Unbiased Identification of Key Residues in Splicing Protein Dib1

Abstract

Pre‐messenger RNA (pre‐mRNA) is transcribed from a DNA segment, and then post‐transcriptionally modified before being translated into protein. To prepare the pre‐mRNA for proper protein production, the spliceosome facilitates the removal of non‐coding introns from the pre‐mRNA and the ligation of exons together, forming the mature messenger RNA (mRNA) in a process known as pre‐messenger RNA splicing. The spliceosome is a large ribonucleoprotein complex composed of 5‐snRNA and over 90 proteins that work in concert to facilitate pre‐mRNA splicing and ensure its accuracy. Due to its complexity and evolutionary importance to survival, mutations of the spliceosome have been linked to diseases. For instance, Burn‐McKeown syndrome is a craniofacial disorder caused by abnormalities in the gene for the U5‐15k protein, a small protein centered within human spliceosomes and an ortholog of Dib1 in yeast.Dib1 is a 17‐kDa thioredoxin‐like protein that is associated with the U5 small nuclear ribonucleoprotein (snRNP) in the U5/U4.U6 complex. Located in the catalytic groove of the pre‐B complex of the spliceosome, Dib1 must vacate the complex so the spliceosome can complete its assembly. However, this “gate‐keeping” mechanism controlling this departure is still unknown. This project intends to discover residues that are crucial to the function of Dib1 by an unbiased method. To identify key residues in Dib1, a randomly mutagenized dib1 plasmid library was constructed. A temperature sensitive (ts) yeast screen was then performed with this library in order to identify Dib1 residues important for cell growth. From this screen, we have identified a new dib1 mutant and will discuss the significance of its location in Dib1 and its relevance to splicing.Support or Funding InformationTrinity University, ASBMB Distinguished Undergraduate Scholarship, NIH‐NIGMS R15GM120720, and The Welch Foundation