SMUG1 promotes telomere maintenance through telomerase RNA end-processing
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE116580 | GEO | 2019/08/11
REPOSITORIES: GEO
Similar Datasets
Project description:Ribonucleoprotein enzymes require specific and dynamic conformations of their RNA constituents for regulated catalysis. Telomerase RNA components (TRs) rely on two conserved domains, a pseudoknot/template sequence and a three-way junction (CR4/5), each of which binds the telomerase reverse transcriptase protein (TERT). Vertebrate TRs evolved a third element, the H/ACA domain, involved in assembly and trafficking of telomerase through binding telomerase cofactors, dyskerin and TCAB1, respectively. Here, we show that telomerase unexpectedly requires TCAB1 for enzyme catalysis and for shaping the conformation of the TR CR4/5 domain. Human and mouse cells lacking TCAB1 exhibit a marked reduction in telomerase activity, but show no defect in enzyme assembly. Instead, loss of TCAB1 causes specific unfolding of critical RNA helices in TR CR4/5 required for catalysis, and impairs TR-TERT association. CR4/5 mutations derived from patients with the telomere disorder dyskeratosis congenita phenocopy the loss of enzyme activity and disruption of TERT binding observed with TCAB1 deletion. These findings show that the H/ACA element acquired by telomerase during vertebrate evolution serves an unanticipated role in controlling folding of the essential TR CR4/5 domain, facilitating optimal TERT engagement and enabling telomerase catalysis through the action of TCAB1 protein tethered at the H/ACA domain.
2018-06-24 | GSE97486 | GEO
Project description:Telomerase is a specialized reverse transcriptase that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-containing pseudoknot (t/PK) and the three-way junction (CR4/5). These two hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are thus essential for telomerase catalytic activity. Here, we probe the structure of hTR in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and ensemble deconvolution analysis. Unexpectedly, approximately 15% of the steady state population of hTR has a CR4/5 conformation lacking features required for hTERT binding. Mutagenesis demonstrates that stabilization of the alternative CR4/5 conformation is detrimental to telomerase assembly and activity. We propose that this misfolded portion of the cellular hTR pool is either slowly refolded or degraded. Thus, kinetic traps for RNA folding that have been so well-studied in vitro may also present barriers for assembly of ribonucleoprotein complexes in vivo.
2023-10-20 | GSE245536 | GEO
Project description:Dyskeratosis congenita is a bone marrow failure syndrome characterized by the presence of short telomeres at presentation. The X-linked form is caused by mutations in the gene DKC1, encoding the protein dyskerin. Dyskerin is required for in the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA) processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, suggesting induced pluripotent stem (iPS) cells from X-linked DC patients may provide information about the mechanisms involved. Here we show that in iPS cells with DKC1 mutations Q31E, A353V and ΔL37 telomere maintenance is compromised with short telomere lengths and decreased telomerase activity. The degree to which telomere lengths are affected by expression of telomerase during reprograming, or with ectopic expression of wild type dyskerin varies, with recurrent mutation A353V showing the most severe effect on telomere maintenance. A353V cells but not Q31E or ΔL37 cells, are refractory to correction by incorporation of a single copy of a wild type DKC1 cDNA into the AAVS1 safe harbor locus. None of the mutant cells show decreased pseudouridine levels in rRNA or defective rRNA processing. Finally transcriptome analysis of the iPS cells shows that WNT signaling is significantly decreased in all mutant cells, raising the possibility that defective WNT signaling may contribute to disease pathogenesis.
2015-03-13 | GSE66849 | GEO
Project description:Reduced oxygen availability (hypoxia) triggers adaptive cellular responses via hypoxia-inducible factor (HIF)-dependent transcriptional activation. Adaptation to hypoxia also involves transcription-independent processes like post-translational modifications, however these mechanisms are poorly characterized. Investigating the involvement of protein SUMOylation in response to hypoxia, we discovered that hypoxia strongly decreases the SUMOylation of Exosome subunit 10 (EXOSC10), the catalytic subunit of the RNA exosome, in a HIF-independent manner. EXOSC10 is a multifunctional exoribonuclease enriched in the nucleolus that mediates the processing and degradation of various RNA species. We demonstrate that the Ubiquitin-specific protease 36 (USP36) SUMOylates EXOSC10 and we reveal SUMO1/sentrin-specific peptidase 3 (SENP3) as the enzyme mediating deSUMOylation of EXOSC10. Under hypoxia, EXOSC10 dissociates from USP36 and translocates from the nucleolus to the nucleoplasm concomitant with its deSUMOylation. Loss of EXOSC10 SUMOylation does not detectably affect rRNA maturation but affects the mRNA transcriptome by modulating the expression levels of hypoxia-related genes. Our data suggest that dynamic modulation of EXOSC10 SUMOylation and localization under hypoxia regulates the RNA degradation machinery to facilitate cellular adaptation to low oxygen conditions.
