| Alternative splicing is an essential eukaryotic RNA processing event, resulting in diversified pools of mRNA isoforms. Alternative splicing can impact many cellular functions and therefore must be carefully regulated. Multiple coordinated interactions between RNA processing machineries occur, necessitating the maintenance of proper interactions at each step during mRNA production.;Understanding the mechanisms of exon pairing is crucial for understanding alternative splicing. Using in vitro kinetic trap assays, we showed that commitment to splice site pairing requires a dedicated ATP hydrolysis step. Thus, our results demonstrate that commitment to splice site pairing occurs independent of U2 snRNP binding.;The ability of the spliceosome to efficiently engage large ligated exon intermediates contradicts established rules of exon size recognition limits. We show for the first time that retention of spliceosomal components at splice sites following an upstream intron removal event is sufficient to maintain efficient splicing. Our results imply that ligated exon intermediates do not require redefinition, thus maintaining the pre-mRNA committed to efficient splicing throughout its entire processing pathway.;Interactions between RNA processing factors aid in efficient and accurate pre-mRNA processing. We demonstrate that alternative splicing and alternative polyadenylation are interconnected, affecting the transcript coding sequence at variable distances from the 3' UTR. The observed changes to the coding region likely have biological consequences, potentially resulting in new protein isoforms or the activation of RNA decay pathways. These findings demonstrate that alternative polyadenylation has a widespread influence on mRNA diversity, exceeding manipulation of mRNA stability, by influencing upstream alternative splicing events. |
