Distinct and evolutionary conserved structural features of the human nuclear exosome complex

Gerlach, Piotr; Schuller, Jan M.; Bonneau, Fabien; Basquin, J.; Reichelt, Peter; Falk, Sebastian; Conti, Elena

doi:10.7554/elife.38686

Cited by 49 publications

(68 citation statements)

References 66 publications

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“…The structure of the exosome is similar in yeast and humans and is composed of 9–11 key protein subunits ( Gerlach et al., 2018 , Januszyk and Lima, 2014 , Makino et al., 2013 , Weick et al., 2018 ). It possesses a catalytically inactive barrel structure of 9-core subunits (EXO-9), arranged as a hexamer (the PH-like ring) capped with a trimeric S1/KH ring.…”

Section: Introductionmentioning

confidence: 99%

Rapid Depletion of DIS3, EXOSC10, or XRN2 Reveals the Immediate Impact of Exoribonucleolysis on Nuclear RNA Metabolism and Transcriptional Control

et al. 2019

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Summary Cell-based studies of human ribonucleases traditionally rely on methods that deplete proteins slowly. We engineered cells in which the 3′→5′ exoribonucleases of the exosome complex, DIS3 and EXOSC10, can be rapidly eliminated to assess their immediate roles in nuclear RNA biology. The loss of DIS3 has the greatest impact, causing the substantial accumulation of thousands of transcripts within 60 min. These transcripts include enhancer RNAs, promoter upstream transcripts (PROMPTs), and products of premature cleavage and polyadenylation (PCPA). These transcripts are unaffected by the rapid loss of EXOSC10, suggesting that they are rarely targeted to it. More direct detection of EXOSC10-bound transcripts revealed its substrates to prominently include short 3′ extended ribosomal and small nucleolar RNAs. Finally, the 5′→3′ exoribonuclease, XRN2, has little activity on exosome substrates, but its elimination uncovers different mechanisms for the early termination of transcription from protein-coding gene promoters.

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Section: Introductionmentioning

confidence: 99%

Rapid Depletion of DIS3, EXOSC10, or XRN2 Reveals the Immediate Impact of Exoribonucleolysis on Nuclear RNA Metabolism and Transcriptional Control

et al. 2019

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“…Elution profiles of apo-stable complexes show that in the absence of RNA, subunits still co-elute, but do so as a lower-molecular weight complex. Available atomic structures of the exosome with and without RNA support the concept that RNA is peripheral to the stability of the complex (Gerlach et al, 2018;Weick et al, 2018).…”

Section: Classification Of Rnp Complexesmentioning

confidence: 98%

Systematic discovery of endogenous human ribonucleoprotein complexes

Mallam

Sae-Lee

Schaub

et al. 2018

Preprint

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RNA-binding proteins (RBPs) play essential roles in biology and are frequently associated with human disease. While recent studies have systematically identified individual RBPs, their higher order assembly into Ribonucleoprotein (RNP) complexes has not been systematically investigated. Here, we describe a proteomics method for systematic identification of RNP complexes in human cells. We identify 1,428 protein complexes that associate with RNA, indicating that over 20% of known human protein complexes contain RNA. To explore the role of RNA in the assembly of each complex, we identify complexes that dissociate, change composition, or form stable protein-only complexes in the absence of RNA. Importantly, these data also provide specific novel insights into the function of well-studied protein complexes not previously known to associate with RNA, including replication factor C (RFC) and cytokinetic centralspindlin complex. Finally, we use our method to systematically identify cell-type specific RNA-associated proteins in mouse embryonic stem cells. We distribute these data as a resource, rna.MAP (rna.proteincomplexes.org) which provides a comprehensive dataset for the study of RNA-associated protein complexes. Our system thus provides a novel methodology for further explorations across human tissues and disease states, as well as throughout all domains of life. ! 3! ! 4! upon CF-MS by comparing chromatographic separations of cellular lysate under control and RNA degrading conditions ( Figure 1A). A statistical framework is then applied to discover RNP complexes by identifying concurrent shifts of known protein complex subunits upon RNA degradation ( Figure 1A).Analysis of DIF-FRAC data answers important questions as to the role of RNA plays in macromolecular complexes. Specifically, we identify RNP complexes that 1) dissociate, 2) form stable protein-only complexes, and 3) change composition in the absence of RNA suggesting specific roles for RNA in each of these cases. Because DIF-FRAC is independent of UV crosslinking, nucleotide incorporation, genetic manipulation or poly(A) RNA capture efficiency, it can therefore be used to investigate a wide variety of cell types, tissues and species. To demonstrate this versatility, we apply DIF-FRAC to mouse embryonic stem cells (mESCs), identifying 1,165 RNA-associated proteins, to show the method is highly adaptable and can be extended to discover RNP complexes in diverse samples.Finally, we created a system-wide resource of 1,428 RNP complexes, many of which are previously unreported as having an RNA component, representing 20% of known human protein complexes. We provide our resource, rna.MAP, to the community as a fully searchable web database at rna.proteincomplexes.org. Results and Discussion Differential fractionation (DIF-FRAC) identifies RNP complexesThe DIF-FRAC strategy builds upon our previous strategy of Co-Fractionation Massspectroscopy (CF-MS) for identifying protein complexes in cellular lysate (Havugimana et al., 2012;Wan et al., 2015). CF-MS chromatograp...

