Minor intron splicing is regulated by FUS and affected by ALS ‐associated FUS mutants
2016; Springer Nature; Volume: 35; Issue: 14 Linguagem: Inglês
10.15252/embj.201593791
ISSN1460-2075
AutoresStefan Reber, Jolanda Stettler, Giuseppe Filosa, Martino Colombo, Daniel Jutzi, Silvia C. Lenzken, Christoph Schweingruber, Rémy Bruggmann, Angela Bachi, Silvia M.L. Barabino, Oliver Mühlemann, Marc‐David Ruepp,
Tópico(s)Neurogenetic and Muscular Disorders Research
ResumoArticle1 June 2016free access Source DataTransparent process Minor intron splicing is regulated by FUS and affected by ALS-associated FUS mutants Stefan Reber Stefan Reber Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland Search for more papers by this author Jolanda Stettler Jolanda Stettler Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Search for more papers by this author Giuseppe Filosa Giuseppe Filosa Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy IFOM-FIRC Institute of Molecular Oncology, Milan, Italy Search for more papers by this author Martino Colombo Martino Colombo Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland Search for more papers by this author Daniel Jutzi Daniel Jutzi Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Search for more papers by this author Silvia C Lenzken Silvia C Lenzken Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy Search for more papers by this author Christoph Schweingruber Christoph Schweingruber Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland Search for more papers by this author Rémy Bruggmann Rémy Bruggmann Interfaculty Bioinformatics Unit and Swiss Institute of Bioinformatics, University of Bern, Bern, Switzerland Search for more papers by this author Angela Bachi Angela Bachi IFOM-FIRC Institute of Molecular Oncology, Milan, Italy Search for more papers by this author Silvia ML Barabino Silvia ML Barabino Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy Search for more papers by this author Oliver Mühlemann Corresponding Author Oliver Mühlemann orcid.org/0000-0003-0657-1368 Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Search for more papers by this author Marc-David Ruepp Corresponding Author Marc-David Ruepp orcid.org/0000-0003-3264-9800 Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Search for more papers by this author Stefan Reber Stefan Reber Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland Search for more papers by this author Jolanda Stettler Jolanda Stettler Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Search for more papers by this author Giuseppe Filosa Giuseppe Filosa Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy IFOM-FIRC Institute of Molecular Oncology, Milan, Italy Search for more papers by this author Martino Colombo Martino Colombo Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland Search for more papers by this author Daniel Jutzi Daniel Jutzi Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Search for more papers by this author Silvia C Lenzken Silvia C Lenzken Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy Search for more papers by this author Christoph Schweingruber Christoph Schweingruber Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland Search for more papers by this author Rémy Bruggmann Rémy Bruggmann Interfaculty Bioinformatics Unit and Swiss Institute of Bioinformatics, University of Bern, Bern, Switzerland Search for more papers by this author Angela Bachi Angela Bachi IFOM-FIRC Institute of Molecular Oncology, Milan, Italy Search for more papers by this author Silvia ML Barabino Silvia ML Barabino Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy Search for more papers by this author Oliver Mühlemann Corresponding Author Oliver Mühlemann orcid.org/0000-0003-0657-1368 Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Search for more papers by this author Marc-David Ruepp Corresponding Author Marc-David Ruepp orcid.org/0000-0003-3264-9800 Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland Search for more papers by this author Author Information Stefan Reber1,2, Jolanda Stettler1,†, Giuseppe Filosa3,4, Martino Colombo1,2, Daniel Jutzi1, Silvia C Lenzken3, Christoph Schweingruber1,2, Rémy Bruggmann5, Angela Bachi4, Silvia ML Barabino3, Oliver Mühlemann 1 and Marc-David Ruepp 1 1Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland 2Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland 3Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy 4IFOM-FIRC Institute of Molecular Oncology, Milan, Italy 5Interfaculty Bioinformatics Unit and Swiss Institute of Bioinformatics, University of Bern, Bern, Switzerland †Present address: Interregionale Blutspende SRK AG, Bern, Switzerland *Corresponding author. Tel: +41 31 631 4348; E-mail: [email protected] *Corresponding