A Budding Yeast Model for Human Disease Mutations in the EXOSC2 Cap Subunit of the RNA Exosome
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PROVIDER: PRJNA684912 | ENA |
REPOSITORIES: ENA
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Project description:RNA exosomopathies, a growing family of tissue-specific diseases, are linked to missense mutations in genes encoding the structural subunits of the conserved 10-subunit exoribonuclease complex, the RNA exosome. Such mutations in the cap subunit gene EXOSC2 cause the novel syndrome SHRF (Short stature, Hearing loss, Retinitis pigmentosa and distinctive Facies). In contrast, exosomopathy mutations in the cap subunit gene EXOSC3 cause pontocerebellar hypoplasia type 1b (PCH1b). Though having strikingly different disease pathologies, EXOSC2 and EXOSC3 exosomopathy mutations result in amino acid substitutions in similar, conserved domains of the cap subunits, suggesting that these exosomopathy mutations have distinct consequences for RNA exosome function. We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by introducing the EXOSC2 mutations in the orthologous S. cerevisiae gene RRP4. The resulting rrp4 mutant cells have defects in cell growth and RNA exosome function. We detect significant transcriptomic changes in both coding and non-coding RNAs in the rrp4 variant, rrp4-G226D, which models EXOSC2 p.Gly198Asp. Comparing this rrp4-G226D mutant to the previously studied S. cerevisiae model of EXOSC3 PCH1b mutation, rrp40-W195R, reveals that these mutants have disparate effects on certain RNA targets, providing the first evidence for different mechanistic consequences of these exosomopathy mutations. Congruently, we detect specific negative genetic interactions between RNA exosome cofactor mutants and rrp4-G226D but not rrp40-W195R. These data provide insight into how SHRF mutations could alter the function of the RNA exosome and allow the first direct comparison of exosomopathy mutations that cause distinct pathologies.
2020-12-14 | GSE163106 | GEO
Project description:The RNA exosome is an evolutionarily conserved exoribonuclease complex that consists of a 3-subunit cap, a 6-subunit barrel-shaped core, and a catalytic base subunit. Missense mutations in genes encoding structural subunits of the RNA exosome cause a growing family of diseases with diverse pathologies, collectively termed RNA exosomopathies. The disease symptoms vary and can manifest as neurological defects or developmental disorders. The diversity of the RNA exosomopathy pathologies suggests that the different missense mutations in structural genes result in distinct in vivo consequences. To investigate these functional consequences and distinguish whether they are unique to each RNA exosomopathy mutation, we generated a collection of in vivo models using budding yeast by introducing pathogenic missense mutations in orthologous S. cerevisiae genes. We then performed a comparative RNA-seq analysis to assess broad transcriptomic changes in each mutant model. Three of the mutant models rrp4-G226D, rrp40-W195R and rrp46-L191H, which model mutations in the genes encoding structural subunits of the RNA exosome, EXOSC2, EXOSC3 and EXOSC5 showed the largest transcriptomic differences. Further analyses revealed shared increased transcripts enriched in translation or ribosomal RNA modification/processing pathways across the three mutant models. Studies of the impact of the mutations on translation revealed shared defects in ribosome biogenesis but distinct impacts on translation. Collectively, our results provide the first comparative analysis of several RNA exosomopathy mutant models and suggest that different RNA exosomopathy mutations result in in vivo consequences that are both unique and shared across each variant, providing more insight into the biology underlying each distinct pathology.
2024-02-01 | GSE246957 | GEO
Project description:We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis that are undetectable in wild-type cells, showing that the nuclear exosome remains functional for CUT degradation. Lack of TRAMP activity stabilises ~1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC). One sample each of Cbc2-associated RNA from wild-type and trf4-deleted cells at 6 hours of meiosis
2014-08-09 | E-GEOD-60221 | biostudies-arrayexpress
Project description:We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis that are undetectable in wild-type cells, showing that the nuclear exosome remains functional for CUT degradation. Lack of TRAMP activity stabilises ~1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC).