2024-01-30 | GSE229894 | GEO
Project description:Mutations in genes encoding components of the telomerase holoenzyme complex result in a spectrum of rare genetic disorders known as telomere diseases, including dyskeratosis congenita (DC). A consistent finding in DC due to pathogenic mutations in DKC1, which encodes dyskerin, is decreased steady-state levels of the non-coding RNA component of telomerase (TERC) and thus impaired telomere maintenance. Dyskerin binds hundreds of other small nucleolar RNAs (snoRNAs). However, the mechanisms by which DKC1 mutations cause variable impacts on these snoRNAs are poorly understood, which is a barrier to understanding disease mechanisms in DC beyond impaired telomere maintenance. Here, using somatic and induced pluripotent stem cells (iPSCs) from DC patients with DKC1 mutations and CRISPR-Cas9-engineered iPSCs, we show that mutations in the N-terminal extension domain (NTE) of dyskerin dysregulate the biogenesis of a subset of snoRNAs, with the most prominent effect on scaRNA13 (small Cajal body-specific RNA 13). In patient iPSCs carrying the del37L dyskerin NTE-domain mutation but not in those with C-terminal mutations, nascent scaRNA13 transcripts showed a discrete population of 3´-extended forms, as seen in the setting of DC-causing mutations in the PARN (polyA-specific ribonuclease) gene. By deep sequencing of RNA 3´ ends, we found that aberrant scaRNA13 transcripts were composed of genomically-encoded extensions and post-transcriptionally oligoadenylated species, mediated by the noncanonical polymerase PAPD5, which counters PARN. NTE domain mutations generated using CRISPR-Cas9 engineering of the endogenous DKC1 recapitulated the scaRNA13 3´-end processing defects seen in del37L patient cells. Conversely, repair of the DKC1 del37L mutation and genetic or pharmacological manipulation of PAPD5 rescued scaRNA13 end processing defects and steady-state levels. Analysis of the human telomerase cryo-EM structure showed that the dyskerin NTE interacts with 3´ end of bound RNA, suggesting that mutations in this domain impair 3´ end protection of nascent scaRNA13 in addition to canonical functions in snoRNA stabilization. Our results provide mechanistic insights into the interplay of dyskerin and the PARN/PAPD5 axis in the biogenesis and accumulation of snoRNAs beyond TERC, which has important implications for our broader understanding of ncRNA dysregulation in human diseases.
2022-08-01 | GSE190173 | GEO
Project description:The RNA exosome is an essential 3’ to 5’ processing exoribonuclease complex that mediates degradation, processing, and quality control of virtually all eukaryotic RNAs. The nucleolar RNA exosome, consisting of a 9-subunit core and a distributive 3ʹ to 5ʹ exonuclease EXOSC10, plays a critical role in processing and degrading nucleolar RNAs, including pre-ribosomal RNA (pre-rRNA). However, how the RNA exosome is regulated in the nucleolus is poorly understood. Here, we report that the nucleolar ubiquitin-specific protease USP36 is a novel regulator of the nucleolar RNA exosome. USP36 binds to the RNA exosome through direct interaction with EXOSC10. Interestingly, USP36 does not significantly regulate the levels of EXOSC10 and other exosome subunits. Instead, it mediates EXOSC10 SUMOylation at Lys (K) 583. Mutating K583 impaired the binding of EXOSC10 to pre-rRNAs and the K583R mutant failed to rescue the defects in rRNA processing and cell growth inhibition caused by knockdown of endogenous EXOSC10, indicating that EXOSC10 SUMOylation is critical for the exosome function in rRNA processing. Furthermore, USP36 itself is a rRNA-binding protein that associates pre-rRNA. These results suggest that USP36 acts as a novel SUMO ligase to mediate EXOSC10 SUMOylation critical for the RNA exosome function in rRNA processing and ribosome biogenesis.
