Retention of spliceosomal components along ligated exons ensures efficient removal of multiple introns

Abstract

The majority of mammalian pre-mRNAs contains multiple introns that are excised prior to export and translation. After intron excision, ligated exon intermediates participate in subsequent intron excisions. However, exon ligation generates an exon of increased size, a feature of pre-mRNA splicing that can interfere with downstream splicing events. These considerations raise the question of whether unique mechanisms exist that permit efficient removal of introns neighboring ligated exons. Kinetic analyses of multiple intron-containing pre-mRNAs revealed that splicing is more efficient following an initial intron removal event, suggesting that either the recruitment of the exon junction complex (EJC) to ligated exons increases the efficiency of multiple intron excisions or that the initial definition of splice sites is sufficient to permit efficient splicing of introns neighboring ligated exons. Knockdown experiments show that the deposition of the EJC does not affect subsequent splicing kinetics. Instead, spliceosomal components that are not involved in the initial splicing event remain associated with the pre-mRNA to ensure efficient removal of neighboring introns. Thus, ligated exons do not require redefinition, providing an additional kinetic advantage for exon defined splice sites.

Transcriptome-wide modulation of splicing by the Exon Junction Complex

“…First, we downregulated EJC associated splicing factors such as Acinus and SRSF1, but none of the EJC-dependent splicing events depended on these factors. Second, Crabb and colleagues have shown that EJC deposition did not affect subsequent splicing kinetics in a reporter with two introns [58]. We also found that minigene vectors from EJC-regulated splicing events showed no difference in splicing with or without a predeposition of EJC (data not shown).…”

The nuclear cap-binding complex interacts with the U4/U6·U5 tri-snRNP and promotes spliceosome assembly in mammalian cells

“…Thus, our data support the notion that CBC helps recruit U1 snRNP to the first 5 ′ ss (Lewis et al 1996b), not by direct interactions with U1 but instead indirectly through the U4/U6·U5 snRNP. Moreover, the U1 snRNP is retained on multiple intron-containing pre-mRNAs after the first splicing event, rendering subsequent splicing more efficient (Crabb et al 2010). Indeed, splicing of both FOS introns 1 and 3 was CBC dependent, suggesting-together with the observed snRNP profiles-that CBC may help retain the U1 snRNP on nascent RNA until all introns are removed.…”

Deep Intron Elements Mediate Nested Splicing Events at Consecutive AG Dinucleotides To Regulate Alternative 3′ Splice Site Choice in Vertebrate 4.1 Genes

“…In various splicing reporters, normal 4.1 pre-mRNAs, and aberrant patient transcripts, the nested intrasplicing step consistently occurred at a composite 3= splice site generated by juxtaposition of the iE branch point with the next AG dinucleotide downstream of the first-step acceptor. We speculate that the second acceptor is selected by a scanning mechanism that would not require de novo exon definition of the ligated sequences in the first step (8). In contrast, each successive step of recursive splicing involves a new composite 5= splice donor site formed when an exon is spliced to a downstream "zero-length exon" containing adjacent 3= and 5= splice sites.…”