[19] RNA lariat debranching enzyme

Wallace and Edmonds first discovered branched nucleic acids in the population of nuclear polyadenylated RNAs. These branched nucleic acids are mostly derived from intron lariats, one of the intermediate structures in pre-mRNA splicing. Pre-mRNA splicing, a eukaryote-specific process, generates two products: the spliced exons and intron lariats. The 2'-5' phosphodiester bonds of intron lariats are then hydrolyzed by the RNA lariat debranching enzyme (Dbr). Dbr is thus a 2'→5'-phosphodiesterase. It specifically hydrolyzes the 2'-5' phosphodiester linkage of RNA intron lariats between the G residues of the 5' splice site and the A residues of the branch point. This cleavage converts the intron lariat into a linearized intron and is the rate-limiting step in intron degradation pathway. Alternative substrates for the debranching assay include total RNA, in vitro transcribed pre-mRNA that has been subjected to in vitro splicing reaction, gel-purified branched RNAs, and in vitro-synthesized branched nucleic acids. When preparing a substrate, it is important to keep the substrate requirements of Dbr in mind. It is also possible to chemically synthesize branched DNA, branched RNA, and branched DNA-RNA chimeras via automated solid-phase methods.