2023-07-20 | PXD031592 | Pride
Project description:The Microprocessor complex (DGCR8/Drosha) is required for microRNA (miRNA) biogenesis but also binds and regulates the stability of several types of cellular RNAs. Of particular interest, DGCR8 controls the stability of mature small nucleolar RNA (snoRNA) transcripts independently of Drosha, suggesting the existence of alternative DGCR8 complex/es with other nucleases to process a variety of cellular RNAs. Here, we found that DGCR8 co-purifies with subunits of the nuclear exosome, preferentially associating with its hRRP6-containing nucleolar form. Importantly, we demonstrate that DGCR8 is essential for the recruitment of the exosome to snoRNAs and to human telomerase RNA. In addition, we show that the DGCR8/exosome complex controls the stability of the human telomerase RNA component (hTR/TERC). Altogether, these data suggests that DGCR8 acts as a novel adaptor to recruit the exosome complex to structured RNAs and induce their degradation. [i] Examination of the RNA binding profile of hRRP6 (also known as EXOSC10) via iCLIP. [ii] HeLa cells were transiently depleted of hRRP6 or DGCR8 using siRNAs. For a control an non-targetting (siNon) siRNA was used. Three biological replicates of each samples were sent for RNA sequencing.
2015-12-10 | E-GEOD-73376 | biostudies-arrayexpress
Project description:Primary telomerase RNA transcripts are processed into shorter mature forms that assemble into a complex with the catalytic subunit and provide the template for telomerase activity. In diverse fungi telomerase RNA 3â end processing involves a single cleavage reaction by the spliceosome akin to the first step of splicing. Longer forms of human telomerase RNA (hTR) have been reported, but how the mature form of precisely 451 nucleotides is generated is still unknown. We now show that the splicing inhibitor isoginkgetin causes accumulation of long hTR transcripts, but find no evidence for a direct role for splicing in hTR processing. Instead, isoginkgetin mimics the effects of inhibiting the RNA exosome. Depletion of exosome components and accessory factors reveals functions for the cap binding complex (CBC) and the nuclear exosome targeting (NEXT) complex in hTR turnover. Whereas longer transcripts are predominantly degraded, shorter precursor RNAs are oligo-adenylated by TRF4-2 and either processed by poly (A) specific ribonuclease (PARN) or degraded by the exosome. Our results reveal that hTR biogenesis involves a kinetic competition between RNA processing and quality control pathways and suggest new treatment options for dyskeratosis congenita caused by mutations in RNA processing factors. We cloned and sequenced 3â ends by RLM-RACE coupled with high-throughput sequencing to gain further insights into hTR processing.
2015-11-25 | E-GEOD-73776 | biostudies-arrayexpress
Project description:Primary telomerase RNA transcripts are processed into shorter mature forms that assemble into a complex with the catalytic subunit and provide the template for telomerase activity. In diverse fungi telomerase RNA 3’ end processing involves a single cleavage reaction by the spliceosome akin to the first step of splicing. Longer forms of human telomerase RNA (hTR) have been reported, but how the mature form of precisely 451 nucleotides is generated is still unknown. We now show that the splicing inhibitor isoginkgetin causes accumulation of long hTR transcripts, but find no evidence for a direct role for splicing in hTR processing. Instead, isoginkgetin mimics the effects of inhibiting the RNA exosome. Depletion of exosome components and accessory factors reveals functions for the cap binding complex (CBC) and the nuclear exosome targeting (NEXT) complex in hTR turnover. Whereas longer transcripts are predominantly degraded, shorter precursor RNAs are oligo-adenylated by TRF4-2 and either processed by poly (A) specific ribonuclease (PARN) or degraded by the exosome. Our results reveal that hTR biogenesis involves a kinetic competition between RNA processing and quality control pathways and suggest new treatment options for dyskeratosis congenita caused by mutations in RNA processing factors.
2015-11-25 | GSE73776 | GEO
Project description:Human telomerase RNA (hTR) is transcribed as a precursor that is then posttranscriptionally modified and processed. A fraction of the transcripts is oligoadenylated by TRAMP and either processed into the mature hTR or degraded by the exosome. Here, we characterize the processing of 3’ extended forms of varying length by PARN and RRP6. We show that tertiary RNA interactions unique to the longer precursor forms favor RNA degradation, whereas H/ACA RNP assembly stimulates productive processing. Interestingly, the H/ACA complex actively promotes processing in addition to protecting the mature 3’ end. Processing occurs in two steps with longer forms being trimmed by RRP6 and shorter forms being preferentially processed by PARN. These results reveal how RNA structure and RNP assembly affect the kinetics of processing and degradation and ultimately determine the amount of functional telomerase produced in cells.
2018-12-26 | GSE111081 | GEO