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Genetic and Biochemical Studies on the Role of Essential Protein Dib1 in Pre‐messenger RNA splicing
Author(s) -
Bowman Emily,
Hernandez Cody,
Schreib Christian,
Lucas Amber,
Maeder Corina
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.594.2
Subject(s) - spliceosome , rna splicing , snrnp , minor spliceosome , small nuclear rna , prp24 , biology , splicing factor , microbiology and biotechnology , genetics , intron , exonic splicing enhancer , rna , messenger rna , gene , non coding rna
Pre‐messenger RNA splicing is a process that involves the removal of non‐coding regions of RNA and the ligation of protein‐coding regions in order to become a mature messenger RNA. This process involves the sequential addition of small nuclear ribonucleic protein particles, or snRNPs, that come together to form a pre‐catalytic spliceosome. Once formed, the U1 and U4 snRNPs leave to create the catalytically active spliceosome. The timing and regulation of this step is critical for splicing which must be accurate to a single nucleotide precision in order to prevent drastic effects further downstream in the translation of mRNA into protein. A key figure in this process is Dib1, a small 17kDa essential protein. Dib1 has been shown to be necessary for cell viability and critical for splicing, although its role in splicing has yet to be characterized. Based on the recent cryo‐EM map of the U4/U6‐U5 triple snRNP complex, Dib1 appears to occupy a space in close proximity of the loop region of the U5 snRNA. This snRNA is expected to interact with the pre‐mRNA in the catalytically active spliceosome. Thus, in order for the spliceosome to be activated, Dib1 would need to depart from its location. To examine the role of Dib1 in splicing we have created multiple alleles of the dib1 gene in order to study their effects on cell viability in S. cerevisae . Using these strains, we are exploring mutant growth phenotypes and the effects on in vitro splicing. In addition, we are examining the effects of the mutations on the protein's stability. Currently we have identified two temperature sensitive mutants through our mutational analysis. Using in vitro splicing assays, both were found to have splicing defects, although their defects differed. In order to determine why the splicing phenotypes differed, we are characterizing the effects of the mutations on the structure of Dib1. The proteins were purified using affinity chromatography followed by anion exchange chromatography. CD analysis of the purified protein was then performed. We will present these findings and discuss the implications on pre‐mRNA splicing.