Substrate recognition by a eukaryotic RNase III: The double-stranded RNA-binding domain of Rnt1p selectively binds RNA containing a 5 '-AGNN-3 ' tetraloop

Rnt1p is an RNase III homolog from budding yeast, required for processing s nRNAs, snoRNAs, and rRNA. Numerous Rnt1p RNA substrates share potential to form a duplex structure with a terminal four-base loop with the sequence AG NN. Using a synthetic RNA modeled after the 25S rRNA 3' ETS cleavage site w e find that the AGNN loop is an important determinant of substrate selectiv ity. When this loop sequence is altered, the rate of Rnt1p cleavage is redu ced. The reduction in cleavage rate can be attributed to reduced binding of the mutant substrate as measured by a gel-shift assay. Deletion of the non conserved N-terminal domain of Rnt1p does not affect cleavage site choice o r the ability of the enzyme to distinguish substrates that contain the AGNN loop, indicating that this region is not required for selective cleavage. Strikingly, a recombinant fragment of Rnt1p containing little more than the dsRBD is able to discriminate between wild-type and mutant loop sequences in a binding assay. We propose that a major determinant of AGNN loop recogn ition by Rnt1p is present in its dsRBD.