Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA227076 | ENA |
REPOSITORIES: ENA
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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
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
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
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: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
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:A 5′, 7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and thus a textbook aspect of co-transcriptional pre-mRNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). Recently, NCBP3 has been proposed to form an alternative, non-canonical CBC, together with NCBP1. NCBP3 has also been shown to interact with the canonical CBC along with the protein SRRT (aka ARS2), in a manner that is mutually exclusive with the RNA export factor, PHAX. Taken together, ambiguities and missing information in the bona fide physiological protein-protein associations of NCBP3 persist. In an effort to clarify the compositions of NCBP1-, 2-, and 3-related macromolecular assemblies, including their intersections and differences, we have applied our recently developed interactome screening platform (PMID: 25938370). Here the experimental design and data processing have been modified and updated to identify interactome differences between targets of affinity capture under a wide range of experimental conditions, followed by label-free quantitative mass spectrometry.
2020-11-09 | PXD016038 | Pride
Project description:During gene expression, RNA export factors are mainly known for driving nucleocytoplasmic transport. While early studies suggested that the Exon Junction Complex (EJC) may provide a binding platform for them, subsequent work proposed that they are only recruited by the Cap-Binding Complex (CBC) to the 5’ end of RNAs, as part of the TREX complex. Using iCLIP, we show that the export receptor Nxf1 and two TREX subunits, Alyref and Chtop, are actually recruited to the whole mRNA co-transcriptionally via splicing but before 3’-end processing. Consequently, Alyref can alter splicing decisions and Chtop regulates alternative polyadenylation. Surprisingly, we observe that eIF4A3 stimulates Alyref deposition on single exon or spliced RNAs close to EJC sites. Additional experiments suggest that Alyref is recruited to the 5’-end of RNAs by CBC before binding RNAs near EJCs. Our study reveals mechanical insights into the co-transcriptional recruitment of mRNA export factors and how this shapes the human transcriptome.
2019-05-01 | GSE113952 | GEO
Project description:During gene expression, RNA export factors are mainly known for driving nucleocytoplasmic transport. While early studies suggested that the Exon Junction Complex (EJC) may provide a binding platform for them, subsequent work proposed that they are only recruited by the Cap-Binding Complex (CBC) to the 5’ end of RNAs, as part of the TREX complex. Using iCLIP, we show that the export receptor Nxf1 and two TREX subunits, Alyref and Chtop, are actually recruited to the whole mRNA co-transcriptionally via splicing but before 3’-end processing. Consequently, Alyref can alter splicing decisions and Chtop regulates alternative polyadenylation. Surprisingly, we observe that eIF4A3 stimulates Alyref deposition on single exon or spliced RNAs close to EJC sites. Additional experiments suggest that Alyref is recruited to the 5’-end of RNAs by CBC before binding RNAs near EJCs. Our study reveals mechanical insights into the co-transcriptional recruitment of mRNA export factors and how this shapes the human transcriptome.
2019-05-01 | GSE113896 | GEO
Project description:During gene expression, RNA export factors are mainly known for driving nucleocytoplasmic transport. While early studies suggested that the Exon Junction Complex (EJC) may provide a binding platform for them, subsequent work proposed that they are only recruited by the Cap-Binding Complex (CBC) to the 5’ end of RNAs, as part of the TREX complex. Using iCLIP, we show that the export receptor Nxf1 and two TREX subunits, Alyref and Chtop, are actually recruited to the whole mRNA co-transcriptionally via splicing but before 3’-end processing. Consequently, Alyref can alter splicing decisions and Chtop regulates alternative polyadenylation. Surprisingly, we observe that eIF4A3 stimulates Alyref deposition on single exon or spliced RNAs close to EJC sites. Additional experiments suggest that Alyref is recruited to the 5’-end of RNAs by CBC before binding RNAs near EJCs. Our study reveals mechanical insights into the co-transcriptional recruitment of mRNA export factors and how this shapes the human transcriptome.
2019-05-10 | GSE130992 | GEO