Branch nucleophile selection in pre-mRNA splicing: Evidence for the bulged duplex model

Charles C. Query, Melissa J. Moore, Phillip A. Sharp

Research output: Contribution to journalArticle

160 Scopus citations

Abstract

Selection of the nucleophile for the first step of nuclear pre-mRNA splicing was probed by site-specific incorporation into splicing substrates of nucleotides modified at the 2' position. The differing abilities of ribose, 2'-deoxyribose, and arabinose nucleotides to base-pair within an RNA·RNA duplex and to contribute a nucleophilic 2'-OH group were exploited to analyze the paired/unpaired disposition of the branch site nucleotide. The results provide direct evidence for a bulged duplex model in which either of two adjacent purines within the consensus branch site sequence may shift into a bulged position and contribute the 2'-OH group for the first step of splicing. Furthermore, the presence of a consensus branch site that cannot present a reactive nucleophile suppresses splicing, including the use of cryptic branch sites elsewhere. We conclude that the branch site region base- pairing with U2 snRNA determines the first step nucleophile and persists at the time of the first transesterification reaction.

Original languageEnglish (US)
Pages (from-to)587-597
Number of pages11
JournalGenes and Development
Volume8
Issue number5
DOIs
StatePublished - Mar 1 1994
Externally publishedYes

Keywords

  • RNA splicing
  • arabinose
  • duplex stability
  • nucleophile
  • site-specific modifications
  • transesterification

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology

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