 5 minutes,
278 followed by 45 cycles of [95oC for 10sec, 54 oC for 10sec, 72 oC for 10sec], followed by a melting
279 curve analysis). Fold changes were calculated using the 2-ΔΔCT method.
280
281 For small nuclear / nucleolar RNAs: Total RNA was collected as above. Polyadenylation-coupled
282 reverse transcription was performed in a standard 20uL Superscript IV reverse transcription
283 reactions, but in 0.5x Superscript IV buffer and with the following additions: 1uL poly(A) buffer
284 (NEB #B0276S), 1uL poly(A) polymerase (NEB # M0276S), 1mM ATP, and 2.5uM anchored
285 oligod(T) primer (5’-GCTGTCAACGATACGCTACGTAACGGCATGACAGTGTTTTTTTTTTTTTTTTTVN-3’).
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286 Samples were incubated at 37oC for 1h, then 85oC for 8 minutes before placing on ice prior to
287 qPCR. qPCRs were run using gene-specific forward primers and a universal reverse primer. All
288 primers were used at a final concentration of 5uM each, and their sequences are listed in
289 Supplemental Table 4.
290 RNA-seq
291 Triplicate RNA samples from si-Control and si-NRDE2-2-treated MDA-MB-231 cells were
292 collected 48h post-transfection using the mirVana RNA isolation kit (Thermo Fisher). Libraries
293 were synthesized using Illumina TruSeq Stranded mRNA sample preparation kits from 500ng of
294 purified total RNA according to the manufacturer’s protocol. The final dsDNA libraries were
295 quantified by Qubit fluorometer, Agilent TapeStation 2200, and RT-qPCR using the Kapa
296 Biosystems library quantification kit according to manufacturer’s protocols. Uniquely indexed
297 libraries were pooled in equimolar ratios and sequenced on an Illumina NextSeq500 with single-
298 end 75bp reads by the Dana-Farber Cancer Institute Molecular Biology Core Facilities. The total
299 read depths for the three control-treated replicates were 61.4M, 63.9M, and 56.1M. The total read
300 depths for the three si-NRDE2-treated replicates were 57.8M, 58.9M, and 46.4M. Sequenced
301 reads were aligned to the hg19 reference genome assembly; >90% of reads were mappable in all
302 samples. Gene counts were quantified using STAR (v2.5.1b) and differential expression testing
303 was performed by DESeq2 (v1.10.1) as part of the VIPER analysis pipeline
304 (https://bitbucket.org/cfce/viper/).
305
306 Intron analysis (IRFinder)
307 Intron retention analysis was performed using the IRFinder tool (version 1.2.3) (Middleton et al.
308 2017) based on the gene annotations for the hg19 reference genome, using the RNA-seq data
309 obtained from triplicate si-Control and si-NRDE2-2-treated cells. All potential introns
310 (n=234,396) were tested, and introns were defined as any region between two exon features in
311 any transcript. The exon coordinates were derived from reference gene annotation files. The
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312 Audic and Claverie test for small numbers of replicates was applied to the quantified intron
313 retention events to determine differential expression between control and NRDE2 knockdown
314 conditions. Significant intron retention events were defined as previously reported (Middleton et
315 al. 2017), according to these criteria: Benjamini-Hochberg-adjusted p < 0.05; IRratio in either
316 control or NRDE2 knockdown > 0.1 (i.e. at least 10% of transcripts exhibit retention of the intron
317 for at least one of the conditions), and retained introns exhibit a coverage (i.e. intron depth) of at
318 least 3 reads after excluding non-measurable intronic regions. Additional details are described in
319 Supplemental Table 3.
320
321 Intron analysis (SeqMonk)
322 Differential expression analysis of 315,405 intronic probes pre-defined in SeqMonk (Babraham
323 Bioinformatics) was performed with DESeq2 on si-Control and si-NRDE2-treated triplicate
324 RNA-seq samples. Introns that passed all three filters (p < 0.01, an average read count difference
325 of >100, and a minimum 3-fold change), were considered significantly deregulated. Removal of
326 seven probes due to overlap with alternative exons resulted in the identification of the 47
327 deregulated introns within 45 different genes shown in Supplemental Table 4. Average read
328 counts from flanking exons, also analyzed by DESeq2 in SeqMonk, were used to calculate intron
329 levels relative to flanking exon expression.
330
331 Flow cytometry
332 For cell cycle analysis, cells were harvested 5 days post-transfection, washed once in PBS, and
333 fixed in 75% ice cold ethanol for >30 minutes. Cells were then washed once in PBS and stained
334 in 10ug/mL Hoechst 33342 (Thermo Fisher) in PBS. DNA content analyzed by flow cytometry
335 (BD Biosciences FACSCalibur). At least 10,000 events were collected per sample.
336
337 Lentiviral production and infection
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338 Generation 2 lentiviral constructs (0.75ug of pMD2.G (Addgene plasmid 12259) and 0.75ug
339 psPAX2) (Addgene plasmid 12260) and 1.5ug of either NRDE2-mGFP expression plasmid
340 (Origene RC224847L2) or a dox-inducible 3xFLAG-CEP131 construct (a kind gift from L. Shi,
341 Tianjin Medical University), were transfected into HEK293T cells in 6-well plates with
342 Lipofectamine 3000. Media was changed after 6h and viral supernatant was collected 36h-48h
343 later, 0.2um filter sterilized and frozen at -80oC. For NRDE2 expression, HEK293T and MDA-
344 MB-231 cells were infected with either control GFP (Origene TR30021V) or NRDE2-GFP
345 lentiviral supernatant. GFP-positive cells were sorted by flow cytometry ~1 week post-infection.
