Reconstitution of a functional 7SK snRNP

John E Brogie; David H Price

doi:10.1093/nar/gkx262

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Reconstitution of a functional 7SK snRNP

Journal article

Open access

Peer reviewed

Reconstitution of a functional 7SK snRNP

John E Brogie and David H Price

Nucleic acids research, Vol.45(11), pp.6864-6880

06/20/2017

DOI: 10.1093/nar/gkx262

PMCID: PMC5499737

PMID: 28431135

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Abstract

The 7SK small nuclear ribonucleoprotein (snRNP) plays a central role in RNA polymerase II elongation control by regulating the availability of active P-TEFb. We optimized conditions for analyzing 7SK RNA by SHAPE and demonstrated a hysteretic effect of magnesium on 7SK folding dynamics including a 7SK GAUC motif switch. We also found evidence that the 5΄ end pairs alternatively with two different regions of 7SK giving rise to open and closed forms that dictate the state of the 7SK motif. We then used recombinant P-TEFb, HEXIM1, LARP7 and MEPCE to reconstruct a functional 7SK snRNP in vitro. Stably associated P-TEFb was highly inhibited, but could still be released and activated by HIV-1 Tat. Notably, P-TEFb association with both in vitro-reconstituted and cellular snRNPs led to similar changes in SHAPE reactivities, confirming that 7SK undergoes a P-TEFb-dependent structural change. We determined that the xRRM of LARP7 binds to the 3΄ stem loop of 7SK and inhibits the methyltransferase activity of MEPCE through a C-terminal MEPCE interaction domain (MID). Inhibition of MEPCE is dependent on the structure of the 3΄ stem loop and the closed form of 7SK RNA. This study provides important insights into intramolecular interactions within the 7SK snRNP.

Methyltransferases - chemistry

Magnesium - chemistry

RNA, Long Noncoding - chemistry

Humans

Protein Multimerization

Protein Structure, Quaternary

Protein Binding

Protein Interaction Domains and Motifs

Ribonucleoproteins, Small Nuclear - chemistry

Ribonucleoproteins - chemistry

Nucleic Acid Conformation

Inverted Repeat Sequences

Details

Title: Subtitle: Reconstitution of a functional 7SK snRNP
Creators: John E Brogie - Biochemistry Department, University of Iowa, Iowa City, IA 52242, USA
David H Price - Biochemistry Department, University of Iowa, Iowa City, IA 52242, USA
Resource Type: Journal article
Publication Details: Nucleic acids research, Vol.45(11), pp.6864-6880
Publisher: England
DOI: 10.1093/nar/gkx262
PMID: 28431135
PMCID: PMC5499737
ISSN: 0305-1048
eISSN: 1362-4962
Grant note: R01 GM035500 / NIGMS NIH HHS
Language: English
Date published: 06/20/2017
Academic Unit: Biochemistry and Molecular Biology
Record Identifier: 9984024530202771

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