Substrate recognition by a eukaryotic RNase III: The double-stranded RNA-binding domain of Rnt1p selectively binds RNA containing a 5 '-AGNN-3 ' tetraloop
R. Nagel et M. Ares, Substrate recognition by a eukaryotic RNase III: The double-stranded RNA-binding domain of Rnt1p selectively binds RNA containing a 5 '-AGNN-3 ' tetraloop, RNA, 6(8), 2000, pp. 1142-1156
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.