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Exploring Dib1's Role in pre‐mRNA Splicing
Author(s) -
Schreib Christian Cody,
Hernandez Cody,
Bowman Emily,
Lucas Amber,
Maeder Corina
Publication year - 2017
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.31.1_supplement.910.9
Subject(s) - spliceosome , rna splicing , snrnp , prp24 , protein splicing , biology , genetics , intron , exonic splicing enhancer , microbiology and biotechnology , rna , minor spliceosome , gene
Pre‐messenger RNA splicing is a molecular process conserved throughout all eukaryotic organisms in which non‐protein coding regions of the pre‐messenger RNA are removed. This process is facilitated by the spliceosome, a large multi‐component molecular machine made of five small nuclear RNAs and ~100 proteins. One such protein is Dib1, a small protein in the U4/U6‐U5 triple snRNP that has been shown to be essential to cell viability and splicing. Previous work suggests that Dib1 leaves the splicing machinery once it assembles, and recent cryo‐EM structures indicate that Dib1 is housed in a central location in the U4/U6‐U5 triple snRNP. To better understand what role Dib1 plays in splicing, we aimed to identify essential regions of the protein by identifying temperature sensitive mutations. Conserved residues found in hydrophobic pockets, basic regions, and loop regions of Dib1 were mutated to alanines utilizing site‐directed mutagenesis. These Dib1 mutants were transformed into a S. cerevisiae dib1Δ strain. Growth phenotypes of these yeast were surveyed using a serial dilution growth assay. Two Dib1 mutations caused decreased cell viability at 37°C. One of these mutations is located in a hydrophobic pocket and the other in an external loop region. The temperature sensitive mutations were further characterized by assaying their effect on splicing efficiency through in vitro splicing assays. Both mutations were found to cause severe splicing defects, blocking splicing prior to the first step. We then assayed the assembly of the spliceosome in the presence of these temperature sensitive Dib1 mutants through native gel analysis. Because splicing is an intricate process that involves many complexes coming on and off the pre‐mRNA, knowing the specific complexes on the pre‐mRNA can tell us when in the process of spliceosome assembly the Dib1 mutations had an effect, and in turn tell us when in the spliceosome assembly Dib1 is essential. The results of these assays suggest that Dib1 plays a vital role in the binding of the U4/U6‐U5 triple snRNP to the pre‐mRNA. Support or Funding Information Arnold and Mabel Beckman Foundation, NIH NIGMS R15GM120720, Welch Foundation