An RNA Processing Activity That Debranches RNA Lariats

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Abstract
The excised introns of pre-messenger RNA's (pre-mRNA's) and intron-containing splicing intermediates are in a lariat configuration in which the 5' end of the intron is covalently joined by a 2',5'-phosphodiester bond to a specific adenosine residue near the 3' end of the intron. A 2',5'-phosphodiesterase activity in HeLa cell extracts has been detected that debranches RNA lariats, converting them to linear RNA molecules by specific cleavage of the 2',5'-phosphodiester bond. This lariat debranching activity is distinct from previously reported 2',5'-phosphodiesterases with regard to its biochemical and substrate requirements as well as its stringent cleavage specificity. The debranching activity is observed only if the RNA lariats generated during in vitro processing are deproteinized and added back to the extract. These results suggest that during the normal in vitro splicing reaction the 2',5'-phosphodiester bond of RNA lariats is protected from cleavage by the lariat debranching activity.