author. Tel: +41 31 631 4627; E-mail: [email protected] The EMBO Journal (2016)35:1504-1521https://doi.org/10.15252/embj.201593791 See also: E Buratti (July 2016) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Fused in sarcoma (FUS) is a ubiquitously expressed RNA-binding protein proposed to function in various RNA metabolic pathways, including transcription regulation, pre-mRNA splicing, RNA transport and microRNA processing. Mutations in the FUS gene were identified in patients with amyotrophic lateral sclerosis (ALS), but the pathomechanisms by which these mutations cause ALS are not known. Here, we show that FUS interacts with the minor spliceosome constituent U11 snRNP, binds preferentially to minor introns and directly regulates their removal. Furthermore, a FUS knockout in neuroblastoma cells strongly disturbs the splicing of minor intron-containing mRNAs, among them mRNAs required for action potential transmission and for functional spinal motor units. Moreover, an ALS-associated FUS mutant that forms cytoplasmic aggregates inhibits splicing of minor introns by trapping U11 and U12 snRNAs in these aggregates. Collectively, our findings suggest a possible pathomechanism for ALS in which mutated FUS inhibits correct splicing of minor introns in mRNAs encoding proteins required for motor neuron survival. Synopsis Mutations in RNA-binding protein FUS are frequently found in familial amyotrophic lateral sclerosis (ALS). This study shows FUS to control minor intron splicing via binding to the U11/12 snRNP and identifies target mRNAs that may contribute to disease pathology. Fused in sarcoma (FUS) interacts with the minor spliceosome and affects minor intron splicing. Loss of FUS results in a strong deregulation of minor intron-containing genes. FUS with an ALS-associated mutation mislocalizes to the cytoplasm and inhibits minor intron splicing. ALS-associated cytoplasmic FUS sequesters U11 and U12 snRNA in cytoplasmic aggregates. Introduction Dysfunctional RNA metabolism is implicated in a wide variety of neurodegenerative diseases, and many mutations leading to neurodegeneration have been identified in proteins with roles in RNA metabolism (Duan et al, 2014; Zhou et al, 2014). For example, mutations in the gene fused in sarcoma (FUS), which encodes an ubiquitously expressed nuclear RNA-binding protein of the hnRNP family, were identified in patients with an inherited form of amyotrophic lateral sclerosis (ALS; Kwiatkowski et al, 2009; Vance et al, 2009). ALS is a fatal, adult-onset neurodegenerative disease that selectively kills brain and spinal cord motor neurons. While most cases appear to be sporadic (sALS), 10% of cases are inherited (familial ALS, fALS). Approximately 4–5% of fALS and some sALS cases are due to mutations in FUS (Lagier-Tourenne & Cleveland, 2009; DeJesus-Hernandez et al, 2010). Fused in sarcoma mRNA consists of 15 exons encoded on chromosome 16 (Aman et al, 1996) and gives rise to a 526-amino acid-long protein. The FUS protein contains a prion-like N-terminal glutamine, glycine, serine and tyrosine rich (Q/G/S/Y) domain, which is conserved within the FET family of proteins (FUS, EWS and TAF15) and is required for homo- or heterodimer formation among them. Furthermore, FUS contains three arginine/glycine/glycine (RGG1–3) domains, a RNA recognition motif (RRM) and a zinc finger (Zn). These domains were associated with DNA and RNA binding. Furthermore, FUS comprises a predicted nuclear export signal (NES) embedded in the RRM and a C-terminal nuclear localization signal (NLS) (Iko et al, 2004; Kwiatkowski et al, 2009; Dormann et al, 2010; Thomsen et al, 2013). Most reported FUS-linked ALS-causing mutations are missense mutations clustered in the highly conserved C-terminal NLS (Dormann et al, 2010). Depending on the mutation, this leads to almost abolished or significantly reduced nuclear import of FUS and to the formation of FUS aggregates in the cytoplasm of neurons and glial cells of ALS patients (Bentmann et al, 2013), indicating that these mutations either lead to a loss of function in the nucleus, to a gain of function in the cytoplasm or to a combination of both (Wang et al, 2013; Sun et al, 2015). Fused in sarcoma has been implicated to function in several steps of gene expression. It regulates the transcription through the interaction with RNA polymerases (RNAP) II and III and several transcription factors (Uranishi et al, 2001; Li et al, 2010; Tan & Manley, 2010; Schwartz et al, 2012). Besides its functions in transcription, FUS was also identified as a splicing regulator based on its presence in spliceosomal complexes (Rappsilber et al, 2002; Zhou et al, 2002) and interactions with several splicing factors (Meissner et al, 2003) as well as with the U1 snRNP (Hackl et al, 