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A budding yeast model for human disease mutations in the EXOSC2 cap subunit of the RNA exosome complex.

ABSTRACT: RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4 The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.

SUBMITTER: Sterrett MC

PROVIDER: S-EPMC8370739 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A Budding Yeast Model for Human Disease Mutations in the EXOSC2 Cap Subunit of the RNA Exosome

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

A Budding Yeast Model for Human Disease Mutations in the EXOSC2 Cap Subunit of the RNA Exosome

Project description:A Budding Yeast Model for Human Disease Mutations in the EXOSC2 Cap Subunit of the RNA Exosome

| PRJNA684912 | ENA

Insight into the RNA Exosome Complex Through Modeling Pontocerebellar Hypoplasia Type 1b Disease Mutations in Yeast.

Project description:Pontocerebellar hypoplasia type 1b (PCH1b) is an autosomal recessive disorder that causes cerebellar hypoplasia and spinal motor neuron degeneration, leading to mortality in early childhood. PCH1b is caused by mutations in the RNA exosome subunit gene, EXOSC3 The RNA exosome is an evolutionarily conserved complex, consisting of nine different core subunits, and one or two 3'-5' exoribonuclease subunits, that mediates several RNA degradation and processing steps. The goal of this study is to assess the functional consequences of the amino acid substitutions that have been identified in EXOSC3 in PCH1b patients. To analyze these EXOSC3 substitutions, we generated the corresponding amino acid substitutions in the Saccharomyces cerevisiae ortholog of EXOSC3, Rrp40 We find that the rrp40 variants corresponding to EXOSC3-G31A and -D132A do not affect yeast function when expressed as the sole copy of the essential Rrp40 protein. In contrast, the rrp40-W195R variant, corresponding to EXOSC3-W238R in PCH1b patients, impacts cell growth and RNA exosome function when expressed as the sole copy of Rrp40 The rrp40-W195R protein is unstable, and does not associate efficiently with the RNA exosome in cells that also express wild-type Rrp40 Consistent with these findings in yeast, the levels of mouse EXOSC3 variants are reduced compared to wild-type EXOSC3 in a neuronal cell line. These data suggest that cells possess a mechanism for optimal assembly of functional RNA exosome complex that can discriminate between wild-type and variant exosome subunits. Budding yeast can therefore serve as a useful tool to understand the molecular defects in the RNA exosome caused by PCH1b-associated amino acid substitutions in EXOSC3, and potentially extending to disease-associated substitutions in other exosome subunits.

| S-EPMC5223504 | biostudies-literature

Structure of a Cytoplasmic 11-Subunit RNA Exosome Complex.

Project description:The RNA exosome complex associates with nuclear and cytoplasmic cofactors to mediate the decay, surveillance, or processing of a wide variety of transcripts. In the cytoplasm, the conserved core of the exosome (Exo10) functions together with the conserved Ski complex. The interaction of S. cerevisiae Exo10 and Ski is not direct but requires a bridging cofactor, Ski7. Here, we report the 2.65 Å resolution structure of S. cerevisiae Exo10 bound to the interacting domain of Ski7. Extensive hydrophobic interactions rationalize the high affinity and stability of this complex, pointing to Ski7 as a constitutive component of the cytosolic exosome. Despite the absence of sequence homology, cytoplasmic Ski7 and nuclear Rrp6 bind Exo10 using similar surfaces and recognition motifs. Knowledge of the interacting residues in the yeast complexes allowed us to identify a splice variant of human HBS1-Like as a Ski7-like exosome-binding protein, revealing the evolutionary conservation of this cytoplasmic cofactor.

| S-EPMC4942675 | biostudies-literature

The human cap-binding complex is functionally connected to the nuclear RNA exosome.

Project description:Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human 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 ZC3H18 protein, to the nuclear exosome targeting (NEXT) complex, thus forming CBC-NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis of 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 products of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5' cap links transcription termination to exosomal RNA degradation through CBCN.

| S-EPMC3923317 | biostudies-literature

A complex prediction: three-dimensional model of the yeast exosome.

Project description:We present a model of the yeast exosome based on the bacterial degradosome component polynucleotide phosphorylase (PNPase). Electron microscopy shows the exosome to resemble PNPase but with key differences likely related to the position of RNA binding domains, and to the location of domains unique to the exosome. We use various techniques to reduce the many possible models of exosome subunits based on PNPase to just one. The model suggests numerous experiments to probe exosome function, particularly with respect to subunits making direct atomic contacts and conserved, possibly functional residues within the predicted central pore of the complex.

| S-EPMC1084189 | biostudies-literature

Comparative analyses of disease-linked missense mutations in the RNA exosome modeled in budding yeast reveal distinct functional consequences in translation

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

The nuclear exosome is active and important during budding yeast meiosis.

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 unannotated 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 that are undetectable in wild-type cells, showing that the nuclear exosome remains functional for CUT degradation, and we further report that the meiotic exosome complex 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.

| S-EPMC4161446 | biostudies-literature

Disease-associated mutations in the p150(Glued) subunit destabilize the CAP-gly domain.

Project description:Point mutations within the CAP-gly domain of the p150(Glued) subunit of the dynactin complex have been identified in patients with distal spinal bulbar muscular atrophy (dSBMA) and Perry's syndrome. Herein, we show by CD and NMR experiments that each mutated CAP-gly domain is folded but less stable than the wild-type (WT) domain. We also demonstrate that the domains harboring these mutations bind to microtubules but fail to bind to EB1. These data indicate that these disease-associated, point mutations affect the stability of this domain and inhibit their interaction with EB1 but do not inhibit their interaction with microtubules.

| S-EPMC2938955 | biostudies-literature

Structure determination of an 11-subunit exosome in complex with RNA by molecular replacement.

Project description:The RNA exosome is an evolutionarily conserved multi-protein complex involved in the 3' degradation of a variety of RNA transcripts. In the nucleus, the exosome participates in the maturation of structured RNAs, in the surveillance of pre-mRNAs and in the decay of a variety of noncoding transcripts. In the cytoplasm, the exosome degrades mRNAs in constitutive and regulated turnover pathways. Several structures of subcomplexes of eukaryotic exosomes or related prokaryotic exosome-like complexes are known, but how the complete assembly is organized to fulfil processive RNA degradation has been unclear. An atomic snapshot of a Saccharomyces cerevisiae 420 kDa exosome complex bound to an RNA substrate in the pre-cleavage state of a hydrolytic reaction has been determined. Here, the crystallographic steps towards the structural elucidation, which was carried out by molecular replacement, are presented.

| S-EPMC3817696 | biostudies-literature

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