Characterizing the impact of splicing protein Dib1 on the pre-messenger RNA interactions with the spliceosome

In eukaryotic organisms, pre-messenger RNA splicing facilitates the removal of non-coding regions of RNA to create a functional mRNA. Pre-mRNA splicing is catalyzed by the spliceosome, a large, dynamic, molecular machine composed of five small nuclear ribonucleoproteins (snRNPs). Dib1, a key protein of the spliceosome, resides in the U4/U6.U5 triple-snRNP. Dib1 is essential for cell viability and is conserved from yeast to humans. Dib1 must depart the splicing complex before the catalytic steps of splicing can occur; however, how Dib1's departure is facilitated is unknown. Dib1 and pre-mRNA are in close proximity in the pre-catalytic spliceosome (Plaschka et al. Nature 2017); characterizing the interactions between Dib1 and pre-mRNA may be necessary to understand Dib1's departure. We are characterizing potential interactions between Dib1 and pre-mRNA using single molecule fluorescence colocalization experiments in collaboration with Dr. Nils Walter at the University of Michigan. An open question is how and whether Dib1 and pre-mRNA are co-localized in the splicing machinery. We are addressing this question using Cy5-labeled Dib1 and Cy3-labeled pre-mRNA. We have labeled purified Dib1 with a Cy5 fluorophore and have been able to splice Cy3-labeled pre-mRNA. Progress and results of the single molecule fluorescence colocalization between Dib1 and pre-mRNA will be discussed. Another area of interest is how temperature-sensitive mutant Dib1, which causes a loss of splicing function in Saccharomyces cerevisiae, may affect the pre-mRNA throughout the splicing cycle. To this end, we are characterizing pre-mRNA conformation in S. cerevisiae extracts containing wild type or mutant Dib1 protein. We are using single molecule fluorescence resonance energy transfer (smFRET) to analyze the conformation of pre-mRNA labeled with a Cy3 fluorophore at the 5′ splice site and a Cy5 fluorophore at the branch point. Comparison of the smFRET analysis will show whether mutant Dib1 causing loss of function is associated with a change of RNA conformation. Progress and results of the smFRET analysis of pre-mRNA will be discussed. Support or Funding Information NIH R15GM120720; Robert A Welch Foundation Grant W-1905 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.