Talk abstracts

Talk on Saturday 10:40-10:55am submitted by Molly Evans

Investigation of debranching enzyme

Molly Evans (Department of Biological Sciences, Carnegie Mellon University), Eduardo Paredes (Department of Chemistry, Carnegie Mellon University), Mark Macbeth (Department of Biological Sciences, Carnegie Mellon University), Subha R. Das (Department of Chemistry, Carnegie Mellon University)

Abstract:
In splicing, exons are joined together and introns removed to generate the correct RNA for translation. The byproduct of splicing is the removed lariat intron. Processing of the lariat intron is performed by debranching enzyme Dbr1, a 2'-5' phosphodiesterase that is well conserved among all eukaryotes. Dbr1 may also have alternative cellular functions, as S. cerevisiae Dbr1 mutants are defective in Ty1 retrotransposition. However, the function of Dbr1 in retrotransposition is unclear. Better systems are needed to characterize Dbr1 and gain more information about the structure and specificity of the enzyme. Previous reports on Dbr1 characterization use non-natural substrates – as the methods of synthesizing the RNA limited the sequence so that the substrates were not the consensus sequences of lariat RNA. Recently, we have developed the solid-phase synthesis of branched RNA that is a mimic of the lariat RNA - including the branched RNA with conserved sequences as well as unnatural branched RNA and DNA-RNA chimeric analogues. The cleavage and binding analyses of these branched RNAs and branched RNA-DNA chimera with Dbr1 offer insight into the substrate specificity of Dbr1. The use of these analogues and Dbr1 and mutants are being investigated for use in protein-RNA crystallization to further our understanding of debranching of RNA and its role in cellular processes.

Keywords: debranching enzyme, branched RNA, splicing