346 For generating stable dox-inducible 3XFLAG-CEP131 expressing lines, infected MDA-MB-231
347 cells were selected and maintained in 1.5ug/mL of Puromycin.
348
349
350 Immunofluorescence and confocal microscopy
351 Antibodies used: γH2AX, Millipore 05-636, 1:500; phospho-H3(Ser10), Cell Signaling
352 Technology #9701, 1:500; α-tubulin, Cell Signaling Technology #3873, 1:2000; 1:500; γ-tubulin,
353 Abcam ab11317, 1:500; Aurora A, Cell Signaling Technology #14475, 1:200; phospho-Aurora
354 A/B/C, Cell Signaling Technology #2914, 1:300; CEP-131, Proteintech 25735-1-AP, 1:300;
355 SKIV2L2, Proteintech 12719-2-AP, 1:600; EXOSC10, Proteintech 11178-1-AP, 1:300;
356 EFTUD2, Proteintech 10208-1-AP, 1:300; eIF4A3, Proteintech 17504-1-AP, 1:300; CEP192,
357 Proteintech 18832-1-AP, 1:300; Centrin-2, Millipore 04-1624, 1:300; S9.6, Kerafast ENH001,
358 1:100.
359
360 Cells were grown on coverslips coated with 0.1% gelatin (Sigma G-9382) in 6-well plates, fixed
361 in chilled 100% methanol for 7 minutes at -20oC (unless otherwise indicated), and washed 3x in
362 PBS. Fixed cells were blocked in 5% BSA (Sigma) in TBST for 1-2h at room temperature and
363 incubated in primary antibodies in 2.5% BSA in TBST overnight at 4oC. The following day, cells
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Jiao et al.
364 were washed 3x in PBS, incubated in Alexa Fluor 488/Alexa Fluor 555/Alexa Fluor 647
365 (Molecular Probes) conjugated secondary antibodies (1:1000 in 2% BSA in TBST) for 1h at room
366 temperature in the dark, washed 3x in PBS, and mounted onto microscope slides with ProLong
367 Diamond Antifade with DAPI (Life Technologies). For fluorescence microscopy, slides were
368 visualized using a Zeiss Axio Observer.Z1 under a Plan-Apochromat 63x / 1.40 Oil DIC
369 objective. Fluorescent images were captured using a Hamamatsu Camera, and processed using
370 the ZEN 2 Blue Edition Software (2011). For analysis of MDA-MB-231 nuclear morphologies
371 and quantification of HEK293T H3S10P-positive mitotic cells, slides were imaged with a
372 monochrome camera on an EVOS FL Imaging System under a 20x fixed objective. For confocal
373 analyses, cells were examined under a LSM 880 Laser Scanning Microscope.
374
375 Live cell imaging
376 RPE-1 cells stably expressing H2B-GFP and α-tubulin-mCherry were seeded in black 24-well
377 plates (ibidi #82406) 48h following siRNA transfections. Two days later, beginning at 96h post-
378 transfection, plates were mounted on a Nikon Ti-E inverted microscope equipped with the Nikon
379 Perfect Focus system, and cells were imaged through a 20x 0.75 NA Plan Apo objective using a
380 Zyla 4.2 sCMOS camera (Andor). Imaging was performed in an environmentally controlled
381 chamber maintained at 37ºC with humidified 5% CO2. Z-stacks (8-um volume at 2-um spacing)
382 were collected at 10-minute intervals over a total of 48h. Mitotic duration was visually scored
383 from the recorded videos using MetaMorph (Molecular Devices), beginning with the first sign of
384 chromosome condensation and ending with decondensed chromosomes in daughter nuclei.
385
386 Western blotting
387 Antibodies used: Monoclonal ANTI-FLAG, M2 (Sigma, F1804); β-actin (Santa Cruz, sc-47778);
388 Cyclin B1 (Cell Signaling Technology #12231); phospho-p53(Ser15) (Cell Signaling Technology
389 #9286); GFP (Abcam, ab290); CEP68 (Proteintech, 15147-1-AP); Antibodies for phospho-
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390 H2AX(Ser139), phospho-H3(Ser10), γ-tubulin, CEP-131, CEP192, Aurora A, SKIV2L2,
391 EFTUD2, eIF4A3, and EXOSC10 are the same ones used for immunofluorescence (above).
392 Cell pellets were resuspended in RIPA buffer, rotated at 4oC for 30 minutes, and centrifuged at
393 13,000rpm for 20 minutes. Laemmli buffer was added to the supernatant and protein samples
394 were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Blots were
395 blocked in 5% milk or BSA (for phospho-proteins), and incubated in primary antibodies
396 overnight at 4oC at 1:2000 dilutions, except for β-actin (1:7500) and DNAPKcs(S2056P)
397 (1:1000). Blots were then washed 3x in TBST, incubated in HRP-conjugated secondary
398 antibodies (Santa Cruz) (~1:10000) for 1hr at room temperature, washed 3x in TBST, and
399 detected by enhanced chemiluminescence (BioRad).