2014-08-09 | GSE60221 | GEO
Project description:Nuclear processing and quality control of eukaryotic RNA is mediated by the multi-subunit RNA exosome, which utilizes accessory factors to regulate its enzymatic activity. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we disclose a physical link between the human nuclear RNA exosome and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA), and then further connects together with the uncharacterized ZC3H18/NHN1 protein to the nuclear exosome targeting (NEXT) complex, forming CBC-NEXT (CBCN). RNA immunoprecipitation analysis using CBCN factors as baits as well as the combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through transcription of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5’cap links transcription termination to exosomal RNA degradation via CBCN.
2013-11-08 | GSE52132 | GEO
Project description:Nuclear RNA degradation pathways are highly conserved across eukaryotes and play important roles in RNA quality control. Key substrates for exosomal degradation include aberrant functional RNAs and cryptic unstable transcripts (CUTs). It has recently been reported that the nuclear exosome is inactivated during meiosis in budding yeast through degradation of the subunit Rrp6, leading to the stabilisation of a subset of meiotic unstable transcripts (MUTs) of unknown function. We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis which are undetectable in wild-type cells, suggesting that the nuclear exosome remains functional, and we further show that the meiotic exosome complex still contains Rrp6. Indeed Rrp6 over-expression is insufficient to suppress MUT transcripts, showing that the reduced amount of Rrp6 in meiotic cells does not directly cause MUT accumulation. Lack of TRAMP activity stabilises ~1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC). CBC mutants display defects in the formation of meiotic double strand breaks (DSBs), and we see similar defects in TRAMP mutants, suggesting that a key function of the nuclear exosome is to prevent saturation of the CBC complex by CUTs. Together, our results show that the nuclear exosome remains active in meiosis and has an important role in facilitating meiotic recombination.
2015-05-15 | PXD001239 | Pride
Project description:Nuclear RNA degradation pathways are highly conserved across eukaryotes and play important roles in RNA quality control. Key substrates for exosomal degradation include aberrant functional RNAs and cryptic unstable transcripts (CUTs). It has recently been reported that the nuclear exosome is inactivated during meiosis in budding yeast through degradation of the subunit Rrp6, leading to the stabilisation of a subset of meiotic unstable transcripts (MUTs) of unknown function. We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis which are undetectable in wild-type cells, suggesting that the nuclear exosome remains functional, and we further show that the meiotic exosome complex still contains Rrp6. Indeed Rrp6 over-expression is insufficient to suppress MUT transcripts, showing that the reduced amount of Rrp6 in meiotic cells does not directly cause MUT accumulation. Lack of TRAMP activity stabilises ~1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC). CBC mutants display defects in the formation of meiotic double strand breaks (DSBs), and we see similar defects in TRAMP mutants, suggesting that a key function of the nuclear exosome is to prevent saturation of the CBC complex by CUTs. Together, our results show that the nuclear exosome remains active in meiosis and has an important role in facilitating meiotic recombination.
2015-05-15 | PXD001223 | Pride
Project description: Not available
| S-EPMC8370739 | biostudies-literature
Project description:Nuclear processing and quality control of eukaryotic RNA is mediated by the multi-subunit RNA exosome, which utilizes accessory factors to regulate its enzymatic activity. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we disclose a physical link between the human nuclear RNA exosome and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA), and then further connects together with the uncharacterized ZC3H18/NHN1 protein to the nuclear exosome targeting (NEXT) complex, forming CBC-NEXT (CBCN). RNA immunoprecipitation analysis using CBCN factors as baits as well as the combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through transcription of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5M-bM-^@M-^Ycap links transcription termination to exosomal RNA degradation via CBCN. In total 10 samples; 4 control IP HeLa, 2 Flag- Ars2 IP, 2 Flag-CBP20 IP; and 2 GFP-RBM7 Ips. The signal intensity data was analyzed with the Affymetrix Tiling Analysis Software (v. 1.1) using parameters: one side upper, 90 bp bandwidth and perfect match only. Output txt files (i.e. result file). TASParam* files include parameter settings and input file information for the TAS analysis for each result file. The description of 'ProcessedDatas_TA.xls' file is provided in the 'README.txt'
2013-11-08 | E-GEOD-52132 | biostudies-arrayexpress
Project description:Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex. Identification of targets for individual exosome subunits in wild-type and mutant yeast cells.
2012-09-25 | E-GEOD-40046 | biostudies-arrayexpress