1994; Yamazaki et al, 2012; Gerbino et al, 2013; Yu et al, 2015). Recent CLIP studies showed that FUS binds to nascent transcripts of many different pre-mRNAs, preferentially to long introns (Hoell et al, 2011; Rogelj et al, 2012), and splicing analysis in the embryonic brains of FUS-knockout mice revealed splicing changes in more than 300 genes (Lagier-Tourenne et al, 2012). However, the molecular mechanism by which FUS influences pre-mRNA splicing remained so far unknown. To address the roles of FUS in RNA metabolism, we performed mass spectrometric analysis to identify high-confidence interactors of FUS. We found that minor spliceosome components are highly enriched among the FUS-interacting proteins. In line with this finding, FUS knockout affects predominantly the removal of minor introns. We subsequently confirmed that FUS is necessary for the efficient splicing of a subset of minor intron-containing mRNAs (so-called U12-type introns), among them members of the voltage-gated sodium channel family that are required for proper muscle function and post-natal maturation of spinal motor neurons (Jurkat-Rott et al, 2010; Porter et al, 1996). Our data reveal that FUS directly regulates the removal of minor introns through the direct interactions with the minor spliceosome at the 5′ splice site of these introns. We further show that the ALS-associated FUS-P525L mutation, which destroys the NLS and results in cytoplasmic retention of FUS (Dormann et al, 2010), fails to promote the efficient splicing of minor introns and causes mislocalization of the minor spliceosome components U11 and U12 snRNA to the cytoplasm. Collectively, our findings identify a role of FUS in splicing of minor introns, many of which occur in genes with neuronal functions, and imply that a loss of this nuclear function might contribute to the development of ALS. Intriguingly, our results suggest a possible mechanistic link between FUS-linked and TDP-43-linked ALS, as well as spinal muscular atrophy (SMA) in the light that all three neurodegenerative diseases exhibit a diminished minor spliceosome function (Lotti et al, 2012; Ishihara et al, 2013). Results Identification of FUS-interacting proteins In order to unravel the molecular pathways involving FUS, a mass spectrometry-based interactome analysis was performed. Since FUS binds RNA (Zinszner et al, 1997), the interactome analysis was carried out with or without RNase treatment of the cell lysates to distinguish between protein-to-protein interactions and interactions mediated by RNA. Immunoprecipitations (IPs) were performed from HEK293T cell lysates transiently expressing Flag-tagged wild-type FUS or Flag-EBFP (enhanced blue fluorescent protein) as a control (Fig 1A). While the control IPs were essentially devoid of co-precipitating proteins (lanes 3, 5 and 7), many proteins were detected on a Coomassie-stained gel in the Flag-FUS IP performed under low stringency conditions (150 mM NaCl washes) and in the absence of RNase (lane 4). Treatment of the lysates with RNase A prior to the IP reduced the number of bands, indicating that many interactions are RNA-mediated (compare lanes 4 and 6). Repetition of the IPs with RNase-treated lysates under more stringent washing conditions (750 mM NaCl) to identify those proteins that associated with FUS with the highest affinity further reduced the number of detectable bands on the gel (lane 8). Figure 1. Mass spectrometric identification of FUS-interacting proteins FLAG-tagged FUS and EBFP (negative control) were immunoprecipitated from the total cell extracts of 293T cells under low stringency (150 mM NaCl) without (lanes 3–4) or with RNase A treatment (lanes 5–6) as well as with RNase A treatment and immunoprecipitation with high stringency washes (750 mM NaCl, lanes 7–8). Purified protein complexes were eluted from the anti-FLAG affinity gel by FLAG peptide, separated by SDS–PAGE and stained with Coomassie. Asterisks indicate the baits (FUS-FLAG and EBFP-FLAG). Venn diagram representing the overlap of the proteins identified under low stringency (with [red] and without RNase A treatment [green]) and high stringency conditions (purple). Gene ontology enrichment analysis of the 40 high-confidence interactors of FUS according to biological process. Gene ontology enrichment analysis of these FUS interactors according to the cellular component. Frequency refers to the percentage of FUS-interacting proteins annotated to a certain GO term in the data set (black bar) and in the human reference set (grey bar). The enrichment value (green triangles) represents the ratio between the frequencies of the specific term in the FUS IPs and in the human genes reference data set. All terms are significantly enriched with a P-value < 0.05. Download figure Download PowerPoint