400
401 Protein / RNA immunoprecipitation
402 HEK293T, HEK293T(3xFLAG-NRDE2), and HEK293T(NRDE2-GFP) cells were grown to
403 confluency in 10cm dishes, washed once in cold PBS, and lysed on ice for 5 minutes in 1x non-
404 denaturing lysis buffer (Cell Signaling Technology #9803) with freshly added HALT protease
405 inhibitor (Thermo Fisher #78430). Lysates were centrifuged at 14,000g for 10 minutes and
406 precleared with Protein G Dynabeads (Thermo Fisher #10003D) for 1h at 4oC. 5uL of anti-FLAG
407 M2 (Sigma F1804) or 1uL of anti-GFP (Abcam ab290) was added per IP (equivalent of one 10cm
408 dish) and rotated overnight at 4oC. The next day, protein G Dynabeads were added and rotated for
409 2h at 4oC. Beads were washed five times in lysis buffer for 5-10 minutes each at 4oC. Protein was
410 eluted in Laemmli sample buffer (Bio-Rad) with 10% 2-mercaptoethanol and subjected to
411 analysis by either Western blotting (see above) or mass spectrometry. For RNA
412 immunoprecipitation, the same procedure was used except all incubations were performed in the
413 presence of Superase Inhibitor (Invitrogen AM2694), and RNA was eluted from beads with
414 Trizol reagent and isolated with Direct-zol RNA Miniprep Plus spin columns (Zymo Research).
415
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416 Tagging the endogenous NRDE2 locus in HEK293T cells using CRISPR/Cas9 technology
417 To insert a 3xFlag tag at the N-terminus of the NRDE2 gene, we used a CRISPR/Cas9
418 recombination strategy as described (Cong et al. 2013; Ran et al. 2013). In short, a guide RNA
419 targeting the 5’end of NRDE2 with the sequence 5’-AGCCTCACTAAGCCCCGCAA-3’ was
420 cloned into the pX458 vector (Addgene, plasmid #48138) following the protocol described here
421 (http://www.genome-engineering.org/crispr/wp-content/uploads/2014/05/CRISPR-Reagent-
422 Description-Rev20140509.pdf). The pX458 vector expresses a Cas-9-GFP fusion protein that can
423 be used as a selection marker. The primers used for the guide RNA cloning were 5’-
424 CACCGAGCCTCACTAAGCCCCGCAA-3’ for the forward primer and 5’-
425 AAACTTGCGGGGCTTAGTGAGGCTC-3’ for the reverse. A repair template that included 1kb
426 upstream of NRDE2 ATG, the in-frame N-terminal 3xFlag tag, and 1kb downstream of the ATG
427 was amplified using standard PCR methods. Then, the repair template was cloned into the HindIII
428 and EcoRV sites of a pUC19 vector. To improve the chances of recombination, a silent mutation
429 was introduced at the PAM sequence in our repair template (from GGG to GGA). Both vectors
430 were transfected into HEK293T cells at 70% confluency using Lipofectamine 2000 (Thermo
431 Fisher) following the company’s protocol for transfections in 24-well plates. The total amount of
432 DNA transfected into cells was 1ug (500ng each vector). After transfection, cells were incubated
433 at 37ºC, 5%CO2 for 48h in DMEM media supplemented with 10% FBS and 100U/mL penicillin,
434 and 100ug/mL streptomycin. Cells were then FACS sorted for GFP-positive cells into two 96-
435 well plates with a single cell per well. After two weeks, clones that grew colonies were then
436 genotyped by PCR for the presence of the 3xFlag sequence. Positive clones were subjected to
437 WB to confirm proper 3xFlag-NRDE2 fusion protein expression.
438 To insert the zsGreen tag at the N-terminus of NRDE2, we followed a similar approach,
439 except the gRNA was cloned into the pX330 vector (Addgene, plasmid #42230), which has no
440 selectable marker. The repair template also had 1kb upstream of ATG, in-frame zsGreen tag, and
441 1kb downstream of the ATG with the silent mutation in the PAM sequence and cloned into
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442 pUC19 as described above. HEK293T cells at 70% confluency were transfected with 1ug (500ng
443 each vector) total DNA using Lipofectamine 2000 in 24-well plates. After transfection, cells were
444 incubated at 37ºC, 5% CO2 for 48h in DMEM media supplemented with 10% FBS and 100U/mL
445 penicillin, and 100ug/mL streptomycin. Cells were then FACS sorted for zsGreen positive cells
446 into two 96-well plates with a single cell per well. After two weeks, clones that grew colonies
447 were then genotyped by PCR for the presence of the zsGreen sequence. Expression of zsGreen-
448 NRDE2 was confirmed by fluorescence microscopy. Primers and sequences of 3xFlag and
449 zsGreen repair templates are available upon request.
450
451 Mass spectrometry
452 Immunoprecipitated samples were run on a 10% polyacrylamide gel; Coomassie Blue-stained
453 bands corresponding to ~25kDa (IgG light chain) and ~50kDa (IgG heavy chain) were excised
454 and removed; the remaining gel slices were cut into ~1mm3 pieces and subjected to a modified in-
455 gel trypsin digestion. Gel pieces were washed and dehydrated with acetonitrile for 10 minutes.
456 followed by removal of acetonitrile. Pieces were then completely dried in a speed-vac.
457 Rehydration of the gel pieces was with 50mM ammonium bicarbonate solution containing 5ng/ul
458 modified sequencing-grade trypsin (Promega, Madison, WI) at 4ºC. After 45 minutes, the excess
459 trypsin solution was removed and replaced with 50mM ammonium bicarbonate solution to just
460 cover the gel pieces. Samples were then placed in a 37ºC room overnight. Peptides were later
461 extracted by removing the ammonium bicarbonate solution, followed by one wash with a solution
462 containing 50% acetonitrile and 1% formic acid. The extracts were then dried in a speed-vac (~1
463 hr) and reconstituted in 5–10ul of HPLC solvent A (2.5% acetonitrile, 0.1% formic acid). A
464 nano-scale reverse-phase HPLC capillary column was created by packing 2.6 um C18 spherical
465 silica beads into a fused silica capillary (100 um inner diameter x ~30 cm length) with a flame-
466 drawn tip. After equilibrating the column each sample was loaded via a Famos auto sampler (LC
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467 Packings) onto the column. A gradient was formed and peptides were eluted with increasing
468 concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid).
