Putative Interaction Between Branch Site and 5' Splice Site During the Exon-Ligation Conformation of Splicing, Poster 56

Pre-mRNA splicing, an essential step in eukaryotic gene expression, is catalyzed by a dynamic assembly of small nuclear RNA (snRNA) and proteins, collectively called the spliceosome. Splicing occurs in two steps but the... [ view full abstract ]

Pre-mRNA splicing, an essential step in eukaryotic gene expression, is catalyzed by a dynamic assembly of small nuclear RNA (snRNA) and proteins, collectively called the spliceosome. Splicing occurs in two steps but the spliceosome only contains one catalytic core, necessitating rearrangement between steps. This investigation explores a putative interaction between the 5' Splice Site and Branch Site during the second, or exon-ligation, conformation of splicing. A genetic approach is taken that involves introducing single mutations, which would disrupt effective splicing, and subsequent double mutations that would rescue the splicing defect if the regions were indeed interacting. The efficacy of splicing is determined using an ACT1-CUP1 reporter that can be assayed via copper growth experiments and radiolabeled primer extension. Initial mutations suggest that the predicted interaction is possible, however, further evidence is needed to confirm the model, including more double mutations and primer extension analyses.