Pre-mRNA splicing and its cotranscriptional connections

Abstract

Transcription of eukaryotic genes by RNA polymerase II (Pol II) yields RNA precursors containing introns that must be spliced out and the flanking exons ligated together. Splicing is catalyzed by a dynamic ribonucleoprotein complex called the spliceosome. Recent evidence has shown that a large fraction of splicing occurs cotranscriptionally as the RNA chain is extruded from Pol II at speeds of up to 5 kb/minute. Splicing is more efficient when it is tethered to the transcription elongation complex, and this linkage permits functional coupling of splicing with transcription. We discuss recent progress that has uncovered a network of connections that link splicing to transcript elongation and other cotranscriptional RNA processing events.

Keywords: RNA pol II; U1 snRNP; exon definition; intron definition; nascent RNA folding; pre-mRNA splicing; transcription elongation.

Figure 1:. Co-transcriptional spliceosome assembly on nascent pre-mRNA attached to RNA Pol II.

U1 snRNP binds the 5'SS (GU), SF1 binds the branch point A (red) and U2AF^65/35 subunits bind the polypyrimidine tract (pY) and 3’ SS (AG) to form E complex on the nascent transcript. A star indicates the 3’OH growing end of the RNA. Note interactions between Pol II and splicing components including helicases that mediate rearrangements within the spliceosome are poorly understood and many are not shown in this simplified depiction. U2 snRNP replaces SF1 to form A complex. Recruitment of the U4/U6.U5-tri-snRNP forms the pre-catalytic B complex. Then structural rearrangements release U1 and U4 snRNPs to form the activated B-complex (B^Act). B^Act catalyzes step 1 in which the 2' OH of the bulged branch point A attacks the 5'SS creating an upstream exon with a free 3'OH and an intron lariat attached to the second exon. Further rearrangements form C-complex that catalyzes Step 2, in which exons are ligated and the intron lariat structure (ILS) is excised and subsequently debranched and degraded. Putative protein:protein contacts between Pol II and splicing factors are not shown for clarity.

Figure 2:

Models for recognition of 5’ and 3’ SS by intron and exon definition with U1 snRNP bound to Pol II. Top panel: An intron looping/scanning model of co-transcriptional spliceosome assembly and intron definition [53]. Tethering of the 5’SS by U1 snRNP bound to Pol II causes intron looping that facilitates recognition of the BP and 3'SS by U2 snRNP and U2AF^65/35 by “scanning” thereby promoting formation of cross intron contacts (green arrows) and “ultrafast” splicing [33,34] before synthesis of exon 2 is complete. Pol II associated U2AF^65/35 could participate in scanning. Bottom panel: Pol II bound U1 snRNP could function in exon definition through the formation of cross-exon contacts (red arrows) with U2 snRNP and U2AF. An exchange of U2 snRNP and/or U2AF contacts between the downstream U1 snRNP (blue) to the upstream U1 snRNP (stippled) on the Pol II surface could accomplish the transition to an intron definition complex (top panel). In an alternative pathway, this transition may occur by direct binding of U6 to the upstream 5’SS [27,28]. Cap binding complex (CBC) is bound to the 5’ 7meG cap that is added co-transcriptionally.

Figure 3:. Potential mechanisms by which transcription speed affects co-transcriptional splicing.

Pol II speed influences co-transcriptional RNA folding, which can in turn promote or repress RBP binding. The RNA structural landscape and RBP interactome in turn influence constitutive and alternative splicing decisions. Fast elongation (top panel) favors less local RNA folding and could thereby favor binding of RBPs with single-stranded binding sites. In this example, the RBP enhances skipping of the alternative exon (red). Slow elongation (bottom panel) favors more local RNA structure, that can reduce RBP binding resulting in exon inclusion.