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PMID: 8223480 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains.

The EMBO journal ·Vol. 12 ·No. 12 ·1993-12-00 ·Pages 4715-26

Cáceres JF, Krainer AR

Abstract

Human pre-mRNA splicing factor SF2/ASF has an activity required for general splicing in vitro and promotes utilization of proximal alternative 5' splice sites in a concentration-dependent manner by opposing hnRNP A1. We introduced selected mutations in the N-terminal RNA recognition motif (RRM) and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and alternative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site selection, demonstrating that these activities involve distinct molecular interactions. Specific protein-RNA contact mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for constitutive splicing, although they are not required for RRM-mediated binding to pre-mRNA. The RS domain is also required for constitutive splicing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM and a central degenerate RRM repeat in binding to RNA. These two domains are sufficient for alternative splicing activity in the absence of an RS domain.

MeSH Terms
Alternative Splicing Amino Acid Sequence Base Sequence DNA Humans Molecular Sequence Data Mutagenesis Nuclear Proteins/chemistry,genetics,metabolism Protein Conformation RNA Precursors/metabolism RNA Splicing RNA-Binding Proteins Serine-Arginine Splicing Factors
Chemicals
Nuclear Proteins RNA Precursors RNA-Binding Proteins Serine-Arginine Splicing Factors DNA
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Cáceres J F
Cold Spring Harbor Laboratory, NY 11724-2208.
Krainer A R
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1993-12-00
Pages
4715-26
Language
English
Region
England
NLM ID
8208664
PMCID
PMC413916
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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