Talk abstracts

Talk on Friday 03:10-03:25pm submitted by Aarti Sahasranaman

Detailed analysis of one step of ribosome biogenesis in Saccharomyces cerevisiae

Aarti Sahasranaman (Department of Biological Sciences, Carnegie Mellon University), John Strahler (Michigan Proteome Consortium, University of Michigan, Ann Arbor), Philip Andrews (Michigan Proteome Consortium, University of Michigan, Ann Arbor), Janine Maddock (Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor), John Woolford (Department of Biological Sciences, Carnegie Mellon University)

Abstract:
Ribosome biogenesis is a complex, dynamic process that must coordinate processing and folding of rRNA precursors with binding of r-proteins. At least 180 assembly factors are required for this process. To understand the mechanism of ribosome assembly, we are studying one particular step- exonucleolytic processing of 27SA₃ pre-rRNA to form 27SB_S pre-rRNA, and generate the 5´ end of 5.8S rRNA. Depletion of seven assembly factors, collectively referred to as the A₃ factors, specifically results in accumulation of 27SA₃ pre-rRNA and reduced levels of 27SB_S pre-rRNA, suggesting that they function in processing 27SA₃ pre-rRNA. Three 5´-3´ exonucleases, Rat1, Xrn1, and Rrp17, are required for this processing step. Six A₃ factors associate with preribosomes in an interdependent manner, and are required for the stable association of Rrp17, but not Rat1 or Xrn1 with preribosomes. In the absence of these A₃ factors, four r-proteins- rpL17, rpL26, rpL35, and rpL37 cannot associate with preribosomes. These four r-proteins are present close to each other in mature ribosomes; rpL17 and rpL26 bind helix 2 formed by base-pairing between the 5´ end of 5.8S rRNA and 25S rRNA. Further investigation of rpL17 revealed that it becomes stably associated with 66S preribosomes after 27SA₃ pre-rRNA processing. In the absence of rpL17, Rat1 cannot stop at the 5´ end of 5.8S rRNA, suggesting that rpL17 serves as the roadblock to Rat1 during 27SA₃ pre-rRNA processing. In A₃ factor mutants, where rpL17 and the other three r-proteins are absent, Rat1 functions as a surveillance tool for turnover of aberrant assembly intermediates. We propose that A₃ factors play a role in bringing together 5.8S and 25S rRNAs to enable formation of helix 2. Binding of rpL17 enabled by A₃ factors, stabilizes this helix and ensures that Rat1 stops at the correct site to generate the 5´ end of 5.8S rRNA. When ribosome assembly is perturbed, Rat1 functions as a quality control system to eliminate defective pre-rRNAs.

Keywords: ribosome assembly, pre-rRNA processing, exonucleases