The Effects of Tail Truncations of Pre‐Messenger RNA Splicing Protein Dib1

Abstract

The spliceosome is a multi‐megadalton ribonucleoprotein complex that catalyzes the removal of non‐coding introns and the ligation of coding exons from pre‐messenger RNA. This process is crucial in the transformation of pre‐messenger RNA into messenger RNA, a template that is readable by the ribosome and allows for the production of a functional protein. Significantly, the spliceosome is constructed anew on every pre‐mRNA. This construction entails over 140 protein factors that associate and dissociate throughout the splicing cycle, causing the conformational rearrangements and transesterification reactions necessary to yield the mature RNA template. Therefore, rather than a singular composition of proteins and RNAs being known as “the spliceosome,” this term actually refers to several different complexes which are distinct in their makeup and their role within the splicing cycle. Construction of a catalytic splicing complex occurs after a series of early assembly steps. Amongst the proteins involved in these early assembly steps is a 16.8 kDa protein, Dib1, that is present in the pre‐catalytic spliceosome but absent from the catalytically active conformation. The loss of Dib1 at this critical juncture hints at a potential role helping to facilitate the transition from pre‐catalytic to catalytic. Previous work on Dib1 has revealed that not only is it required for splicing, but the human ortholog, hDim1, has been shown to possess autocleavage activity in vitro where the last 14 amino acids are cleaved from its C‐terminal tail. Thus, we are interested in whether the C‐terminus impacts the function of the protein as it pertains to its role in splicing. In order to elucidate the importance of this region, we have constructed a series of C‐terminal truncations of varying lengths within Dib1 in order to assess any resulting splicing defects. These defects have been monitored through the use of growth assays and splicing assays to ascertain not only the overall importance of this region, but also the specific length required to ensure splicing abilities similar to wildtype Dib1 within the model organism S. cerevisiae. Overall, we observe that Dib1 can tolerate a large deletion before affecting cell growth and splicing.