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• W2892078811 abstract "•Splicing of NOP56 pre-mRNA is controlled in cis by the intron-hosted snoRNA snoRD86•Different structural conformations of snoRD86 dictate alternative splicing events•Excess snoRNP core proteins reduce NOP56 protein and instead induce a SPA-lncRNA•Cellular levels of box C/D snoRNAs and protein partners are tightly coordinated Box C/D snoRNAs constitute a class of abundant noncoding RNAs that associate with common core proteins to form catalytic snoRNPs. Most of these operate in trans to assist the maturation of rRNAs by guiding and catalyzing the 2′-O-methylation of specific nucleotides. Here, we report that the human intron-hosted box C/D snoRNA snoRD86 acts in cis as a sensor and master switch controlling levels of the limiting snoRNP core protein NOP56, which is important for proper ribosome biogenesis. Our results support a model in which snoRD86 adopts different RNP conformations that dictate the usage of nearby alternative splice donors in the NOP56 pre-mRNA. Excess snoRNP core proteins prevent further production of NOP56 and instead trigger the generation of a cytoplasmic snoRD86-containing NOP56-derived lncRNA via the nonsense-mediated decay pathway. Our findings reveal a feedback mechanism based on RNA structure that controls the precise coordination between box C/D snoRNP core proteins and global snoRNA levels. Box C/D snoRNAs constitute a class of abundant noncoding RNAs that associate with common core proteins to form catalytic snoRNPs. Most of these operate in trans to assist the maturation of rRNAs by guiding and catalyzing the 2′-O-methylation of specific nucleotides. Here, we report that the human intron-hosted box C/D snoRNA snoRD86 acts in cis as a sensor and master switch controlling levels of the limiting snoRNP core protein NOP56, which is important for proper ribosome biogenesis. Our results support a model in which snoRD86 adopts different RNP conformations that dictate the usage of nearby alternative splice donors in the NOP56 pre-mRNA. Excess snoRNP core proteins prevent further production of NOP56 and instead trigger the generation of a cytoplasmic snoRD86-containing NOP56-derived lncRNA via the nonsense-mediated decay pathway. Our findings reveal a feedback mechanism based on RNA structure that controls the precise coordination between box C/D snoRNP core proteins and global snoRNA levels. Small nucleolar RNAs (snoRNAs) are abundant noncoding RNAs that are enriched in the nucleoli of eukaryotic cells. They are classified as either box C/D or box H/ACA snoRNAs based on the presence of conserved sequence elements, which mediate the interaction with distinct sets of evolutionarily conserved and essential core protein partners. The complex between a snoRNA and its core proteins—the snoRNP—constitutes a mature functional particle that is directed to its target RNA, primarily pre-rRNA, via antisense guide elements in the snoRNA (Dupuis-Sandoval et al., 2015Dupuis-Sandoval F. Poirier M. Scott M.S. The emerging landscape of small nucleolar RNAs in cell biology.Wiley Interdiscip. Rev. RNA. 2015; 6: 381-397Crossref PubMed Scopus (151) Google Scholar, Matera et al., 2007Matera A.G. Terns R.M. Terns M.P. Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs.Nat. Rev. Mol. Cell Biol. 2007; 8: 209-220Crossref PubMed Scopus (579) Google Scholar, Watkins and Bohnsack, 2012Watkins N.J. Bohnsack M.T. The box C/D and H/ACA snoRNPs: key players in the modification, processing and the dynamic folding of ribosomal RNA.Wiley Interdiscip. Rev. RNA. 2012; 3: 397-414Crossref PubMed Scopus (320) Google Scholar). Upon substrate binding, the snoRNP can site-specifically modify its target nucleotide; box C/D and H/ACA snoRNPs catalyze 2′-O-methylation and pseudouridinylation, respectively (Ganot et al., 1997Ganot P. Bortolin M.L. Kiss T. Site-specific pseudouridine formation in preribosomal RNA is guided by small nucleolar RNAs.Cell. 1997; 89: 799-809Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, Kiss-László et al., 1996Kiss-László Z. Henry Y. Bachellerie J.P. Caizergues-Ferrer M. Kiss T. Site-specific ribose methylation of preribosomal RNA: a novel function for small nucleolar RNAs.Cell. 1996; 85: 1077-1088Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar). Moreover, both types of snoRNAs are believed to assist the correct folding of their target RNAs, and a subset of box C/D snoRNAs are critical for the processing of pre-rRNA (Dupuis-Sandoval et al., 2015Dupuis-Sandoval F. Poirier M. Scott M.S. The emerging landscape of small nucleolar RNAs in cell biology.Wiley Interdiscip. Rev. RNA. 2015; 6: 381-397Crossref PubMed Scopus (151) Google Scholar, Watkins and Bohnsack, 2012Watkins N.J. Bohnsack M.T. The box C/D and H/ACA snoRNPs: key players in the modification, processing and the dynamic folding of ribosomal RNA.Wiley Interdiscip. Rev. RNA. 2012; 3: 397-414Crossref PubMed Scopus (320) Google Scholar). Dysregulation of snoRNAs is a signature for many cancers (Gong et al., 2017Gong J. Li Y. Liu C.J. Xiang Y. Li C. Ye Y. Zhang Z. Hawke D.H. Park P.K. Diao L. et al.A pan-cancer analysis of the expression and clinical relevance of small nucleolar RNAs in human cancer.Cell Rep. 2017; 21: 1968-1981Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, Jorjani et al., 2016Jorjani H. Kehr S. Jedlinski D.J. Gumienny R. Hertel J. Stadler P.F. Zavolan M. Gruber A.R. An updated human snoRNAome.Nucleic Acids Res. 2016; 44: 5068-5082Crossref PubMed Scopus (148) Google Scholar, Williams and Farzaneh, 2012Williams G.T. Farzaneh F. Are snoRNAs and snoRNA host genes new players in cancer?.Nat. Rev. Cancer. 2012; 12: 84-88Crossref PubMed Scopus (261) Google Scholar), and in some cases, it has even been suggested to be a requirement for oncogenesis (Crea et al., 2016Crea F. Quagliata L. Michael A. Liu H.H. Frumento P. Azad A.A. Xue H. Pikor L. Watahiki A. Morant R. et al.Integrated analysis of the prostate cancer small-nucleolar transcriptome reveals SNORA55 as a driver of prostate cancer progression.Mol. Oncol. 2016; 10: 693-703Crossref PubMed Scopus (42) Google Scholar, Su et al., 2014Su H. Xu T. Ganapathy S. Shadfan M. Long M. Huang T.H. Thompson I. Yuan Z.M. Elevated snoRNA biogenesis is essential in breast cancer.Oncogene. 2014; 33: 1348-1358Crossref PubMed Scopus (125) Google Scholar, Zheng et al., 2015Zheng D. Zhang J. Ni J. Luo J. Wang J. Tang L. Zhang L. Wang L. Xu J. Su B. Chen G. Small nucleolar RNA 78 promotes the tumorigenesis in non-small cell lung cancer.J. Exp. Clin. Cancer Res. 2015; (Published online May 15, 2015)https://doi.org/10.1186/s13046-015-0170-5Crossref Scopus (61) Google Scholar, Zhou et al., 2017Zhou F. Liu Y. Rohde C. Pauli C. Gerloff D. Köhn M. Misiak D. Bäumer N. Cui C. Göllner S. et al.AML1-ETO requires enhanced C/D box snoRNA/RNP formation to induce self-renewal and leukaemia.Nat. Cell Biol. 2017; 19: 844-855Crossref PubMed Scopus (100) Google Scholar), underscoring a critical need for cells to control snoRNA levels. Human cells express several hundred different variants of each of the two snoRNA types, many of which are known, or predicted, to guide the modification of specific nucleotides in pre-rRNA. The remaining snoRNAs—the so-called orphans—have no assigned targets (Dupuis-Sandoval et al., 2015Dupuis-Sandoval F. Poirier M. Scott M.S. The emerging landscape of small nucleolar RNAs in cell biology.Wiley Interdiscip. Rev. RNA. 2015; 6: 381-397Crossref PubMed Scopus (151) Google