Poster abstracts

Poster number 82 submitted by Crystal Young

Elucidating the roles of Rrp5 and Rok1 in 40S ribosome maturation

Crystal Young (Chemistry Department, University of Michigan), Katrin Karbstein (Chemistry Department and Department of Biological Chemistry, University of Michigan)

Abstract:
Ribosomes catalyze protein synthesis in all cells. Even though assembly of ribosomes is well studied and understood in bacteria, assembly in eukaryotes is much more complex and requires >170 accessory proteins. These proteins facilitate processing and folding of the four rRNAs and their assembly with the 78 ribosomal proteins, most of which have no prokaryotic homologs. However, the exact function of most ribosome assembly factors remains unknown. Three of the four rRNAs, the 18S, 5.8S and 25S rRNAs, are co-transcribed in a single transcript, which is then cleaved in a series of well-ordered steps to release the mature rRNAs. While the spatial and temporal ordering of these cleavage steps has been determined, the mechanisms by which they occur have yet to be elucidated.
Data in our lab suggest that prior to and immediately after cleavage at site A2 (which separates 18S rRNA from the 5.8S and 25S rRNA), the rRNA is in a conformation different from that required for the last cleavage step. In order to achieve this final structure, RNA must be removed after A2 cleavage. Our model predicts that the interaction between two essential assembly factors, Rrp5 and Rok1, is responsible for this conformational change. The c-terminus of Rrp5, an rRNA binding protein, has been shown to be essential for A2 cleavage. Additionally, Rrp5 has been shown to have both a physical and genetic interaction with Rok1, a putative DEAD-box RNA helicase.^1,2 We hypothesize that Rok1 interacts with the rRNA via Rrp5 and uses its ATPase activity to facilitate the essential rRNA conformational change.
Preliminary northern analysis results suggest that Rok1 depleted cells accumulate the expected 18S rRNA precursors. In biochemical analyses of Rok1, ATPase inhibition assays in the presence of AMPPNP or ADP indicate that Rok1 binds ADP at least 500 fold tighter than ATP. In vivo, an accessory protein such as Rrp5 may be required for increased ATP affinity and Rok1 activation. In further support of the Rok1-Rrp5 interaction, pull-down experiments with recombinant proteins show that Rok1 binds Rrp5 directly.
Future directions include development of an in vitro helicase assay using the expected in vivo duplex substrate, Rok1 and Rrp5.

References:
1 Torchet, C.; Jacq, C.; Hernann-Le Denmat, S. RNA 1998, 4, 1636.
2 Vos, H.R.; Bax, R. et al. Nucleic Acids Research 2004, 32, 5827.

Keywords: Ribosome Assembly, 18S rRNA Maturation, Rok1 and Rrp5