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PMID: 2524033 Published · ppublish English Journal Article

Evidence for nuclear factors involved in recognition of 5' splice sites.

Nucleic acids research ·Vol. 17 ·No. 7 ·1989-04-11 ·Pages 2655-74

Zapp ML, Berget SM

Abstract

To investigate soluble factors involved in pre-messenger RNA splicing we have fractionated nuclear extract by simple centrifugation to produce a supernatant pellet pair. Factors larger than 15S including U2, U4, U5, and U6 snRNPs fractionate with the pellet; U1 snRNPs distribute equally in pellet and supernatant. Each fraction is individually incompetent for splicing and spliceosome assembly; mixing restores wild type activity and assembly. The pellet fraction directs an aberrant assembly pathway in which proper 3', but improper 5' splice site recognition occurs. Complexes formed with the pellet fraction are distinguishable from wild-type complexes using native gel electrophoresis. Pellet complexes contain U1 snRNP antigens and their formation requires ATP, U1 snRNPs, U2 snRNPs, and sequences at the 3' end of the intron - properties shared with the initial steps of normal assembly and directed by sequences at the 3' end of the intron. In contrast, pellet complex assembly shows no dependence on the presence of a 5' splice junction within precursor RNA. Furthermore, binding of factors to the 5' splice junction is deficient in pellet assemblies. Thus, the pellet lacks a factor required for proper recognition of 5' splice sites. This factor can be supplied by the supernatant. Complementation occurs when supernatant U1 RNA is destroyed, suggesting that the supernatant factor recognizing 5' splice sites is not U1 snRNPs.

MeSH Terms
Adenosine Triphosphate/physiology Cell-Free System HeLa Cells Humans RNA Precursors/physiology RNA Splicing RNA, Messenger/physiology Ribonucleoproteins/isolation & purification,physiology Ribonucleoproteins, Small Nuclear Subcellular Fractions/analysis
Chemicals
RNA Precursors RNA, Messenger Ribonucleoproteins Ribonucleoproteins, Small Nuclear Adenosine Triphosphate
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Zapp M L
Verna and Marrs McClean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030.
Berget S M
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/17.7.2655
Published
1989-04-11
Pages
2655-74
Language
English
Region
England
NLM ID
0411011
PMCID
PMC317649
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/17.7.2655 PMC Full Text View journal details
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