2008 Rustbelt RNA Meeting
Assembly and processing of the small ribosomal subunit (40S) in yeast requires multiple steps and many associated factors, as the construction of this essential piece of metabolic machinery is closely regulated. Cleavage steps of the precursor ribosomal RNA (rRNA) are strictly ordered, though the regulation of this ordering remains only partially elucidated. Nob1 is an essential, conserved protein involved in ribosome biogenesis in Saccharomyces cerevisiae. Specifically, cells depleted in Nob1 show a defect in the final step of processing of the 40S small ribosomal subunit rRNA: Cleavage of 20S pre-rRNA to form the mature 3’ end of 18S rRNA does not occur (1). Because Nob1 contains a predicted PIN domain that is required for this cleavage (2), it has been proposed that Nob1 is the endonuclease responsible for maturation of the 20S rRNA to 18S rRNA. However, Nob1 binds the rRNA precursor at a very early stage, and the mechanism by which the endonuclease activity is regulated to only cleave at the final step remains unknown.
Here, we show that recombinant Nob1 binds to rRNA fragments from the 3’ end of the 20S rRNA. Nob1 binds ten-fold more tightly to a substrate (20S)-mimic than product (18S)-mimic. Analysis of Nob1 affinities for a collection of rRNA fragments indicates that sequences upstream and downstream of this region cooperate in Nob1 binding. Structure probing with DMS indicates that these RNAs fold into different conformations in the absence of Nob1. Additionally, Nob1 protects a different subset of residues in these RNAs. These results suggest that conformational changes in the RNA regulate Nob1 binding and activity in generating the mature 3’ end of 18S rRNA.
(1) Fatica, A., Oeffinger, M., Dlakic, M., and Tollervey, D. (2003) Mol. Cell. Biol. 23, 1798-1807.
(2) Fatica, A., Tollervey, D., and Dlakic, M. (2004) RNA 10, 1698-1701.
Keywords: ribosome assembly, conformational change, endonuclease regulation