Branch nucleophile selection in pre-mRNA splicing: evidence for the bulged duplex model.
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.
Query
C C
CC
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139-4307.
Moore
M J
MJ
Sharp
P A
PA
D001089:Arabinose / Q000378:metabolism; D001483:Base Sequence; D004951:Esterification; D008969:Molecular Sequence Data; D012326:RNA Splicing; D012333:RNA, Messenger
D012333:RNA, Messenger; D001089:Arabinose