Scholar, Jorjani et al., 2016Jorjani H. Kehr S. Jedlinski D.J. Gumienny R. Hertel J. Stadler P.F. Zavolan M. Gruber A.R. An updated human snoRNAome.Nucleic Acids Res. 2016; 44: 5068-5082Crossref PubMed Scopus (148) Google Scholar). In humans, the few snoRNAs that are involved in pre-rRNA processing are transcribed from independent promoters, whereas the majority—the “modifying” snoRNAs—are hosted within introns of highly transcribed protein-coding RNA or long noncoding RNA (lncRNA) genes. Hence, the production of a mature intron-hosted snoRNA generally depends on the transcription and splicing of its primary host transcript. Excised and debranched introns are normally degraded by exonucleases, but snoRNP or pre-snoRNP formation protects the snoRNA. The “byproduct” from this production scheme is a spliced transcript, either a mRNA or a lncRNA, which may or may not be functional (Brown et al., 2008Brown J.W. Marshall D.F. Echeverria M. Intronic noncoding RNAs and splicing.Trends Plant Sci. 2008; 13: 335-342Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, Dupuis-Sandoval et al., 2015Dupuis-Sandoval F. Poirier M. Scott M.S. The emerging landscape of small nucleolar RNAs in cell biology.Wiley Interdiscip. Rev. RNA. 2015; 6: 381-397Crossref PubMed Scopus (151) Google Scholar). We, and others, have demonstrated that snoRNA host genes are particularly disposed to producing alternatively spliced transcript variants that are subjected to degradation by the cytoplasmic nonsense-mediated decay (NMD) pathway (Colombo et al., 2017Colombo M. Karousis E.D. Bourquin J. Bruggmann R. Mühlemann O. Transcriptome-wide identification of NMD-targeted human mRNAs reveals extensive redundancy between SMG6- and SMG7-mediated degradation pathways.RNA. 2017; 23: 189-201Crossref PubMed Scopus (94) Google Scholar, Lykke-Andersen et al., 2014Lykke-Andersen S. Chen Y. Ardal B.R. Lilje B. Waage J. Sandelin A. Jensen T.H. Human nonsense-mediated RNA decay initiates widely by endonucleolysis and targets snoRNA host genes.Genes Dev. 2014; 28: 2498-2517Crossref PubMed Scopus (117) Google Scholar, Thoren et al., 2010Thoren L.A. Nørgaard G.A. Weischenfeldt J. Waage J. Jakobsen J.S. Damgaard I. Bergström F.C. Blom A.M. Borup R. Bisgaard H.C. Porse B.T. UPF2 is a critical regulator of liver development, function and regeneration.PLoS ONE. 2010; 5: e11650Crossref PubMed Scopus (51) Google Scholar, Weischenfeldt et al., 2008Weischenfeldt J. Damgaard I. Bryder D. Theilgaard-Mönch K. Thoren L.A. Nielsen F.C. Jacobsen S.E. Nerlov C. Porse B.T. NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements.Genes Dev. 2008; 22: 1381-1396Crossref PubMed Scopus (196) Google Scholar). Thus, the exon-coded mRNA, or lncRNA, output from snoRNA host genes can be uncoupled from snoRNA levels via alternative splicing. Moreover, intron-hosted snoRNAs are often situated near or in exons that are subject to alternative splicing events (Lykke-Andersen et al., 2014Lykke-Andersen S. Chen Y. Ardal B.R. Lilje B. Waage J. Sandelin A. Jensen T.H. Human nonsense-mediated RNA decay initiates widely by endonucleolysis and targets snoRNA host genes.Genes Dev. 2014; 28: 2498-2517Crossref PubMed Scopus (117) Google Scholar). This suggests that alternative splicing can also be used by cells to modulate snoRNA production, although an equally valid possibility is that snoRNAs can be directly involved in the regulation of alternative splicing of their cognate pre-mRNA. The present study illustrates a case in which the latter is true. Box C/D snoRNAs have an average length of ∼80 nt and generally contain two repeats of the sequence 5′-RUGAUGA-3′ (where R is a purine), named the C and C′ boxes, and two repeats of the sequence 5′-CUGA-3′, named the D and D′ boxes. The