469 As peptides eluted they were subjected to electrospray ionization and then entered into an LTQ
470 Orbitrap Velos Pro ion-trap mass spectrometer (Thermo Fisher). Peptides were detected,
471 isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each
472 peptide. Peptide sequences (and hence protein identity) were determined by matching protein
473 databases with the acquired fragmentation pattern by the software program, Sequest (Thermo
474 Fisher). All databases include a reversed version of all the sequences and the data was filtered to
475 between a 1-2% peptide false discovery rate.
476
477
478 Author contributions
479 A.L.J., R.P, N.T.U., J.R.H., D.P., S.K., and F.J.S. designed the experiments. A.L.J., R.P., N.T.U.,
480 J.R.H., M.E.P., and B.D.A. performed experiments and analyzed results. A.L.J., R.P., J.R.H.,
481 S.K., and F.J.S. wrote/edited the manuscript.
482 Acknowledgements
483 We thank members of the Slack, Kennedy and Pellman laboratories for insightful discussions,
484 experimental suggestions, and critical reading of the manuscript. We also thank: Zach Herbert
485 and the Dana Farber Molecular Biology Core Facilities for RNA-Seq and gene expression
486 analysis; the Harvard Chan Bioinformatics Core for computational guidance; K. Schukken and
487 the Foijer laboratory (University Medical Center Groningen, Netherlands) for generously sharing
488 cell lines used for live imaging; L. Shi (Tianjin Medical University, China) for generously sharing
489 the inducible CEP131 lentiviral construct; Ross Tomaino and the Taplin Mass Spectrometry
490 Facility for mass spectrometry analyses. N.T.U. is a HHMI fellow of the Damon Runyon Cancer
491 Research Foundation (DRG-2238-15). This work was supported by the National Institute of
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Jiao et al.
492 Health (R01AG033921) to F.J.S., funding from the Ludwig Center at Harvard and a pilot grant
493 from the Harvard Medical School Epigenetics Initiative to F.J.S and S.K.
494
495 Conflicts of Interest
496 The authors declare no conflicts of interest.
497
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614 Tables
615 Supplemental Table 1. This table lists proteins co-purified with NRDE2-GFP in HEK293T cells
616 detected by mass spectrometry following GFP immunoprecipitation (IP). Control IP was
617 performed with the same antibody using HEK293T cells expressing GFP alone. All NRDE2-
618 interacting proteins listed were detected at >10% coverage for at least one replicate. A maximum
619 of one peptide detected in the control sample was allowed.
620
621 Supplemental Table 2. Differential gene expression analysis from RNA-seq data from si-Control
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622 and si-NRDE2-2-treated MDA-MB-231 cells collected 48h post-transfection.
623
624 Supplemental Table 3. Intron retention analysis by IRFinder from RNA-seq data from si-
625 Control and si-NRDE2-2-treated MDA-MB-231 cells collected 48h post-transfection.
626
627 Supplemental Table 4. Differentially retained introns analyzed in Seqmonk from RNA-seq data
628 from si-Control and si-NRDE2-2-treated MDA-MB-231 cells collected 48h post-transfection.
629
630 Supplemental Table 5. Primers used in this study.
631
632
633 Figure Legends
634 Figure 1. NRDE2 is an essential gene required for mitotic progression and genome stability.
635 (a) MTT assays for cell proliferation following transfection with control or NRDE2-targeting
636 siRNAs. 20nM siRNAs were used in all experiments. Error bars = SD (n=3). (b, c)
637 Representative FACS analyses of DNA content (b) and Western blots (c) of MDA-MB-231 cells
638 collected 5 days post-transfection (n=3). (d) Mitotic duration was quantified by time-lapse
639 microscopy at 10min resolution using RPE-1 cells expressing H2B-GFP (for chromatin
640 visualization) and α-tubulin-mCherry (for mitotic spindle visualization) at 5-6 days post-
641 transfection (n=50 cells per condition). See also Supplemental Movies S1-S3. (e) Representative
642 time course analysis of γH2AX levels by Western blot (n=3). (f) Categories and quantification of
643 aberrant nuclear morphologies visualized by DAPI 4 days post si-NRDE2 transfection in MDA-
644 MB-231 cells. “Multiple” refers to nuclear abnormalities falling under multiple categories, the
645 most common being polylobed + micronuclei. White: DNA. Green: α-tubulin. >300 cells were
646 scored per condition per replicate (n=3). Bar=10um. * p < 0.05, two-tailed t-test.
647
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648
649 Figure 2. NRDE2 associates with the RNA exosome, the EJC, and the U5 snRNP. (a)
650 Selected proteins co-purifying with NRDE2-GFP. Known components of the RNA exosome, the
651 EJC, and the U5 snRNP detected with >10% coverage in at least one IP replicate are shown; see
652 also Supplemental Fig. 2. The full list of proteins is shown in Supplemental Table 1. (b) Extracts
653 prepared from HEK293T cells and HEK293T cells stably expressing NRDE2-GFP were
654 subjected to immunoprecipitation with anti-GFP antibodies; samples were analyzed by Western
655 blotting with the indicated antibodies. (n=2, representative blots are shown) (c) HEK293T cells
656 stably expressing NRDE2-GFP were fixed in 3% paraformaldehyde, immunostained with the
657 indicated antibodies, and counter-stained with DAPI (blue). Apotome-enabled images were taken
658 on a Zeiss Axio Observer.Z1 and processed using ZEN software. Approximately 100 cells were
659 analyzed, and representative images are shown. Bar = 2um. (d) RNA immunoprecipitation with
660 anti-GFP antibodies from HEK293T cells stably expressing NRDE2-GFP followed by qPCR.
