Abstract
Splicing of human pre-mRNA is reciprocally coupled to 3′ end formation by terminal exon definition, which occurs co-transcriptionally. It is required for the final maturation of most human pre-mRNAs and is therefore important to understand. We have used several strategies to block splicing at specific stages in vivo and studied their effect on 3′ end formation. We demonstrate that a terminal splice acceptor site is essential to establish coupling with the poly(A) signal in a chromosomally integrated β-globin gene. This is in part to alleviate the suppression of 3′ end formation by U1 small nuclear RNA, which is known to bind pre-mRNA at the earliest stage of spliceosome assembly. Interestingly, blocks to splicing that are subsequent to terminal splice acceptor site function, but before catalysis, have little observable effect on 3′ end formation. These data suggest that early stages of spliceosome assembly are sufficient to functionally couple splicing and 3′ end formation, but that on-going intron removal is less critical.
Highlights
IntroductionThe splicing process has two catalytic steps with the first releasing the 50 exon and the second joining the two exons together with consequent release of an intron lariat [1]
Most human pre-mRNAs contain multiple introns that are removed by splicing
Our findings are consistent with the following model for the interconnections between splicing and 30 end processing
Summary
The splicing process has two catalytic steps with the first releasing the 50 exon and the second joining the two exons together with consequent release of an intron lariat [1]. It is performed by a set of five small nuclear RNAs (snRNAs) in concert with 100 s of protein factors. Intronic sequences, such as the 50 and 30 splice site, intervening branch-point and polypyrimidine tract, play a vital role [2]. Splicing of most introns in higher eukaryotes is thought to occur by definition of the comparatively shorter exons, across which 30 and 50 splice sites are recognized by U2 and U1, respectively [3]
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