Comprehensive Mechanistic Analysis of the RNA‐Lariat Debranching Enzyme

Abstract

RNA‐lariat Debranching Enzyme (Dbr1p) hydrolyzes the 2′‐5′ phosphodiester bond between the intronic, adenosine branch point and the 5′ end of the intron. Intron lariat debranching is essential for processing of snoRNAs and miRNAs and for efficient intron turnover. Interestingly, Dbr1p null mutants have been shown to be defective in Ty1 retrotransposition in Saccharomyces cerevisiae. Additionally, siRNA knockdown of DBR1 in a eukaryotic cell line has inhibited HIV‐1 replication, making characterization and structure determination imperative to the rational design of a wide range of inhibitors.We present structural, mechanistic and comparative analyses of two Dbr1 proteins. While S. cerevisiae Dbr1p has been characterized biochemically, complete mechanistic understanding has been limited by the unavailability of physiologically relevant substrates, as well as the lack of a high‐resolution Dbr1p structure. Our comprehensive analysis has utilized in vitro cleavage assays with a variety of branched substrates, site‐directed mutagenesis, inductively coupled plasma atomic emission spectroscopy (ICP‐AES) and fluorescence anisotropy to characterize Dbr1p kinetics, and to identify a unique versatility in substrate recognition and a hetero‐dinuclear active site.Funding is provided by the CMU Center for Nucleic Acids Science and Technology, NSF GRFP #0750271 and the UNCF/Merck Graduate Fellowship.