661 Data are normalized to control immunoprecipitations with the same anti-GFP antibody in
662 untagged HEK293T cells. Error bars = SD (n=3).
663
664 Figure 3. NRDE2 suppresses intron retention in a subset of short, GC-rich introns with
665 weak 5’ and 3’ splice sites. (a) Summary of intron retention analysis results using IRFinder
666 (version 1.2.3) (Middleton et al. 2017) based on gene annotations for the hg19 reference genome;
667 see Methods for details. (b) Box plots of intron lengths, GC content, 5’ and 3’ splice site strengths
668 for all introns (n=234,396), retained introns (n=2,172), NRDE2-induced IR sequences (n=15),
669 and NRDE2-suppressed IR sequences (n=163). Splice site strengths were calculated based on the
670 maximum entropy model (Yeo and Burge 2004). Grey numbers indicate p-values evaluated with
671 the two-sided Mann-Whitney U test. (c) Bar graphs depicting the log2 expression change
672 (evaluated by DESeq2) of genes exhibiting IR events either induced (teal) or suppressed (purple)
673 by NRDE2. A subset of gene names are shown; all genes are listed in Supplemental Table 3.
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674
675 Figure 4. NRDE2 is required for CEP131 pre-mRNA splicing and protein expression. (a)
676 qPCR analysis of intron retention in CEP131, TUBGCP2, and KIF2C. Primers detecting spliced
677 transcripts are located on exons flanking the retained intron. Primers detecting unspliced
678 transcripts flanked either the 5’ splice junctions or 3’ splice junctions. Error bars = SD (n=3). * p
679 < 0.05, two-tailed t-test. (b) Genes displaying >2-fold increased IR following NRDE2 depletion
680 are shown where the IR ratio in NRDE2-depleted cells is >0.5. The IR ratio measures the fraction
681 of transcripts retaining a given intron (Middleton et al. 2017). (c) Integrated Genomics Viewer
682 (IGV) visualization of RNA-seq data from si-Control and si-NRDE2-2 samples at the CEP131
683 locus. The retained intron is highlighted in pink. (d) RNA immunoprecipitation with anti-GFP
684 antibodies from HEK293T cells stably expressing NRDE2-GFP followed by qPCR. Data are
685 normalized to control immunoprecipitations with the same anti-GFP antibody in untagged
686 HEK293T cells. Error bars = SD (n=3). (e) Representative fluorescent images and quantification
687 of CEP131-immunostained HEK293T cells 6 days post-si-transfection. > 70 cells were scored per
688 condition. * p < 0.05, two-tailed t-test.
689
690 Figure 5. NRDE2 is required for centrosome maturation, integrity, and function. (a-d)
691 MDA-MB-231 cells were fixed and stained with the indicated antibodies four days post-
692 transfection. Maximum intensity z-projections of representative image stacks are shown. Mean
693 foci intensities were measured in a 1-2um2 circle (depending on the marker) drawn around the
694 brightest point of each marker. > 20 foci were quantified per condition per replicate (n ≥ 3). (e)
695 (left) Maximum intensity z-projections of confocal image stacks of mitotic MDA-MB-231 cells
696 fixed and stained with the centriole marker centrin-2 four days post-transfection. (right)
697 Quantification of centriole number and the distance between centriole pairs; at least 70 centriole
698 pairs were analyzed per condition. All intensity and distance measurements were performed using
699 the Zen Blue software (Zeiss). Bar = 5um. n.s, not significant. * p < 0.05, two-tailed t-test.
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700
701 Supplemental Figure 1. NRDE2 depletion results in DNA damage and activation of p53. (a)
702 Confirmation of NRDE2 mRNA knockdown by qPCR in MDA-MB-231 breast cancer cells and
703 H23 lung cancer cells. RNA was collected 3-4 days post-transfection. (b) Western blots from
704 HEK293T cells expressing endogenously tagged 3XFLAG-NRDE2 confirm efficient si-NRDE2-
705 mediated protein knockdown (n=3, representative blots are shown). (c) Left: cell counts of HeLa
706 cells 8 days post-transfection. Right: qPCR analysis of NRDE2 expression at two days post-
707 transfection. NRDE2 OE: NRDE2-GFP overexpression. Control cells expressed only GFP. (d)
708 Representative immunofluorescence images of DAPI, anti-γH2AX-stained and anti-H3(S10P)-
709 stained HEK293T cells revealing increased DNA damage, micronuclei, and mitotic cells
710 following NRDE2 depletion. Bar = 20um. (e) Quantification of immunofluorescence images as in
711 (d), where cells were scored as mitotic if they displayed both positive staining for H3(S10P) as
712 well as condensed chromosomes (by DAPI staining); >250 cells were scored per condition per
713 replicate (n=3). (f) Representative western blot analysis of p53 activation following NRDE2
714 knockdown in HEK293T cells (n=2). (a-d) All error bars represent SD of three independent
715 experiments. * p < 0.05, two-tailed t-test.
