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PMID: 9016643 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Functional analysis of the polypyrimidine tract in pre-mRNA splicing.

Nucleic acids research ·Vol. 25 ·No. 4 ·1997-02-15 ·Pages 888-96

Coolidge CJ, Seely RJ, Patton JG

Abstract

The polypyrimidine tract is one of the important cis-acting sequence elements directing intron removal in pre-mRNA splicing. Progressive deletions of the polypyrimidine tract have been found to abolish correct lariat formation, spliceosome assembly and splicing. In addition, the polypyrimidine tract can alter 3'-splice site selection by promoting alternative branch site selection. However, there appears to be great flexibility in the specific sequence of a given tract. Not only the optimal composition of the polypyrimidine tract, but also the role of the tract in introns with no apparent polypyrimidine tracts or where changes in the tract are apparently harmless are uncertain. Accordingly, we have designed a series of cis-competition splicing constructs to test the functional competitive efficiency of a variety of systematically mutated polypyrimidine tracts. An RT/PCR assay was used to detect spliced product formation as a result of differential branch point selection dependent on direct competition between two opposing polypyrimidine tracts. We found that pyrimidine tracts containing 11 continuous uridines are the strongest pyrimidine tracts. In such cases, the position of the uridine stretch between the branch point and 3'-splice site AG is unimportant. In contrast, decreasing the continuous uridine stretch to five or six residues requires that the tract be located immediately adjacent to the AG for optimal competitive efficiency. The block to splicing with decreasing polypyrimidine tract strength is primarily prior to the first step of splicing. While lengthy continuous uridine tracts are the most competitive, tracts with decreased numbers of consecutive uridines and even tracts with alternating purine/pyrimidine residues can still function to promote branch point selection, but are far less effective competitors in 3'-splice site selection assays.

MeSH Terms
Introns Polydeoxyribonucleotides/genetics Polymerase Chain Reaction Pyrimidine Nucleotides/genetics RNA Precursors/genetics RNA Splicing RNA, Messenger/genetics Saccharomyces cerevisiae/genetics Transcription, Genetic
Chemicals
Polydeoxyribonucleotides Pyrimidine Nucleotides RNA Precursors RNA, Messenger
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Coolidge C J
Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235, USA.
Seely R J
Patton J G
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/25.4.888
Published
1997-02-15
Pages
888-96
Language
English
Region
England
NLM ID
0411011
PMCID
PMC146492
Subset
IM
Grants
NHLBI NIH HHS · HL07751 · United States
NIGMS NIH HHS · R01 GM50418 · United States
External Links
PubMed source DOI: 10.1093/nar/25.4.888 PMC Full Text View journal details
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