Downloaded from rnajournal.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press 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: Current address: Department of Biological Sciences, University at Albany, The State University of New York, Albany, NY, 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 Downloaded from rnajournal.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Jiao et al. 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. 21 22 23 24 25 26 2 Downloaded from rnajournal.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Jiao et al. 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 3 Downloaded from rnajournal.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Jiao et al. 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 4 Downloaded from rnajournal.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Jiao et al. 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.