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

ABSTRACT: 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'

ORGANISM(S): Homo sapiens

SUBMITTER: Edouard BERTRAND

PROVIDER: E-GEOD-52132 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 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

CBC-ARS2 stimulates 3'-end maturation of multiple RNA families and favors cap-proximal processing

Project description:The nuclear cap-binding complex (CBC) stimulates multiple steps in several RNA maturation pathways, but how it functions in humans is incompletely understood. For small capped RNAs like pre-snRNAs, the CBC recruits PHAX. Here, we identify the CBCAP complex, composed of CBC, Ars2 and PHAX, and show that both CBCAP and CBC-Ars2 complexes can be reconstituted from recombinant proteins. Ars2 stimulates PHAX binding to the CBC as well as snRNAs 3'-end processing, hereby coupling maturation with export. In vivo, CBC and Ars2 bind similar capped non-coding and coding RNAs, and stimulate their 3M-bM-^@M-^Y-end processing. Strongest effects are displayed for cap-proximal polyadenylation sites, thereby favoring premature transcription termination. Ars2 functions in part through the mRNA 3M-bM-^@M-^Y-end cleavage factor CLP1, which binds RNA Polymerase II through PCF11. Ars2 is thus a major CBC effector that stimulates functional and cryptic 3'-end processing sites. In total 12 samples; 4 control IP, 2 Flag- Ars2 IP and 2 Flag-CBP20 IP; 2 control FFL siRNA and 2 siRNA Ars2

2013-11-08 | E-GEOD-52131 | biostudies-arrayexpress

CBC-ARS2 stimulates 3'-end maturation of multiple RNA families and favors cap-proximal processing

Project description:The nuclear cap-binding complex (CBC) stimulates multiple steps in several RNA maturation pathways, but how it functions in humans is incompletely understood. For small capped RNAs like pre-snRNAs, the CBC recruits PHAX. Here, we identify the CBCAP complex, composed of CBC, Ars2 and PHAX, and show that both CBCAP and CBC-Ars2 complexes can be reconstituted from recombinant proteins. Ars2 stimulates PHAX binding to the CBC as well as snRNAs 3'-end processing, hereby coupling maturation with export. In vivo, CBC and Ars2 bind similar capped non-coding and coding RNAs, and stimulate their 3’-end processing. Strongest effects are displayed for cap-proximal polyadenylation sites, thereby favoring premature transcription termination. Ars2 functions in part through the mRNA 3’-end cleavage factor CLP1, which binds RNA Polymerase II through PCF11. Ars2 is thus a major CBC effector that stimulates functional and cryptic 3'-end processing sites.

2013-11-08 | GSE52131 | GEO

Structural analysis of human ARS2, a platform for co-transcriptional RNA sorting

Project description:ARS2 is a highly conserved metazoan protein involved in numerous aspects of nuclear RNA metabolism. As a direct partner of the nuclear cap-binding complex (CBC) it mediates interactions with diverse RNA processing and transport machineries in a transcript-dependent manner. Here we present the human ARS2 crystal structure, which exhibits similarities and metazoan-specific differences to the plant homologue SERRATE, most notably an additional RRM domain. We present biochemical, biophysical and cellular interactome data comparing wild type and mutant ARS2 that identify regions critical for interactions with FLASH (involved in histone mRNA biogenesis), NCBP3 (a putative cap-binding protein involved in mRNA export) and single-stranded RNA. We show that FLASH and NCBP3 have overlapping binding sites on ARS2 and that CBC-ARS2-NCBP3 form a ternary complex that is mutually exclusive with CBC-ARS-PHAX (involved in snRNA export). Our results support that mutually exclusive higher order CBC-ARS2 complexes are critical in determining Pol II transcript fate.

2018-09-03 | PXD009035 | Pride

ARS2 instructs early transcription termination-coupled RNA decay by recruiting ZC3H4 to nascent transcripts [TT-Seq]

Project description:The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif in the transcription restriction factor ZC3H4. This interaction recruits ZC3H4 to chromatin to elicit Pol II termination, independent of other early termination pathways defined by the cleavage and polyadenylation (CPA) and Integrator (INT) complexes. ZC3H4, in turn, directly connects to the ZCCHC8 component of the nuclear exosome targeting (NEXT) complex, hereby facilitating rapid degradation of the nascent RNA. Hence, ARS2 instructs the coupled transcription termination and degradation of the transcript onto which it is bound. This contrasts with ARS2 function at CPA-instructed termination sites where the protein exclusively partakes in RNA suppression via post-transcriptional decay.

