Poster abstracts

Poster number 67 submitted by Binal Shah

Mechanistic insights into how RNA chaperone activity facilitates eukaryotic ribosome biogenesis

Binal N. Shah (Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064), Timea Gerczei (Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, Current Add Dept of Chemistry, Ball State University, Muncie, IN 47306), Anthony J. Manzo (Department of Chemistry, University of Michigan, Ann Arbor, MI 48109), Nils G. Walter (Department of Chemistry, University of Michigan, Ann Arbor, MI 48109), Carl C. Correll (Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064)

Abstract:
To satisfy the high demand for ribosome synthesis in rapidly growing eukaryotic cells, short duplexes between the U3 small nucleolar RNA (snoRNA) and the precursor ribosomal RNA (pre-rRNA) must form quickly and with high yield(1-3). These interactions, designated the U3-ETS and U3-18S duplexes, are essential to initiate the processing of small subunit rRNA. Previously, we developed assays designed to mimic these RNA-RNA interactions and showed qualitatively that these duplexes form only after addition of one of two proteins: Imp3p or Imp4p(4). Here, we developed fluorescence-based assays to determine whether these proteins assemble and possess RNA chaperone activity sufficient to satisfy the high U3-ETS duplex yield and rapid U3-18S duplex formation (t_1/2 ~ 85 sec) expected in vivo. In the absence of protein, the U3-ETS duplex is weak due to short duplex length. A kinetic barrier limits formation of the U3-18S duplex because the U3-stem structure must unfold to expose its base-pairing site. Both proteins assemble with the U3 snoRNA into a chaperone complex, which is the smallest relevant complex to ensure sufficient U3-pre-rRNA duplex formation and yield. This complex stabilizes the U3-ETS duplex K_d by two orders of magnitude with a negligible effect on the duplex k_on. The chaperone complex also stimulates U3-18S duplex formation presumably opens up the U3-stem structure to expose the 18S base-pairing site. Our findings demonstrate that formation of the U3-18S duplex will occur too slowly (t_1/2 >> 85 sec) in the absence of protein for any concentration of pre-rRNA. In contrast to the absence of protein, assembly of the chaperone complex sufficiently accelerates U3-18S duplex formation to satisfy the need for rapid formation of this duplex under a wide range of pre-rRNA concentrations. The activities of this chaperone complex result in high U3-ETS duplex yield and a fast formation of the U3-18S duplex, thereby ensuring that the U3-pre-rRNA interactions limit neither ribosome biogenesis nor rapid cell growth.

References:
References
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(2) Sharma, K.; Tollervey, D. Mol Cell Biol 1999, 19, 6012-9.
(3) Osheim, Y. N.; French, S. L.; Keck, K. M.; Champion, E. A.; Spasov, K.; Dragon, F.; Baserga, S. J.; Beyer, A. L. Molecular Cell 2004, 16, 943-54.
(4) Gerczei, T.; Correll, C. C. Proc Natl Acad Sci U S A 2004, 101, 15301-6.

Keywords: chaperone, conformational change, ribosome biogenesis