Comprehensive RNP profiling in cells identifies U1 snRNP complexes with cleavage and polyadenylation factors active in telescripting.

Polyadenylation Proteomics RNA Ribonucleoprotein, U1 Small Nuclear / genetics mRNA Cleavage and Polyadenylation Factors
3′-end processing Crosslinking Mass spectrometry Polyadenylation RNA-seq RNPs Telescripting Transcription elongation Transcription termination U1 snRNP

Journal

Methods in enzymology
ISSN: 1557-7988
Titre abrégé: Methods Enzymol
Pays: United States
ID NLM: 0212271

Informations de publication

Date de publication:
2021
Historique:
entrez: 29 6 2021
pubmed: 30 6 2021
medline: 3 7 2021
Statut: ppublish

Résumé

Full-length transcription in the majority of protein-coding and other genes transcribed by RNA polymerase II in complex eukaryotes requires U1 snRNP (U1) to co-transcriptionally suppress transcription-terminating premature 3'-end cleavage and polyadenylation (PCPA) from cryptic polyadenylation signals (PASs). This U1 activity, termed telescripting, requires U1 to base-pair with the nascent RNA and inhibit usage of a downstream PAS. Here we describe experimental methods to determine the mechanism of U1 telescripting, involving mapping of U1 and CPA factors (CPAFs) binding locations in relation to PCPA sites, and identify U1 and CPAFs interactomes. The methods which utilizes rapid reversible protein-RNA and protein-protein chemical crosslinking, immunoprecipitations (XLIPs) of components of interest, and RNA-seq and quantitative proteomic mass spectrometry, captured U1-CPAFs complexes in cells, providing important insights into telescripting mechanism. XLIP profiling can be used for comprehensive molecular definition of diverse RNPs.

Identifiants

DOI: 10.1016/bs.mie.2021.04.017 PMID: 34183128
pubmed: 34183128
pii: S0076-6879(21)00176-2
doi: 10.1016/bs.mie.2021.04.017
pii:
doi:

Substances chimiques

Ribonucleoprotein, U1 Small Nuclear 0
mRNA Cleavage and Polyadenylation Factors 0
RNA 63231-63-0

Types de publication

Journal Article Research Support, N.I.H., Extramural

Langues

eng

Sous-ensembles de citation

IM

Pagination

325-347

Subventions

Organisme : NIGMS NIH HHS
ID : R01 GM112923
Pays : United States

Informations de copyright

Copyright © 2021 Elsevier Inc. All rights reserved.

Auteurs

Zhiqiang Cai (Z)

Department of Biochemistry and Biophysics, School of Medicine, Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA, United States.

Byung Ran So (BR)

Department of Biochemistry and Biophysics, School of Medicine, Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA, United States.

Gideon Dreyfuss (G)

Department of Biochemistry and Biophysics, School of Medicine, Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA, United States. Electronic address: gdreyfuss@hhmi.upenn.edu.

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Classifications MeSH

questionsmedicales.fr › Phénomènes génétiques › Régulation de l'expression des gènes › Maturation post-transcriptionnelle des ARN › Maturation de l'extrémité 3' des ARN › Polyadénylation › Comprehensive RNP profiling in cells...
questionsmedicales.fr › Disciplines des sciences naturelles › Chimie › Biochimie › Protéomique › Comprehensive RNP profiling in cells...
questionsmedicales.fr › Acides nucléiques, nucléotides et nucléosides › Acides nucléiques › ARN › Comprehensive RNP profiling in cells...
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Nucléoprotéines › Protéines de liaison à l'ARN › Facteurs d'épissage des ARN › Petites ribonucléoprotéines nucléaires U1 › Comprehensive RNP profiling in cells...
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Nucléoprotéines › Protéines de liaison à l'ARN › Facteurs de clivage et de polyadénylation de l'ARN messager › Comprehensive RNP profiling in cells...