Poster abstracts

Poster number 110 submitted by Madhumitha Ramesh

Understanding the roles of eukaryote-specific rRNA expansion segments in ribosome biogenesis

Madhumitha Ramesh (Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA), John L. Woolford (Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA)

Abstract:
A universal core secondary structure for ribosomal RNA (rRNA) has been identified across kingdoms of life. (1,2) However, yeast rRNA, and eukaryotic rRNA in general, have extra blocks of sequences relative to that of prokaryotic rRNA, called “expansion segments” (ES). These ES vary in their length and sequence, both within and among different organisms. This variability seems to preclude ES from playing crucial roles in the function of the ribosome. (3)

Isolated attempts to understand the function of ES have been made before. Previous reports indicate that some, but not all ES can tolerate insertions. (4) Also, deletion mutational analysis of two ES has been shown to affect ribosome biogenesis (5). Despite these reports, a thorough study of the precise roles of these ES remains to be done, possibly because of the limitations in the availability of convenient systems to study rRNA mutants.

We set out to systematically investigate the potential roles played by expansion segments of the 25S rRNA in Saccharomyces cerevisiae ribosome biogenesis. We began with deleting five of the eukaryote-specific ES in yeast large subunit rRNA. We used a temperature sensitive PolI mutant yeast strain in which transcription of rDNA is driven from a plasmid-borne rDNA copy with a GAL promoter (6). Also, we developed yeast strains derived from this strain in which one can pull down pre-ribosomes using affinity purification. The phenotype of the mutants was first assayed by studying their growth. Following this, primer extension assays and affinity purifications were used to zoom in on the precise ribosome assembly phenotype that these mutants exhibit.

This systematic study will help us unravel the yet unexplored functions of these eukaryote-specific ES and pave the way for a deeper understanding of the mechanisms of ribosome biogenesis in general.

References:
1. Veldman, G.M., et al. (1981) Nucleic Acids Res. 9:6935-6952
2. Clark, C. G., et al. (1984) Nucleic Acids Res. 12: 6197-6220.
3. Gerbi, S.A. (1996). In ‘Ribosomal RNA Structure, Evolution, Processing, and Function in Protein Biosynthesis’, R.A. Zimmerman, and A.E. Dahlberg, eds. (Boca Raton, FL, CRC Press), pp. 71-87
4. Musters, W., et al. 1989) Mol. Cell. Biol. 9:3989-3996
5. Jeeninga, R. E., et al. (1997). RNA. 3: 476-488
6. Nogi, Y., et al. (1991). Proc. Natl. Acad. Sci. USA 88: 3962-3966

Keywords: rRNA , ribosome biogenesis, expansion segments