2023-06-19 | GSE228383 | GEO

ARS2 instructs early transcription termination-coupled RNA decay by recruiting ZC3H4 to nascent transcripts [RNA-Seq]

2023-06-19 | GSE212383 | GEO

ARS2 instructs early transcription termination-coupled RNA decay by recruiting ZC3H4 to nascent transcripts

2023-06-19 | GSE212208 | GEO

ARS2 is a general suppressor of pervasive transcription [RNAseq]

Project description:Termination of transcription is important for establishing gene punctuation marks. It is also critical for suppressing many of the pervasive transcription events occurring throughout eukaryotic genomes and coupling their RNA products to efficient decay. In human cells, the ARS2 protein has been implicated in such function as its depletion causes transcriptional read-through of selected gene terminators and because it physically interacts with the ribonucleolytic nuclear RNA exosome. Here, we study the role of ARS2 on transcription and RNA metabolism genome-wide. We show that ARS2 depletion negatively impacts levels of promoter-proximal RNA polymerase II (RNAPII) at protein-coding (pc) genes, Moreover, our results reveal a general role of ARS2 in transcription termination-coupled RNA turnover at short transcription units like snRNA-, replication dependent histone (RDH)-, promoter upstream transcript (PROMPT)- and enhancer RNA (eRNA)-loci. Depletion of the ARS2 interaction partner ZC3H18 mimics the ARS2 depletion, although to a milder extent, whereas depletion of the exosome core subunit RRP40 only impacts RNA abundance post-transcriptionally. Interestingly, ARS2 is also involved in transcription termination events within first introns of pc genes. Our work therefore establishes ARS2 as a general suppressor of pervasive transcription with the potential to regulate protein-coding gene expression.

2017-07-31 | GSE99059 | GEO

ARS2 is a general suppressor of pervasive transcription

Project description:Termination of transcription is important for establishing gene punctuation marks. It is also critical for suppressing many of the pervasive transcription events occurring throughout eukaryotic genomes and coupling their RNA products to efficient decay. In human cells, the ARS2 protein has been implicated in such function as its depletion causes transcriptional read-through of selected gene terminators and because it physically interacts with the ribonucleolytic nuclear RNA exosome. Here, we study the role of ARS2 on transcription and RNA metabolism genome-wide. We show that ARS2 depletion negatively impacts levels of promoter-proximal RNA polymerase II (RNAPII) at protein-coding (pc) genes, Moreover, our results reveal a general role of ARS2 in transcription termination-coupled RNA turnover at short transcription units like snRNA-, replication dependent histone (RDH)-, promoter upstream transcript (PROMPT)- and enhancer RNA (eRNA)-loci. Depletion of the ARS2 interaction partner ZC3H18 mimics the ARS2 depletion, although to a milder extent, whereas depletion of the exosome core subunit RRP40 selectively impacts RNA abundance post-transcriptionally. Interestingly, ARS2 is also involved in transcription termination events within first introns of pc genes. Our work therefore establishes ARS2 as a general suppressor of pervasive transcription with the potential to regulate protein-coding gene expression.

2017-07-31 | GSE99344 | GEO

Mutually Exclusive CBC-Containing Complexes Contribute to RNA Fate.

Project description:The nuclear cap-binding complex (CBC) stimulates processing reactions of capped RNAs, including their splicing, 3'-end formation, degradation, and transport. CBC effects are particular for individual RNA families, but how such selectivity is achieved remains elusive. Here, we analyze three main CBC partners known to impact different RNA species. ARS2 stimulates 3'-end formation/transcription termination of several transcript types, ZC3H18 stimulates degradation of a diverse set of RNAs, and PHAX functions in pre-small nuclear RNA/small nucleolar RNA (pre-snRNA/snoRNA) transport. Surprisingly, these proteins all bind capped RNAs without strong preferences for given transcripts, and their steady-state binding correlates poorly with their function. Despite this, PHAX and ZC3H18 compete for CBC binding and we demonstrate that this competitive binding is functionally relevant. We further show that CBC-containing complexes are short lived in vivo, and we therefore suggest that RNA fate involves the transient formation of mutually exclusive CBC complexes, which may only be consequential at particular checkpoints during RNA biogenesis.

2017-03-03 | GSE94427 | GEO

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