The gel lanes were then excised and processed for in-gel tryptic digestion before liquid chromatography–tandem mass spectrometry analysis (LC-MS/MS). Proteins detected in the control IP, which represent unspecific interactors with the anti-Flag matrix, were eliminated from further analysis. A total of 587 potential interactors were identified, 444 of which were only detected under low stringency conditions and in the absence of RNase, and 97 of those were also identified in the RNase-treated sample (Fig 1B). Of these 97 proteins, 40 were even detected under high stringency conditions, identifying them as relatively stable and RNA-independent interactors of FUS with high confidence. The results derive from two biological replicates for each condition and only proteins detected in both were considered. The proteins identified under all three conditions are listed in Appendix Table S1. Gene ontology (GO) analysis was performed for the 40 proteins co-immunoprecipitating with FUS under all three conditions to reveal the most enriched biological processes and cellular components involving these genes. Statistically highly significantly enriched were biological processes related to RNA metabolism, in particular RNA splicing (Fig 1C). This aspect is also evident in the cellular component analysis, where the terms "spliceosomal complex" and "U12-type spliceosomal complex" are highly enriched (Fig 1D). FUS interacts with the U11 snRNP Because of the GO analysis pointing towards spliceosomal complex and previous evidence for an interaction of FUS with U1 snRNP (Hackl et al, 1994; Yamazaki et al, 2012; Sun et al, 2015; Yu et al, 2015), we performed RNA-IPs from HeLa nuclear extract using anti-FUS antibodies, or anti-BSA antibodies as negative control, followed by RT–qPCR to test for U snRNA enrichment. Consistent with the published data, U1 snRNA was strongly enriched in the FUS IP. Intriguingly, and in line with the GO analysis of the mass spectrometry results (Fig 1D), the most enriched U snRNA was the U11 snRNA, a member of the minor spliceosome (also called U12-type spliceosome) and constituent of the U11/U12 di-snRNP (Fig 2A and Appendix Fig S1A). These results are in accordance with the previous findings reporting the presence of FUS in the human spliceosomal complexes E, A and B, in which the U1 and U11 snRNPs are present, but not in the Bact and C complexes, a stage at which U1 and U11 snRNPs have left the pre-mRNA (Hartmuth et al, 2002; Deckert et al, 2006; Behzadnia et al, 2007). The results further indicate that the observed enrichment of U5 snRNA in our FUS-RNA-IPs is unlikely to be direct, because U5 snRNA is present throughout the entire splicing cycle, whereas FUS is confined to the early spliceosome assembly stages (Will & Luhrmann, 2011). We therefore did not further investigate the U5 snRNA association with FUS, but instead focused on the interaction of the minor spliceosome component U11 snRNA with FUS. Figure 2. FUS interacts with the U11 snRNP HeLa nuclear extracts were subjected to immunoprecipitation with anti-FUS antibodies or BSA antibodies (negative control) and the co-precipitated snRNAs as well as 7SL RNA were quantitated by RT–qPCR. The respective RNA amounts detected in the IPs are expressed as percentage of the input. The precipitated RNA levels measured in the negative control were subtracted from those in the FUS-RNA-IP. The data before background subtraction are shown in Appendix Fig S1. Error bars indicate standard deviations (SD) of three biological replicates, each measured in duplicate. Enrichment of FUS after U11 snRNP affinity purification with a biotinylated antisense oligonucleotide (AS-U11) complementary to U11 snRNA from HeLa nuclear extracts. As control, incubation of the magnetic streptavidin beads with AS-U11 was omitted. After biotinylated antisense oligonucleotide pull-down, the purified complexes were eluted from the beads and subjected to 4–12% NuPAGE gels. The blots were incubated with mouse anti-FUS-IRDye800CW, goat anti-hnRNPH, rabbit anti-U11-59K and rabbit anti-SmD3, followed by the detection with species-specific IRDye680LT- or IRDye800CW-labelled secondary antibodies to confirm the presence of FUS, hnRNPH, U11-59K and SmD3 in the AS-U11-enriched fraction. Input corresponds to 9% of the used material for pull-down. Download figure Download PowerPoint The interaction of FUS with U11 snRNP was validated by performing pull-downs using biotinylated antisense oligonucleotides against U11 snRNA. The U11 snRNP pull-downs were not only enriched for the U11/U12 di-snRNP-specific factor U11-59K and the common spliceosomal Sm ring component SmD3, but also for FUS and for hnRNP H (Fig 2B). hnRNP H is required for optimal U11 snRNP binding to certain transcripts (McNally et al, 