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 InformationNIH R15GM120720; Robert A Welch Foundation Grant W‐1905This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.