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PMID: 2959470 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly.

The EMBO journal ·Vol. 6 ·No. 8 ·1987-08-00 ·Pages 2415-24

Bindereif A, Green MR

Abstract

We have studied the assembly, composition and structure of splicing complexes using biotin-avidin affinity chromatography and RNase protection assays. We find that U1, U2, U4, U5 and U6 snRNPs associate with the pre-mRNA and are in the mature, functional complex. Association of U1 snRNP with the pre-mRNA is rapid and ATP independent; binding of all other snRNPs occurs subsequently and is ATP dependent. Efficient binding of U1 and U2 snRNPs requires a 5' splice site or a 3' splice site/branch point region, respectively. Both sequence elements are required for efficient U4, U5 and U6 snRNP binding. Mutant RNA substrates containing only a 5' splice site or a 3' splice site/branch point region are assembled into 'partial' splicing complexes, which contain a subset of these five snRNPs. RNase protection experiments indicate that in contrast to U1 and U2 snRNPs, U4, U5 and U6 snRNPs do not contact the pre-mRNA. Based upon the time course of snRNP binding and the composition of sucrose gradient fractionated splicing complexes we suggest an assembly pathway proceeding from a 20S (U1 snRNP only) through a 40S (U1 and U2 snRNPs) to the functional 60S splicing complex (U1, U2, U4, U5 and U6 snRNPs).

MeSH Terms
Chromatography, Affinity/methods Kinetics Plasmids RNA Precursors/genetics,isolation & purification,metabolism RNA Splicing Ribonucleoproteins/genetics,isolation & purification,metabolism Ribonucleoproteins, Small Nuclear Templates, Genetic Transcription, Genetic
Chemicals
RNA Precursors Ribonucleoproteins Ribonucleoproteins, Small Nuclear
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Bindereif A
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Green M R
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34 references, click to expand
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1987-08-00
Pages
2415-24
Language
English
Region
England
NLM ID
8208664
PMCID
PMC553648
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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