C and D boxes are highly conserved in all box C/D snoRNAs and form a kink-turn structure (the C/D box) through non-conventional base-pairing (Kiss-László et al., 1998Kiss-László Z. Henry Y. Kiss T. Sequence and structural elements of methylation guide snoRNAs essential for site-specific ribose methylation of pre-rRNA.EMBO J. 1998; 17: 797-807Crossref PubMed Scopus (178) Google Scholar, Krogh et al., 2016Krogh N. Jansson M.D. Häfner S.J. Tehler D. Birkedal U. Christensen-Dalsgaard M. Lund A.H. Nielsen H. Profiling of 2′-O-Me in human rRNA reveals a subset of fractionally modified positions and provides evidence for ribosome heterogeneity.Nucleic Acids Res. 2016; 44: 7884-7895Crossref PubMed Scopus (147) Google Scholar, Watkins et al., 2000Watkins N.J. Ségault V. Charpentier B. Nottrott S. Fabrizio P. Bachi A. Wilm M. Rosbash M. Branlant C. Lührmann R. A common core RNP structure shared between the small nucleoar box C/D RNPs and the spliceosomal U4 snRNP.Cell. 2000; 103: 457-466Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, Watkins et al., 2002Watkins N.J. Dickmanns A. Lührmann R. Conserved stem II of the box C/D motif is essential for nucleolar localization and is required, along with the 15.5K protein, for the hierarchical assembly of the box C/D snoRNP.Mol. Cell. Biol. 2002; 22: 8342-8352Crossref PubMed Scopus (174) Google Scholar). The C′ and D′ boxes may form a similar structure (the C′/D′ box), but they are less conserved and possibly of no relevance in some snoRNAs (Kiss-László et al., 1998Kiss-László Z. Henry Y. Kiss T. Sequence and structural elements of methylation guide snoRNAs essential for site-specific ribose methylation of pre-rRNA.EMBO J. 1998; 17: 797-807Crossref PubMed Scopus (178) Google Scholar, Krogh et al., 2016Krogh N. Jansson M.D. Häfner S.J. Tehler D. Birkedal U. Christensen-Dalsgaard M. Lund A.H. Nielsen H. Profiling of 2′-O-Me in human rRNA reveals a subset of fractionally modified positions and provides evidence for ribosome heterogeneity.Nucleic Acids Res. 2016; 44: 7884-7895Crossref PubMed Scopus (147) Google Scholar). In the snoRNP with bound core proteins, the snoRNA adopts a common compact pseudo-symmetric structure, starting with a basal stem followed by the C/D box, antisense guide sequences, the C′/D′ box, and a stem-loop (Lin et al., 2011Lin J. Lai S. Jia R. Xu A. Zhang L. Lu J. Ye K. Structural basis for site-specific ribose methylation by box C/D RNA protein complexes.Nature. 2011; 469: 559-563Crossref PubMed Scopus (97) Google Scholar, Ye et al., 2009Ye K. Jia R. Lin J. Ju M. Peng J. Xu A. Zhang L. Structural organization of box C/D RNA-guided RNA methyltransferase.Proc. Natl. Acad. Sci. USA. 2009; 106: 13808-13813Crossref PubMed Scopus (48) Google Scholar). In addition, a box C/D snoRNP consists of one copy of NHP2L1 bound to the C/D box, a heterodimer of the paralogous proteins NOP58 and NOP56 contacting the C/D and C′/D′ boxes, respectively, and two copies of FBL (Fibrillarin; a 2′-O-methyltransferase), one on each antisense guide (Cahill et al., 2002Cahill N.M. Friend K. Speckmann W. Li Z.H. Terns R.M. Terns M.P. Steitz J.A. Site-specific cross-linking analyses reveal an asymmetric protein distribution for a box C/D snoRNP.EMBO J. 2002; 21: 3816-3828Crossref PubMed Scopus (95) Google Scholar, Lin et al., 2011Lin J. Lai S. Jia R. Xu A. Zhang L. Lu J. Ye K. Structural basis for site-specific ribose methylation by box C/D RNA protein complexes.Nature. 