716
717 Supplemental Figure 2. NRDE2 associates with RNA processing proteins enriched in pre-
718 mRNA splicing functions. (a) Domain structure of the NRDE2 protein. Amino acid positions are
719 numbered. The nuclear localization signal was predicted using NucPred(Brameier et al. 2007). (b,
720 c) STRING network visualization and GO term enrichment analyses of all NRDE2-interacting
721 proteins with >15 peptides detected among the averaged replicate NRDE2-GFP
722 immunoprecipitated samples. Purple edges represent experimentally verified protein-protein
723 interactions.
724
725
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726 Supplemental Figure 3. Endogenous NRDE2 co-localizes with components of the
727 spliceosome, the EJC, and the RNA exosome. (a) HEK293T cells expressing endogenously
728 tagged zsGreen-NRDE2 were fixed in 3% paraformaldehyde, immunostained with the indicated
729 antibodies, and counter-stained with DAPI (blue). Apotome-enabled images were taken on a
730 Zeiss Axio Observer Z1 and processed using ZEN software. Approximately 100 cells were
731 analyzed, and representative images are shown. Bar = 2um. (b) Extracts prepared from HEK293T
732 cells and HEK293T cells expressing an endogenously tagged 3xFLAG-NRDE2 allele were
733 subjected to immunoprecipitation with anti-FLAG antibodies; samples were analyzed by Western
734 blotting with anti-FLAG (to detect 3XFLAG-NRDE2), and anti-SKIV2L2. (n=3, representative
735 blots are shown).
736
737 Supplemental Figure 4. qPCR analysis of gene expression changes identified by RNA-seq in
738 NRDE2-depleted cells. qPCR validation of RNA-seq data (red bars) reveals highly reproducible
739 gene expression changes following NRDE2 depletion at a later time point (96h, white bars), with
740 an additional siRNA (si-NRDE2-1, grey bars), and in a different cell line (RPE-1 cells, black
741 bars). Error bars represent SD of two independent experiments.
742
743 Supplemental Figure 5. A small subset of introns are retained in NRDE2-depleted cells.
744 (a) IGV visualizations of RNA-seq data from si-Control and si-NRDE2-treated cells at
745 representative genes with intron retention events (highlighted in pink). STAT5B was identified by
746 both IRFinder and SeqMonk analyses; TACC3 was identified in SeqMonk; CALCOCO1 and
747 SLC4A2 were identified by IRFinder. (b) Box plots of intron lengths, GC content, 5’ and 3’ splice
748 site strengths for all introns annotated in IRFinder (n=234,396), compared to NRDE2-suppressed
749 IR sequences (n=43) calculated based on differential intron expression and relative intron/exon
750 levels determined in SeqMonk. Splice site strengths (MaxEnt scores) were calculated based on
751 the maximum entropy model(Yeo and Burge 2004). Grey numbers indicate p-values evaluated
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Jiao et al.
752 with the two-sided Mann-Whitney U test. n.s, not significant (p > 0.05). (c) Read distributions of
753 triplicate RNA-seq samples mapped by STAR (v2.5.1b) reveal a similar distribution of reads
754 from intronic, exonic, and UTR regions across all samples.
755
756 Supplemental Figure 6. Intron retention in CEP131 results in premature stop codons.
757 SnapGene view of the upstream exon (exon 16, orange) and the retained intron (intron 16, grey)
758 in CEP131 pre-mRNA. Red asterisks indicate in-frame stop codons.
759
760 Supplemental Figure 7. NRDE2 regulates CEP131 protein expression and unspliced
761 CEP131 RNA decay. (a) Representative immunostaining and quantification of CEP131 foci 4
762 days post-transfection in MDA-MB-231 cells. More than 30 cells were scored per condition. Bar
763 = 20um. (b) Representative Western blot analysis of MDA-MB-231 cell extracts collected 3, 4,
764 and 5 days post-transfection (n=2). (c) Experimental outline and qPCR analysis of CEP131 pre-
765 mRNA stability. CEP131 expression was first normalized within each sample to ACTB mRNA;
766 all data points were then normalized to DMSO-treated 72h samples. In control-treated cells,
767 unspliced CEP131 pre-mRNA is less stable than ACTB mRNA, resulting in a downward slope
768 following Actinomycin D treatment. In si-NRDE2-treated cells, unspliced CEP131 pre-mRNA is
769 more stable than ACTB mRNA, resulting in an upward slope. Error bars = SD (n=3). (d) qPCR
770 analysis of total CEP131 RNA (measured using a primer set in exon 2 of CEP131 mRNA) after
771 four days post-transfection in MDA-MB-231 cells. Error bars = SEM (n=3). * p < 0.05, two-
772 tailed t-test.
773
774 Supplemental Figure 8. Reduced CEP131 protein is sufficient to cause centrosome
775 dysfunction and aberrant nuclear morphologies. (a) Representative immunostaining and
776 quantification of CEP131 foci 4 days post-transfection in MDA-MB-231 cells. More than 30 cells
777 were scored per condition. Bar = 20um. (b) Representative immunostaining and quantification of
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Jiao et al.
778 mitotic Aurora Kinase A (AURKA) foci 4 days post-transfection in MDA-MB-231 cells. Only
779 cells in prometaphase / metaphase were scored. More than 70 cells were scored per condition. Bar
780 = 5um. (c) Categories and quantification of aberrant nuclear morphologies visualized by DAPI 4
781 days post si-CEP131 transfection in MDA-MB-231 cells. “Multiple” refers to nuclear
782 abnormalities falling under multiple categories. White: DNA. Green: α-tubulin. >300 cells were
783 scored per condition per replicate (n=3). Bar=10um. * p < 0.05, two-tailed t-test.