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“…As illustrated in Figure 4B, the amino acid changes present in EXOSC5 in patients occur in regions of the protein that are evolutionarily conserved between humans, zebrafish and budding yeast, although only Leu206 is strictly conserved as Leu191 in budding yeast. To begin to explore how these amino acid substitutions could alter the structure of EXOSC5 or the contacts that EXOSC5 makes with other RNA exosome subunits within the core complex, we took advantage of the RNA exosome structural model (41). Each of the amino acids altered is located in a different region of the Kcal/mol according to mCSM) and exert a destabilizing effect on the protein-protein interaction with EXOSC8 (-0.7 Kcal/mol according to mCSM).…”

Section: Functional Consequences Of Pathogenic Amino Acid Changes Inmentioning

confidence: 99%

Biallelic variants in the RNA exosome geneEXOSC5are associated with developmental delays, short stature, cerebellar hypoplasia and motor weakness

Slavotinek

Misceo

Htun

et al. 2020

Preprint

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The RNA exosome is an essential ribonuclease complex involved in the processing and degradation of both coding and noncoding RNAs. We present three patients with biallelic variants in EXOSC5, which encodes a structural subunit of the RNA exosome. The common clinical features of these patients comprise failure to thrive, short stature, feeding difficulties, developmental delays that affect motor skills, hypotonia and esotropia. Brain MRI revealed cerebellar hypoplasia and ventriculomegaly. The first patient had a deletion involving exons 5-6 of EXOSC5 and a missense variant, p.Thr114Ile, that were inherited in trans, the second patient was homozygous for p.Leu206His, and the third patient had paternal isodisomy for chromosome 19 and was homozygous for p.Met148Thr. We employed three complementary approaches to explore the requirement for EXOSC5 in brain development and assess the functional consequences of pathogenic variants in EXOSC5. Loss of function for the zebrafish ortholog results in shortened and curved tails and bodies, reduced eye and head size and edema. We modeled pathogenic EXOSC5 variants in both budding yeast and mammalian cells. Some of these variants show defects in RNA exosome function as well as altered interactions with other RNA exosome subunits. Overall, these findings expand the number of genes encoding RNA exosome components that have been implicated in human disease, while also suggesting that disease mechanism varies depending on the specific pathogenic variant. IntroductionThe RNA exosome is a ribonuclease complex composed of ten, evolutionarily conserved subunits that form a ring-like structure with 3'-5' exonuclease activity that is critical for the processing and degradation of a variety of RNAs both in the nucleus and cytoplasm (1-3). The exosome subunits are designated as exosome component (EXOSC) proteins in humans, mice (M. musculus) and other mammals and ribosomal RNA processing (Rrp) proteins in zebrafish (D. rerio), fruit flies (D. melanogaster) and budding yeast (S. cerevisiae), where the complex was first identified and studied (4, 5). The EXOSC4-9 subunits and their orthologs form a central, six-subunit ring that is covered by a three-subunit cap composed of EXOSC1-3. The ring-like complex creates a central channel through which RNA substrates are threaded to the catalytic subunit, DIS3 or DIS3L, at the base (6-10). In addition to DIS3 or DIS3L, the 9-subunit exosome complex also associates with another catalytic subunit, EXOSC10 at the cap. Notably, DIS3 and EXOSC10 are predominantly nuclear (7,(9)(10)(11), whereas DIS3L is mostly cytoplasmic (12).Although all six structural core ring subunits contain an RNase PH-like domain, they are all catalytically inactive due to amino acid substitutions that replace key catalytic residues (13). Thus, RNA substrates of the RNA exosome access the DIS3 catalytic subunit primarily via interactions with the non-catalytic core channel (14-16). Consistent with the critical function of the RNA exosome in the processing and decay of numerous ce...

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Distinct and evolutionary conserved structural features of the human nuclear exosome complex

Cited by 49 publications

References 66 publications

Rapid Depletion of DIS3, EXOSC10, or XRN2 Reveals the Immediate Impact of Exoribonucleolysis on Nuclear RNA Metabolism and Transcriptional Control

Rapid Depletion of DIS3, EXOSC10, or XRN2 Reveals the Immediate Impact of Exoribonucleolysis on Nuclear RNA Metabolism and Transcriptional Control

Systematic discovery of endogenous human ribonucleoprotein complexes

Biallelic variants in the RNA exosome geneEXOSC5are associated with developmental delays, short stature, cerebellar hypoplasia and motor weakness

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