RNA lariat debranching enzyme as tool for analyzing RNA structure

The primary transcripts (pre-mRNAs) of most structural genes in higher eukaryotes contain intervening sequences (introns) that are removed by splicing. The lariat debranching enzyme is an RNA processing activity, originally identified in mammalian cell extracts, that specifically hydrolyzes the 2′-5′ phosphodiester bond in RNA lariats. Analysis of yeast pre-mRNA mutants supports a role for the enzyme in the intron degradation pathway. The 2′-5′ phosphodiester bond is resistant to digestion by most nucleases. The high substrate specificity of the lariat debranching enzyme has made it a powerful tool for the analysis of RNA structure. The enzyme is relatively stable and maintains activity following fractionation over a number of conventional chromatographic resins. In all instances, the activity fractionates as a single component. RNA lariats (and branched linear RNAs) have anomalous electrophoretic mobilities on denaturing polyacrylamide gels. Following enzymatic debranching, the linear RNA product migrates more rapidly than the lariat. The 2′-5′ phosphodiester bond completely blocks reverse transcriptase in a primer-extension experiment. The position of the primerextension stop can therefore be used to localize the RNA branch.