2011; 469: 559-563Crossref PubMed Scopus (97) Google Scholar, Szewczak et al., 2002Szewczak L.B. DeGregorio S.J. Strobel S.A. Steitz J.A. Exclusive interaction of the 15.5 kD protein with the terminal box C/D motif of a methylation guide snoRNP.Chem. Biol. 2002; 9: 1095-1107Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, Ye et al., 2009Ye K. Jia R. Lin J. Ju M. Peng J. Xu A. Zhang L. Structural organization of box C/D RNA-guided RNA methyltransferase.Proc. Natl. Acad. Sci. USA. 2009; 106: 13808-13813Crossref PubMed Scopus (48) Google Scholar). The antisense guide sequences are unique for each box C/D snoRNA and are exposed for base-pairing with the target RNA in the mature snoRNP structure (Lin et al., 2011Lin J. Lai S. Jia R. Xu A. Zhang L. Lu J. Ye K. Structural basis for site-specific ribose methylation by box C/D RNA protein complexes.Nature. 2011; 469: 559-563Crossref PubMed Scopus (97) Google Scholar, Ye et al., 2009Ye K. Jia R. Lin J. Ju M. Peng J. Xu A. Zhang L. Structural organization of box C/D RNA-guided RNA methyltransferase.Proc. Natl. Acad. Sci. USA. 2009; 106: 13808-13813Crossref PubMed Scopus (48) Google Scholar). In addition to the core proteins, formation of a mature box C/D snoRNP requires multiple assembly factors and involves rearrangements of the RNP conformation (Bizarro et al., 2014Bizarro J. Charron C. Boulon S. Westman B. Pradet-Balade B. Vandermoere F. Chagot M.E. Hallais M. Ahmad Y. Leonhardt H. et al.Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control.J. Cell Biol. 2014; 207: 463-480Crossref PubMed Scopus (47) Google Scholar and references therein). A comprehensive model for the multistep box C/D snoRNP assembly process has been proposed and involves an initial protein complex including the core proteins NOP58 and NHP2L1. This complex is rearranged and associates with the box C/D pre-snoRNA and one copy of FBL to establish a catalytically inactive snoRNP assembly complex. During the ultimate maturation steps, the last core protein, NOP56, enters the complex together with a second copy of FBL (Bizarro et al., 2014Bizarro J. Charron C. Boulon S. Westman B. Pradet-Balade B. Vandermoere F. Chagot M.E. Hallais M. Ahmad Y. Leonhardt H. et al.Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control.J. Cell Biol. 2014; 207: 463-480Crossref PubMed Scopus (47) Google Scholar). In this model, NOP56 is therefore decisive for the final maturation and activation of catalytically active box C/D snoRNPs. In this study, we demonstrate that a snoRNA can function as a cis-acting modulator of alternative splicing. Using HEK293 cells as the model system, we show that the orphan snoRD86, situated in the NOP56 pre-mRNA, controls NOP56 protein levels in response to the availability of both box C/D snoRNP assembly factors and NOP56 itself. Dysregulation of NOP56 levels leads to altered ribosome biogenesis and cell-cycle progression, demonstrating the importance of a balanced expression of NOP56. We have previously demonstrated that NOP56 protein expression is dictated by alternative splicing of its snoRNA hosting pre-mRNA and involves the NMD pathway (Lykke-Andersen et al., 2014Lykke-Andersen S. Chen Y. Ardal B.R. Lilje B. Waage J. Sandelin A. Jensen T.H. Human nonsense-mediated RNA decay initiates widely by endonucleolysis and targets snoRNA host genes.Genes Dev. 2014; 28: 2498-2517Crossref PubMed Scopus (117) Google Scholar). Specifically, the NOP56 pre-mRNA, which hosts five snoRNAs within its introns (Figure 1A, top), gives rise to two major splice variants: (1) the protein-coding mRNA (pc-mRNA) encoding NOP56 and (2) the NMD substrate mRNA (ns-mRNA). After triggering NMD, the ns-mRNA gives rise to a stable 3′ fragment derived by SMG6-mediated endonucleolytic cleavage (Figure 1A, bottom right). pc- and ns-mRNA differ due to the usage of alternative splice donors up- (uSD, pc-mRNA) and downstream (dSD, ns-mRNA) of one of the encoded box C/D snoRNAs, snoRD86 (Figures 1A and 1B). The 3′ fragment has snoRD86 residing at its 5′ terminus, a poly(A) tail at its 3′ end, and is enriched in the cytoplasm (Lykke-Andersen et al., 2014Lykke-Andersen S. Chen Y. Ardal B.R. Lilje B. Waage J. Sandelin A. Jensen T.H. Human nonsense-mediated RNA decay initiates widely by endonucleolysis and targets snoRNA host genes.Genes Dev. 2014; 28: 2498-2517Crossref PubMed Scopus (117) Google Scholar). We dubbed this lncRNA “snoRD86-cSPA” due to its resemblance to nuclear 5′-snoRNA-ended and 3′-polyadenylated (SPA) lncRNAs (Wu et al., 2016Wu H. Yin Q.F. Luo Z. Yao R.W. Zheng C.C. Zhang J. Xiang J.F. Yang L. Chen L.L. Unusual processing generates SPA lncRNAs that sequester multiple RNA binding proteins.Mol. Cell. 2016; 64: 534-548Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, Wu et al., 2017Wu H. Yang L. Chen L.L. The diversity of long noncoding RNAs and their generation.Trends Genet. 2017; 33: 540-552Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), with the “c” reflecting the cytoplasmic localization of snoRD86-cSPA. Quantification of published poly(A)-enriched RNA sequencing (RNA-seq) data from human embryonic kidney Flp-In T-Rex (HEK293FT) cells (Lykke-Andersen et al., 2014Lykke-Andersen S. Chen Y. Ardal B.R. Lilje B. Waage J. Sandelin A. Jensen T.H. Human nonsense-mediated RNA decay initiates widely by endonucleolysis and targets snoRNA host genes.Genes Dev. 2014; 28: 2498-2517Crossref PubMed Scopus (117) Google Scholar), using the Cufflinks software suite (Trapnell et al., 2012Trapnell C. Roberts A. Goff L. Pertea G. Kim D. Kelley D.R. Pimentel H. Salzberg S.L. Rinn J.L. Pachter L. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.Nat. Protoc. 2012; 7: 562-578Crossref PubMed Scopus (170) Google Scholar), revealed that NOP56 is among the most highly expressed genes at steady state (Figure 1C, left). Notably, snoRD86-cSPA is very abundant, constituting ∼80% of the total exon-coded NOP56 RNA (Figure 1C, right). This observation was supported by quantifications of exon-coded NOP56 RNA species by northern blotting analysis using two different probes (Figures 1D and 1E), reverse transcription (RT)-PCR using primers flanking the alternatively spliced region with subsequent cloning and sequencing of PCR products (Figure S1A; data not shown), and quantitative RT PCR (RT-qPCR) specifically detecting pc- and ns-mRNA (Figures 1D and S1B). Under control conditions, snoRD86-cSPA was clearly detected by northern blotting analysis to be present at higher levels than pc- and ns-mRNA combined (Figure 1E, lanes 1 and 3). Depletion of the NMD-specific endonuclease SMG6 (Figure S1D) led to the accumulation of ns-mRNA and disappearance of snoRD86-cSPA (Figure 1E, lanes 1 and 2). Treatment with the translation inhibitor cycloheximide, which also inhibits NMD, had a similar effect (Figures 1E, lanes 3–5, S1A, and quantified in S1B and S1C). This supported the conclusion that ns-mRNA is a substrate for the translation-dependent NMD pathway, which, via endonucleolytic cleavage by SMG6, produces snoRD86-cSPA. Consistently, snoRD86-cSPA could be specifically degraded by treatment with the 5′-3′ exonuclease Terminator (Figure S1E, compare lanes 1 and 2), demonstrating the presence of a monophosphate at its 5′ end. Subcellular fractionation confirmed that snoRD86-cSPA is almost exclusively cytoplasmic at steady state (Figure S1F). Moreover, analysis of published FBL RNA immunoprecipitation sequencing data from the PA1 cell line (Wu et al., 2016Wu H. Yin Q.F. Luo Z. Yao R.W. Zheng C.C. Zhang J. Xiang J.F. Yang L. Chen L.L. Unusual processing generates SPA lncRNAs that sequester multiple RNA binding proteins.Mol. Cell. 2016; 64: 534-548Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) demonstrated that snoRD86-cSPA is bound by FBL (Figure S1G). Finally, northern blotting analysis