Poster abstracts
Poster number 115 submitted by John Pettersson
Single-molecule analysis of backbone branched RNAs and lariat debranching enzyme
John R. Pettersson (Department of Chemistry Carnegie Mellon University ), Sourav K. Dey (Department of Chemistry Carnegie Mellon University ), Linda A. Peteanu (Department of Chemistry Carnegie Mellon University ), Subha R. Das (Department of Chemistry Carnegie Mellon University )
Abstract:
The spliceosome processes pre-mRNA by removing introns and ligating exons together. In this process, the excised intron is in the form of a lariat that includes a 2’-5’ phosphodiester bond. The lariat debranching enzyme (Dbr1p) specifically hydrolyzes the 2’-5’ phosphodiester bond producing a linear RNA that can be further processed into snoRNAs, miRNAs and other biologically active RNAs. Dbr1p has not been fully characterized due to the limited access to substrates for the enzyme. We have developed a method to synthesize backbone branched RNAs (bbRNAs) that include a 2’-5’ phosphodiester linkage which mimics the branchpoint and serve as a substrate for Dbr1p, as well as click-branched RNA lariat analogues (cbRNA) that are not cleaved by the enzyme. This access to bbRNAs, cbRNA, and recombinant Dbr1p has enabled us to combine biochemical bulk studies with single-molecule spectroscopy. We use single-molecule TIRF and confocal microscopy to analyze the conformation and dynamics of the RNAs and to further examine the interactions between bbRNAs, cbRNAs and Dbr1p.
Keywords: RNA, Spectroscopy, Enzyme