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Substrate recognition by a eukaryotic RNase III: The double-stranded RNA-binding domain of
 

Summary: Substrate recognition by a eukaryotic RNase III:
The double-stranded RNA-binding domain of
Rnt1p selectively binds RNA containing
a 59-AGNN-39 tetraloop
ROLAND NAGEL and MANUEL ARES, JR.
Center for the Molecular Biology of RNA, Sinsheimer Laboratories, University of California, Santa Cruz,
Santa Cruz, California 95064, USA
ABSTRACT
Rnt1p is an RNase III homolog from budding yeast, required for processing snRNAs, snoRNAs, and rRNA. Numerous
Rnt1p RNA substrates share potential to form a duplex structure with a terminal four-base loop with the sequence
AGNN. Using a synthetic RNA modeled after the 25S rRNA 39 ETS cleavage site we find that the AGNN loop is an
important determinant of substrate selectivity. When this loop sequence is altered, the rate of Rnt1p cleavage is
reduced. 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 nonconserved N-terminal domain of Rnt1p does not affect cleavage site choice or 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 recognition by Rnt1p is present in its dsRBD.
Keywords: dsRBD; pre-rRNA processing; endoribonuclease
INTRODUCTION

  

Source: Ares Jr., Manny - Department of Molecular, Cell, and Developmental Biology, University of California at Santa Cruz

 

Collections: Biology and Medicine