of RNA from various human cell lines and the African green monkey kidney cell line Cos-7 demonstrated that the ratio between the NOP56 mRNA variants and the snoRD86-cSPA varies greatly (Figure S1H). This likely reflects that alternative splicing of the NOP56 pre-mRNA is subject to considerable regulation. Characterization of the RNA output from the NOP56 locus and the conspicuous positioning of snoRD86 between the regulated splice donors made us hypothesize that NOP56 protein levels are autoregulated and that the snoRD86 sequence might act as a sensor for box C/D snoRNP core proteins to switch between the pc- and ns-mRNA variants (Figure 2A). To address this theory, we used small interfering RNAs (siRNAs) to deplete the box C/D snoRNP core proteins NOP56, NOP58, and FBL (Figure 2B). Since snoRNP core proteins are essential (Gautier et al., 1997Gautier T. Bergès T. Tollervey D. Hurt E. Nucleolar KKE/D repeat proteins Nop56p and Nop58p interact with Nop1p and are required for ribosome biogenesis.Mol. Cell. Biol. 1997; 17: 7088-7098Crossref PubMed Scopus (232) Google Scholar, Newton et al., 2003Newton K. Petfalski E. Tollervey D. Cáceres J.F. Fibrillarin is essential for early development and required for accumulation of an intron-encoded small nucleolar RNA in the mouse.Mol. Cell. Biol. 2003; 23: 8519-8527Crossref PubMed Scopus (84) Google Scholar, Schimmang et al., 1989Schimmang T. Tollervey D. Kern H. Frank R. Hurt E.C. A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability.EMBO J. 1989; 8: 4015-4024Crossref PubMed Scopus (236) Google Scholar, Watkins et al., 2000Watkins N.J. Ségault V. Charpentier B. Nottrott S. Fabrizio P. Bachi A. Wilm M. Rosbash M. Branlant C. Lührmann R. A common core RNP structure shared between the small nucleoar box C/D RNPs and the spliceosomal U4 snRNP.Cell. 2000; 103: 457-466Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar), and long-term depletion is expected to cause cell death, we tested the impact of core protein depletion on cell-cycle parameters. Importantly, our depletion conditions only mildly impacted the cell-cycle profile and cell morphology (Figures S2A and S2B) and thus proved feasible for further study. Conversely, depletion of NHP2L1 led to rapid cell death (data not shown), thus rendering further analysis of this condition meaningless. Next, we evaluated the accompanying NOP56 exon-coded RNA levels (Figures 2C, 2D, S2C, and S2D). First, depletion of NOP56 using a siRNA targeting the common exon 10 served as a control for the RNA detection methods, and, as expected, levels of pc-mRNA and snoRD86-cSPA were substantially reduced (Figures 2C and S2C, compare lanes 1 and 2, quantification in Figures 2D and S2D). When NMD was inhibited by depletion of SMG6, ns-mRNA accumulated ∼3-fold (Figures 2C and S2C, compare lanes 1 and 5, quantification by RT-qPCR in Figure 2D). Finally, and in accordance with the stated hypothesis, depletion of either NOP58 or FBL led to an ∼3-fold rise in levels of pc-mRNA (Figures 2C and S2C, compare lane 1 with lanes 3 and 4, quantification by RT-qPCR in Figure 2D). Concurrently, ns-mRNA (Figures 2D and S2C) and snoRD86-cSPA (Figure 2C, quantification in Figure S2D) levels dropped significantly. Taken together, these results supported our initial hypothesis. In keeping with the accumulation of the protein-coding NOP56 pc-mRNA, quantitative western blotting analysis revealed 6- to 7-fold elevated NOP56 protein levels upon depletion of NOP58 or FBL (Figures 2E and S1D). We note that NOP58 protein levels also increased upon depletion of NOP56 or FBL (Figures 2B and S1D; see Discussion). We conducted NOP56 immunofluorescence (IF) experiments to determine the subcellular localization of accumulated NOP56. Upon depletion