A mutational analysis of the polypyrimidine tract of introns. Effects of sequence differences in pyrimidine tracts on splicing

R. F. Roscigno, M. Weiner, M. A. Garcia-Blanco

Research output: Contribution to journalArticle

128 Scopus citations

Abstract

The polypyrimidine (py) tract of introns is required for efficient spliceosome assembly and splicing of pre-mRNAs. A detailed mutational analysis of the py tract of an adenovirus 2 intron was carried out. Utilizing a 'precursor in pieces' vector system, it was possible to synthesize py tract mutant pre-mRNAs that were otherwise identical. The mutant pre-mRNAs were analyzed for in vitro splicing, for formation of splicing complexes, and for binding to proteins in the HeLa nuclear extract. Chimeric pre-mRNAs that contained the yeast branch point consensus sequence (UAC-UAAC) and altered py tracts were also analyzed. Mutational analysis showed the following. First, any mutation in the py tract that affected splicing did so by interferring with complex A formation in spliceosome assembly. Second, introduction of purines into the py tract is detrimental only if the length of the tract is shortened and if there is a reduction in the number of consecutive uracil residues. Third, uracil and cytosine do not have equivalent functions in the py tract. Our results with chimeric pre-mRNAs also show that a strong py tract can partially replace a weak branch point sequence and a strong branch point sequence can partially replace a weak py tract. Finally, the one surprising finding obtained when examining protein binding was that a mutant pre-mRNA did not bind to heterogeneous nuclear ribonucleoprotein C proteins and yet spliced close to wild type level.

Original languageEnglish (US)
Pages (from-to)11222-11229
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number15
StatePublished - Jan 1 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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