784
785 Supplemental Figure 9. Reduced CEP131 expression is not necessary for the cell cycle
786 arrest, DNA damage, and proliferative defects in NRDE2-depleted cells. (a) MDA-MB-231
787 cells carrying an integrated dox-inducible 3XFLAG-CEP131 construct were transfected with
788 either control or si-NRDE2-2, in the presence or absence of 10ng/mL doxycycline. Cell lysates
789 were collected four days post-transfection for Western blot analysis. (b) Cell counts of MDA-
790 MB-231 cells six days post-transfection. Cells were split 24h post-transfection, seeded at 16,000
791 cells/well in 24-well format, and counted five days later.
792
793 Supplemental Figure 10. NRDE2 is required for the centrosomal recruitment of AURKA,
794 centrosome protein expression, and mitotic spindle organization. (a) Representative confocal
795 z-projections of mitotic MDA-MB-231 cells fixed and stained with AURKA antibodies four days
796 post-transfection; foci intensities were quantified using Zen Blue (Zeiss) software. Only cells in
797 prometaphase/metaphase were scored. More than 40 AURKA foci were analyzed per condition.
798 The experiment was repeated three times with similar results. (b) qPCR analysis of AURKA
799 expression in MDA-MB-231 cells 4 days post-transfection. Error bars = SD (n=2). (c-e)
800 Representative western blot analysis of MDA-MB-231 cells (n>2). AURKA expression was
801 assessed four days post-transfection. CEP192 expression was assessed five days post-transfection.
802 (f) qPCR analysis of CEP192 expression in MDA-MB-231 cells from RNA was isolated four
803 days post-transfection. Error bars = SD (n=2). (g) Representative confocal images and
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Jiao et al.
804 quantification of abnormal mitotic spindles of MDA-MB-231 cells fixed four days post-
805 transfection. Yellow arrowheads indicate spindle poles with abnormal phenotypes; grey
806 arrowheads point to unaligned chromosomes. Error bars = SD (n=3). >40 cells in
807 prometaphase/metaphase were scored per condition per replicate. * p < 0.05, two-tailed t-test.
808
809 Supplemental Figure 11. NRDE2 does not affect global R-loop accumulation. Representative
810 immunofluorescence images of S9.6-immunostained, methanol-fixed MDA-MB-231 cells taken
811 three days post-transfection. Bar = 20um. Mean S9.6 intensities were measured within nuclei
812 (manually selected areas defined by DAPI staining) in >90 cells per condition (n=2). n.s, not
813 significant, two-tailed t-test.
814
815 Supplemental Figure 12. Summary of results. NRDE2 associates with RNA splicing and decay
816 factors, and promotes the splicing of short, GC-rich introns with weak 5’ and 3’ splice sites.
817 CEP131 is among the genes most affected by increased intron retention in NRDE2-depleted cells.
818 NRDE2 binds preferentially to unspliced CEP131 transcripts, and suppresses a specific intron
819 retention event in CEP131 pre-mRNA. Green boxes depict CEP131 exons; EJC, exon junction
820 complex; dotted grey lines indicate protein-protein and protein-RNA interactions identified in this
821 study. While the NRDE2-dependent expression of CEP131 protein likely contributes to the
822 essential role of NRDE2 in the maintenance of genomic stability, other mis-splicing events (e.g.
823 in TUBGCP2 and KIF2C) and disrupted expression of additional centrosome proteins (e.g. γ-
824 tubulin and CEP192) cumulatively result in impaired centrosome function. Centrosome defects
825 are a major source of mitotic delays, chromosome missegregation, DNA damage, and
826 proliferative arrest/cell death.
827
828
829
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Jiao et al.
830 Supplementary Movies 1-3. Representative mitoses in control-treated (Movie S1) and si-
831 NRDE2-treated (Movies S2, S3) RPE-1 cells stably expressing H2B-GFP and α-tubulin-mCherry.
832 All movies are compiled 20x time lapse images taken 5-6 days post-transfection with 10min
833 resolution (see accompanying .mov files).