of NOP58 or FBL, the nucleoplasmic and cytoplasmic NOP56 staining increased (Figure 2F, quantification in Figure 2G, cytoplasmic staining was not quantified). Moreover, co-staining with specific markers for nucleoli (nucleolin) and Cajal bodies (coilin) suggested that depletion of either of the box C/D core proteins NOP56 and NOP58 and, to a lesser extent, FBL caused changes in the nucleolar constitution, whereas Cajal bodies appeared largely unchanged (Figures S2E and S2F). In sum, depletion of either of the box C/D core proteins NOP58 and FBL leads to nucleoplasmic accumulation of NOP56 and altered nucleolar organization. To further examine snoRD86’s role in alternative splicing of the NOP56 pre-mRNA, we constructed a reporter gene consisting of a tetracycline-inducible cytomegalovirus (CMV) promoter followed by NOP56 exons 7 to 12 and their respective introns (the corresponding exon-coded RNAs are displayed in Figure 3A). The reporter was integrated into HEK293FT cells, and its tetracycline-inducible expression was verified by northern blotting analysis with probes directed against either the 5′ or the 3′ end of the gene (Figure S3A). As for the endogenous NOP56 exon-coded RNAs, the reporter versions of (1) pc-mRNA accumulated when NOP58 or FBL were depleted (Figures 3B, S3B, and S3C, compare lanes 2 and 3 to lane 1), (2) ns-mRNA could primarily be detected upon depletion of SMG6 (Figures 3B, S3B, and S3C, compare lane 4 to lanes 1–3), and (3) snoRD86-cSPA displayed reduced levels upon depletion of NOP58, FBL, or SMG6 (Figures 3B and S3B, compare lane 1 to lanes 2–4). Moreover, Terminator could degrade the reporter snoRD86-cSPA, whereas pc- and ns-mRNA were resistant (Figure S3B, compare lanes 5–8 to 1–4). With a workable reporter system in hand, we next constructed three mutants in which SNORD86 was (1) deleted (ΔD86), (2) replaced by its reverse complementary sequence (D86rc), or (3) replaced with an unrelated box C/D snoRNA gene SNORD71 (D71) (Figure S3D). All NOP56 reporter cell lines were tested for equal copy numbers to assure that the expression levels of reporter RNAs were directly comparable (Figure S3E). siRNA-mediated knockdown of NOP58 (Figure S3F) was used as a proxy for sensitivity toward box C/D snoRNA core proteins. Additionally, we reasoned that knockdown of SMG6 (Figure S3F) would serve to get the best possible estimate of ns-mRNA levels. RT-qPCR analysis demonstrated that whereas NOP58 depletion led to a 2.5-fold increase in wild-type (WT) pc-mRNA levels, no accumulation was observed for the three tested mutants (Figure 3C, top, compare columns 1 and 2 for each version of the reporter gene). Thus, snoRD86 is required in cis for the regulation of NOP56 pre-mRNA splicing. Accordingly, overexpression of snoRD86 in trans from a heterologous host gene did not change the splicing of endogenous NOP56 pre-mRNA (Figures S3G–S3I). Importantly, under control conditions, the expression of pc-mRNA from both the ΔD86 and the D86rc constructs was similar to that of the WT (Figure 3C, top, compare first columns for each version of the reporter gene). The same was also true for ns-mRNA upon SMG6 depletion (Figure 3C, bottom, compare first or third columns for each version of the reporter gene). Therefore, it is evident that both splice donors are at" @default.
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• W2892078811 date "2018-10-01" @default.
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• W2892078811 title "Box C/D snoRNP Autoregulation by a cis-Acting snoRNA in the NOP56 Pre-mRNA" @default.
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• W2892078811 doi "https://doi.org/10.1016/j.molcel.2018.08.017" @default.
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