Abstract

Splicing of precursor mRNAs is a hallmark of eukaryotic cells and is carried out by a large macromolecular complex known as the spliceosome, together with dozens of associated proteins. During this process, the spliceosome recognizes sequence motifs at the 3′ and 5′ splice sites, excises introns, and ligates exons through two consecutive transesterification reactions. Each of these steps can be tightly regulated to ensure the correct production of mRNA isoforms necessary for proper cellular function. Although pre-mRNA splicing has been studied for many years, the molecular mechanisms underlying splice-site recognition and the progression of splicing remain incompletely understood. The accurate recognition of 5′ splice sites is determined by their sequence motifs and is essential for the precise definition of intron boundaries by the spliceosome. In this study, the molecular determinants of 5′ splice-site recognition, together with the progression of the two-step transesterification reaction, were investigated using the pET-STAT3 minigene carrying targeted mutations at the 5′ splice-site of STAT3 exon 21. A shifted artificial U1 snRNA was used to compensate for impaired endoge-nous U1 snRNA binding caused by these mutations. Splicing intermediates were traced and semi-quantitatively analysed by enriching STAT3 exon 21 RNA using a biotinylated probe and streptavidin magnetic beads. This approach enabled monitoring of the utilization of modified splice-sites during intermediate stages of splicing and provided insight into how splice-site mutations affect the stepwise chemistry of intron removal. …víceméně