The Roles of the Essential Proteins Dib1, Prp31, Prp6 and the U5 snRNA During Splicing
Pre-mRNA splicing is a conserved biological process essential to all eukaryotes. Splicing defects lead to a variety of disorders in humans including Retinitis Pigmentosa and Burn-McKeown Syndrome, defining the importance of splicing in health science fields. Pre-mRNA splicing requires complex machinery composed of five small nuclear RNAs (snRNAs) complexed with ~90 proteins that form small nuclear ribonucleoproteins. Spliceosome assembly occurs over several dynamic steps to form a pre-catalytic B complex before splicing reactions can occur.
Splicing cannot begin until the pre-mRNA and U5 snRNA can interact. Analysis of cryo-EM structures (Plaschka, et al. 2017) as well as biochemical and genetic studies reveal three essential proteins in the B complex blocking a pre-mRNA-U5 snRNA interaction. At the center of the catalytic site sits Dib1, a 17-kDa protein with a thioredoxin-like (TRXL) domain. Prp6 is a large, 102-kDa U4/U6.U5 triple-snRNP protein whose N-terminal region appears to interact with the hydrophobic pocket located on one side of the Dib1 TRXL domain. Prp31 is another large, 56-kDa protein that appears to interact with a basically charged region of Dib1 opposite of the hydrophobic pocket via acidic residues on its extended loop region. Crosslinking studies (Dix, et al. 1998) revealed that the U5 snRNA loop 1 interacts with a 16-kDa protein and cryo-EM studies of B complexes revealed U5 loop 1 may interact with Dib1.
Prp6, Prp31, and Dib1 must all depart the spliceosome before splicing can occur. The interactions between these proteins and the U5 snRNA may serve to stabilize the spliceosome and prevent the premature splicing of pre-mRNA. Here, we aim to identify temperature sensitive (ts) growth mutants of Prp6, Dib1, Prp31 and U5 snRNA that inhibit or abolish splicing and identify the mutants’ roles in the assembly of the B complex and the U4/U6. U5 triple snRNP. Studies are performed in S. cerevisiae, an excellent model to study splicing. Our findings of key residues in Prp6, Prp31 and U5 snRNA that are important for interactions with Dib1 will be presented as well as the effects of these novel mutants on splicing and spliceosome assembly.
Federation of American Societies for Experimental Biology
Embry, A., Potts, C., Jamison, D., Turner, C., & Maeder, C. (2019) The roles of the essential proteins dib1, prp31, prp6, and the u5 snRNA during splicing. FASEB Journal, 33(1-supplement).