Poster abstracts

Poster number 90 submitted by Crystal Young

Characterizing Rok1, a putative RNA helicase involved in 18S processing

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

Abstract:
Ribosomes are the machinery responsible for catalyzing protein synthesis in all cells. While the process by which these essential complexes form is well studied and understood in prokaryotes, in eukaryotes the formation is much more complex and therefore not entirely known. What makes the eukaryotic ribosome more complicated is the observation that assembly requires >170 accessory proteins in order to process, fold and bind 78 ribosomal proteins to the 5.8S, 25S and 5S ribosomal RNAs (rRNAs) that make up the large subunit and the 18S rRNA that comprises the small subunit(1). Of these four rRNAs, three (5.8S, 25S and 18S) are co-transcribed in a single transcript known as the 35S rRNA(1). In order for the mature rRNAs to be formed from the 35S rRNA, a series of processes and cleavages must occur. In the maturation of the 18S rRNA, for example, cleavage must occur at the site known as A2, which separates the 18S segment from the rest of the 35S rRNA. We hypothesize that in order for this step to occur, an inhibitory helix formed by base pairs alternative to the mature structure must be disrupted. Our model suggests that Rok1, an ATP-dependent RNA helicase, interacts with the rRNA via Rrp5, an rRNA binding protein, and uses its ATPase activity to unwind this inhibitory duplex. Prior studies have shown that Rok1 is essential for viability and that the depletion of the Rok1 protein blocks synthesis of the 18S rRNA, therefore providing support to the model suggested above(2,3). To prove our hypothesis, we will focus on characterizing Rok1 and its interactions with Rrp5. Thus far, we have exemplified Rok1’s ATPase activity and conducted ATPase inhibition studies that suggest that Rok1 binds ADP more tightly than ATP. In addition, we have shown that Rok1 binds rRNA. Future directions include the addition of Rok1, Rrp5 and rRNA to all assays simultaneously in hopes of developing an in vitro helicase assay that will help us to better understand the maturation of 18S rRNA.

References:
1 Fromont-Racine, M.; Senger, B.; Saveanu, C.; Fasiolo, F. Gene 2003, 313, 17.
2 Song, Y.; Kim, S.; Kim, J. Gene 1995, 166, 151.
3 Venema, J.; Bousquet-Antonelli, C.; Gelugne, J.; Caizergues-Ferrer, M.; Tollervey, D. Mol. Cell. Biol. 1997, 17, 3398.

Keywords: ribosomal assembly, 18S rRNA