2006) and is one of the conserved FUS interactors (Appendix Table S1, Appendix Fig S1B). In agreement with the previously published data (Sun et al, 2015), we also co-purified FUS in U1 snRNP pull-downs (Appendix Fig S2). To confirm that these interactions take place in situ and do not arise from rearrangements in the extracts after cell lysis, we performed proximity ligation assays (PLA): HeLa cells fixed with paraformaldehyde and permeabilized with Triton X-100 were incubated with antibodies recognizing FUS together with antibodies directed against U1 snRNP and U11/U12 di-snRNP-specific proteins, respectively (Appendix Fig S3). The PLA confirmed that FUS co-localized in cells within < 40 nm distance to U1 snRNP-specific factors U1A and U1C as well as to the U11/12 di-snRNP-specific proteins U11-59K and U11-31K, consistent with the observed association of FUS with the U1 and the U11/12 di-snRNP. Expression and splicing of minor intron-containing genes is strongly disturbed in the absence of FUS To test the influence of FUS on gene expression and splicing at a genome-wide level and to identify the potential targets of FUS, we generated FUS-knockout SH-SY5Y (FUS KO SH-SY5Y) cells and assessed the alterations in splicing by high-throughput sequencing. These FUS KO SH-SY5Y cells were generated by targeting the first intron of the FUS gene with CRISPR/Cas9 and co-transfection of a donor plasmid harbouring a Zeocin resistance cDNA for homologous recombination. The Zeocin cDNA is preceded by a chimeric intron containing the strong 3′ splice site from the rabbit β-globin intron 2, resulting in the in-frame splicing of the FUS exon 1 to the Zeocin cassette. The Zeocin cassette is followed by the strong SV40 polyadenylation signal that leads to premature polyadenylation of the FUS mRNA and the expression of the Zeocin resistance marker (Fig 3A). The absence of FUS mRNA isolated from individual Zeocin-resistant cell clones was verified by RT–qPCR (data not shown), and for the two selected FUS KO clones, the absence of FUS protein was demonstrated by Western blotting (Fig 3B). From these two clonal cell lines and from wild-type SH-SY5Y cells, we then extracted total RNA and performed mRNA-seq. Figure 3. FUS knockout in SH-SY5Y neuroblastoma cells Scheme of the FUS-knockout strategy in SH-SY5Y neuroblastoma cells. The first intron of the FUS gene was targeted with CRISPR/Cas9 to introduce a DNA cassette consisting of a chimeric intron with a strong 3′ splice site (red line), a spacer sequence (olive-green box), the coding sequence of the Sh ble gene, which confers Zeocin resistance (ZeoR, green box), and the SV40 polyadenylation signal (blue box). Upon transcription from the FUS promoter, the first exon of FUS is spliced in frame to this ZeoR-encoding exon and the SV40 polyadenylation signal causes the premature polyadenylation of the FUS mRNA. Western blot confirming the absence of FUS in the two selected clones. Extracts from wild-type (wt) and FUS-knockout SH-SY5Y cells (clones A4 and A5) were subjected to SDS–PAGE, transferred to a nitrocellulose membrane and FUS (green) and tyrosine tubulin (red; loading control) were detected using respective primary and secondary antibodies. Reads from the mRNA-seq of the SH-SY5Y neuroblastoma cells mapped to the FUS gene. The first four exons and the intervening introns of the FUS gene are depicted in purple (boxes indicate exons, and thin lines indicate introns; for better visualization, intron lengths are reduced by a factor of five compared to exons). The mapped reads to this locus are shown on a log10 scale for the wild-type cells (red), FUS KO clone A4 (green) and FUS KO clone A5 (blue). In the two FUS KO clones, hardly any reads are detected downstream of the introduced ZeoR cassette. Source data are available online for this figure. Source Data for Figure 3 [embj201593791-sup-0003-SDataFig3B.pdf] Download figure Download PowerPoint The sequencing data confirmed that in the FUS KO cells, transcription of the FUS gene is terminated as intended in intron 1 where the artificial Zeocin-encoding exon with the SV40 polyadenylation signal was introduced (Fig 3C). To address whether minor intron-containing genes are differentially expressed in the FUS KO SY-SY5Y cells, we performed a gene-level analysis on the mRNA-seq data. Among the differentially expressed genes, those harbouring a minor intron were indeed enriched (Fig 4A). Two-thirds of the differentially expressed minor intron-containing genes were downregulated and one-third was upregulated, respectively (Fig 4B and Appendix Fig S4A). The top-30 downregulated and top-30 upregulated genes are shown in Appendix Tables S3 and S4, respectively. Furthermore, we validated selected genes from these lists with a special focus on genes