834
835
836
837
838
839
840
841
842
843
844
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Fig. 1
A B
H23 HEK293T 15 MDA-MB-231 10 15 si-Control si-NRDE2-1 si-NRDE2-2 8 10 10 si-Control 6 si-NRDE2-1 2N 2N 2N 4N 4 5 si-NRDE2-2 5 4N ative A560 Rel ative ative A560 Rel ative ative A560 Rel ative 2 0 0 0 4N 2 4 6 7 3 5 7 3 5 6 7 counts Cell Days post-transfection Days post-transfection Days post-transfection DNA content
72h 96h 120h C D * E 400
si-Control si-NRDE2-1 si-NRDE2-2
(mins) 300 si-Controlsi-NRDE2-1si-NRDE2-2si-Controlsi-NRDE2-1si-NRDE2-2si-Controlsi-NRDE2-1si-NRDE2-2
Cyclin B1 s 200 β-Actin γH2AX 100 H3(S10P) Time in Mitosi in Time 0 β-Actin β-Actin
si-Control si-NRDE2-2 0.6 F nucl ei
Normal Nuclear Bud Micronuclei Bilobed Polylobed Multiple 0.4 abnormal th wi th
0.2 f c ells o ion rac t
F 0.0 si-Control si-NRDE2-2 si-Control si-NRDE2-2 Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press
Fig. 2
A B 5% Input GFP IP
NRDE2-GFP IP peptide counts (coverage) A B NRDE2 62 (24.3%) 113 (33.8%)
RNA Exosome SKIV2L2 91 (38.3%) 136 (43.5%) Untagged NRDE2-GFP Untagged NRDE2-GFP EXOSC10 20 (15.6%) 25 (21.9%) - NRDE2-GFP EXOSC7 3 (10.7%) 7 (25.1%) Exon Junction Complex EIF4A3 26 (31.4%) 10 (18.2%) MAGOH 6 (20.5%) 10 (47.3%) - SKIV2L2 ACIN1 17 (11.3%) 31 (20.3%) PNN 25 (21.9%) 25 (23.7%) - EXOSC10 Spliceosome (U5 snRNP) SNRNP200 11 (5.5%) 25 (10.5%) EFTUD2 17 (15.1%) 21 (18.1%) - EFTUD2 PRPF8 21 (8.1%) 41 (14.8%) - IgG SNRNP40 6 (24.4%) 5 (14.3%) - EIF4A3
- β-Actin C SKIV2L2 EXOSC10 EFTUD2 EIF4A3 α-tubulin
D
25 ) rol ) 20 snRNAs snoRNAs
t vs. con 15 NRDE2-GFP
hme n 10 nr ic DE2 RNA-IP RNA-IP NR DE2 5 d E ol d (F Merge 0
U1 U2 U4 U5 U6 U3 U8 U11 U12 U22 Interphase Metaphase Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Fig. 3
A B <2e-16 n.s. (p=0.07) <2e-16 n.s <2e-16 0.0005 n.s. (p=0.06) n.s. <2e-16 0.005 3e-10 <2e-16
Total=2,172
All IR events NRDE2-induced IR NRDE2-suppressed IR
C
1.5 1.0 1.0 0.5 0.5 0.0 0.0 -0.5 -0.5 Log2 Fold Chang e Log2 Fold Chang e -1.0 -1.5 -1.0
GSS NT5C PIF1 KAT2ASPAG5 MED12 ZMIZ2 IRAK1TANC1 AP2A2 ACY1 CLK2 CD58 CRY1 NSMF BOD1 JAG1 MOV10 SPPL2B DOCK5 TCIRG1 RNF213 RNF215 AFMIDPOMT2 ZNF692 DALRD3 RECQL4 PCED1A ADAM15 HNRNPM FAHD2A Genes with NRDE2-induced IR (n=14) Genes with NRDE2-suppressed IR (n=138) Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press
Fig. 4
A B
5’ Splice Junctions 3’ Splice Junctions 0.8 1.5 1.5 0.8
pli ced 0.6 * * * * * * 0.6 1.0
Unsplice d 1.0 Un s ed / Ra tio ed / 0.40.4 IR IR
pli c si-Control pli c si-Control IR Rati o si-Control S 0.5 S 0.5 si-NRDE2-2 0.20.2 ssi-NRDi-NRDE2-2E2-2 Rela tive 0.0 Rela tive 0.0 0.00.0 CEP131 TUBGCP2 KIF2C CEP131 TUBGCP2 KIF2C
PADI1 EML6 NPR2EML6 AKNA si-Control si-NRDE2-2 CEP131CEP131 AMPD2AMPD2DNAH2 CALCOCO1 GenesGenes with with retained retained introns introns
C D
100 *
[0-25] 80 nrichme nt
60 old E si-Control
F * Actin (total) [0-25] 40 CEP131 (spliced) CEP131 (unspliced) RN A-IP 20 si-NRDE2-2
E2 NRD E2 0 CEP131
E
si-Control si-NRDE2-2 * 40000
ensi ty 30000 in t
oci oci 20000 CEP131
10000 EP 131 f C 0 DAPI
si-Control si-NRDE2-2 Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press
Fig. 5
A DAPI CEP131 α-tubulin merge * 60000 en sity in t
oci 40000 si-Control
EP 131 f 20000 C c
0 Mi toti si-NRDE2-2 si-Control si-NRDE2-2 B DAPI p-AURKA α-tubulin merge 40000 * en sity
in t 30000 oci oci f
si-Control 20000 288 P) 10000
UR KA (T 0 A si-NRDE2-2
si-Control si-NRDE2-2 C DAPI γ-tubulin α-tubulin merge 50000 *
40000 en sity nt 30000
oci I Interphase si-Control 20000 * Mitotic
ubulin F 10000 -T 0 si-NRDE2-2
si-Control si-Control si-NRDE2-2 si-NRDE2-2
D DAPI CEP192 α-tubulin merge * 14
2 foci 19 2 12 10 8
si-Control 6 4 2 0 r of mitotic CEP mitotic of Numbe r si-NRDE2-2 si-Control si-NRDE2-2
E centrin-2 DAPI merge n.s. * 15 12 (u m)
10 8 ce di st an ce r mitotic cell mitotic pe r si-Control i oc i
5 riolar 4 en t 2 f rin- 2 er c 0 0 Int Ce nt
si-NRDE2-2 si-Control si-Control si-NRDE2-2 si-NRDE2-2 Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press
Human Nuclear RNAi-Defective 2 (NRDE2) is an essential RNA splicing factor
Alan L. Jiao, Roberto Perales, Neil T. Umbreit, et al.
RNA published online December 11, 2018 originally published online December 11, 2018 Access the most recent version at doi:10.1261/rna.069773.118
Supplemental http://rnajournal.cshlp.org/content/suppl/2018/12/11/rna.069773.118.DC1 Material
P
Accepted Peer-reviewed and accepted for publication but not copyedited or typeset; accepted Manuscript manuscript is likely to differ from the final, published version.
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