To interrogate this mechanism, we developed a CRISPR interference-based assay that enables the analysis of factors located over particular DNA regions that are associated with particular regions of the pre-mRNA. However, the mechanism that brings the two splice sites into proximity to facilitate co-transcriptional splicing is unknown. This implies that the formation of the commitment complex occurs almost instantly following the synthesis of the 3′SS. Intron lengthening was accompanied by only a minor compromise in splicing efficiency 25, 27, 28: In mammalian cells, short and long introns are generally spliced rapidly irrespective of length 23, 28. During vertebrate evolution introns lengthened by thousands of nucleotides, whereas the average exon length has remained about 150 nucleotides 25, 26. The 5′SS and the PPT interact with U1 snRNP and U2AF2, respectively, immediately after emerging from within pol II 18, 19.Ī study of splicing kinetics revealed that a large fraction of intron removal is complete within seconds 20, 21 to several minutes in living cells 13, 22, 23, 24. U1 snRNP and U2AF2 associate with the pol II CTD, and these interactions have functional effects on splicing 16, 17. The C-terminal domain (CTD) of pol II is necessary for activation of transcription and for efficient pre-mRNA processing 14, 15. This is termed co-transcriptional splicing. Recent studies indicate that most pre-mRNAs undergo splicing while being transcribed by RNA polymerase II (pol II) 9, 10, 11, 12, although there are exceptions 13. The commitment complex then advances into the pre-spliceosome (complexes A and B), which transitions to other complexes that catalyze intron removal and exon ligation in two steps (complex C) 6. Both splice sites are thus defined at this early stage of the reaction. In this complex, the U1 snRNP binds the 5′SS via base pairing between U1 snRNA and the 5′SS, and the 3′SS and the PPT are associated with a heterodimer of U2AF1 (U2AF35) and U2AF2 (U2AF65) 7, 8. The first step in spliceosome assembly is the formation of the commitment complex. The PPT and the branch site are located upstream of intronic 3′ ends 6. The splicing reaction is governed by four main regulatory consensus sequences: the 5′ and the 3′ splice sites (5′SS and 3′SS, respectively), which are located at exon–intron boundaries, the polypyrimidine tract (PPT), and the branch site sequence. Splicing is carried out within the spliceosome, a multi-component complex composed of five nuclear ribonucleoprotein (snRNP) complexes-U1, U2, U4, U5, and U6-and many additional proteins 4, 5. The splicing machinery recognizes either exons or introns as the spliced unit, through mechanisms called exon definition and intron definition, respectively 3. Splicing is the mRNA maturation reaction where introns are removed from pre-mRNA and exons are ligated together 1, 2.
0 kommentar(er)