with indicated roles in neuronal development, function and survival by RT–qPCR (Appendix Fig S4B and C). Figure 4. FUS depletion deregulates splicing of minor intronsFrom four biological replicates of FUS KO SH-SY5Y clones A4 and A5 as well as wild-type cells, poly(A)-selected cDNAs were prepared and sequenced on a Illumina HiSeq3000 machine and the data were analysed as described in 4. Analysis of RNA-seq results focusing on the transcripts of genes containing at least one minor intron. Genes were divided into two categories depending on whether they were differentially expressed upon FUS KD (DEG) or whether their expression was not affected (non-DEG). The percentage of genes with at least one minor intron is depicted for both categories. Compared to the non-DEG, the % of minor intron-containing genes is significantly enriched in the DEG. Effect size: 1.65, P-value: 3.49e-10. Histogram of differentially expressed minor intron-containing genes. Genes are grouped according to the level of differential expression. Approximately two-thirds of the affected genes are downregulated in the absence of FUS. Bar plot showing the percentage of differentially spliced introns among all expressed introns. Major and minor introns are displayed separately. More than 30% of minor introns are differentially spliced under FUS knockout, whereas only a small subset of the major introns are affected. The cumulative plot shows the abundance of minor splice sites among the most differentially expressed splice sites in the RNA-seq data from the FUS KO SH-SY5Y cells. The most differentially expressed sites (ranked from left to right) are depicted on the x-axis, while the y-axis shows the number of minor splice sites among them. The black line represents the distribution observed in the RNA-seq data. This distribution was compared to what would be expected if minor and major splice sites were equally affected by the FUS knockout. This hypothetical distribution (shown in green) was computed with a hypergeometric function. This analysis reveals a high enrichment for minor splice sites, indicating that the usage of minor splice sites is more often altered upon FUS KO relative to the usage of major splice sites. Download figure Download PowerPoint Performing a custom analysis on splicing efficiency (described in 4), we detected more than 400 introns that are differentially spliced in the FUS-depleted cells. Whereas only a very small fraction of major introns were found to be differentially spliced in the FUS-depleted cells, more than 30% of the minor introns that are expressed in SH-SY5Y cells exhibit differential splicing (Fig 4C). In fact, minor introns are highly overrepresented among the differentially spliced introns compared to the numbers one would expect if major intron splicing and minor intron splicing were affected to the same extent by FUS depletion (Fig 4D). The finding that splicing of minor introns is much stronger affected than the splicing of major introns by the absence of FUS is in line with the preference of FUS towards the minor spliceosome constituent U11 snRNP and the GO analysis of the spectrometry results. While the gene-level analysis showed both up- and downregulation of minor intron-containing genes in response to FUS depletion, the majority of the minor introns detected by our differential splicing analysis are spliced more efficiently, which would suggest that FUS mainly functions as a splicing inhibitor. However, the underrepresentation of transcripts whose splicing is promoted by FUS can be attributed to their rapid degradation by the nuclear exosome that was shown to specifically degrade mRNAs with retained minor introns (Niemela et al, 2014). Additionally, mRNAs with a retained intron that escape decay by the nuclear exosome and are exported to the cytoplasm very likely will harbour premature termination codons and hence be degraded by the nonsense-mediated mRNA decay (NMD) pathway (Schweingruber et al, 2013). Hence, the actual number of affected minor introns might be even higher than estimated by our splicing efficiency analysis, because aberrant mRNAs with retained minor introns would most likely undergo a rapid degradation and therefore not be easily detectable in the FUS-depleted SH-SY5Y cells. To examine whether these effects of FUS on splicing of minor introns can also be observed in mice, we re-analysed published mRNA-seq data from mouse brain (Lagier-Tourenne et al, 2012). Corroborating the results from the human neuroblastoma cells, we found that the splicing of minor introns tends to be more affected by FUS depletion than the splicing of major introns also in mouse brain (Appendix Fig S4D). The abundance of many minor intron-containing mRNAs was al
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