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PMID: 8972845 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

In vitro reconstitution of mammalian U1 snRNPs active in splicing: the U1-C protein enhances the formation of early (E) spliceosomal complexes.

Nucleic acids research ·Vol. 24 ·No. 23 ·1996-12-01 ·Pages 4614-23

Will CL, Rümpler S, Klein Gunnewiek J, van Venrooij WJ, Lührmann R

Abstract

We have established an in vitro reconstitution/splicing complementation system which has allowed the investigation of the role of mammalian U1 snRNP components both in splicing and at the early stages of spliceosome formation. U1 snRNPs reconstituted from purified, native snRNP proteins and either authentic or in vitro transcribed U1 snRNA restored both early (E) splicing complex formation and splicing-activity to U1-depleted extracts. In vitro reconstituted U1 snRNPs possessing an m3G or ApppG cap were equally active in splicing, demonstrating that a physiological cap structure is not absolutely required for U1 function. However, the presence of an m7GpppG or GpppG cap was deleterious to splicing, most likely due to competition for the m7G cap binding proteins. No significant reduction in splicing or E complex formation was detected with U1 snRNPs reconstituted from U1 snRNA lacking the RNA binding sites of the U1-70K or U1-A protein (i.e., stem-loop I and II, respectively). Complementation studies with purified HeLa U1 snRNPs lacking subsets of the U1-specific proteins demonstrated a role for the U1-C, but not U1-A, protein in the formation and/or stabilization of early splicing complexes. Studies with recombinant U1-C protein mutants indicated that the N-terminal domain of U1-C is necessary and sufficient for the stimulation of E complex formation.

MeSH Terms
Animals Binding Sites Binding, Competitive HeLa Cells Humans Mutation RNA/chemistry,metabolism RNA Caps RNA Splicing Recombinant Proteins Ribonucleoprotein, U1 Small Nuclear/chemistry,metabolism Spliceosomes/metabolism Structure-Activity Relationship Xenopus
Chemicals
RNA Caps Recombinant Proteins Ribonucleoprotein, U1 Small Nuclear RNA
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Will C L
Institut für Molekularbiologie und Tumorforschung, Philipps Universität Marburg, Germany.
Rümpler S
Klein Gunnewiek J
van Venrooij W J
Lührmann R
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41 references, click to expand
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/24.23.4614
Published
1996-12-01
Pages
4614-23
Language
English
Region
England
NLM ID
0411011
PMCID
PMC146292
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/24.23.4614 PMC Full Text View journal details
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