Dr. Sanjaya Abeysirigunawardena

"Post-transcriptional RNA modification and Ribosome assembly"

Ribosome is essential for all forms of life. Ribosome biogenesis is a complex process that requires synchronization of various cellular events including transcription of ribosomal RNA, ribosomal protein binding, RNA processing and the post-transcriptional and -translational modifications of ribosomal RNA and proteins respectively.[1] Interestingly, several studies have suggested a correlation between nucleotide modifications and antibiotic resistance.[2] For example, mutant RsmG lacking methyltransferase activity causes low-level streptomycin resistance and gives rise to weakly hyper-accurate ribosomes.[3] The main mission of our lab is to understand the molecular basis for the correlation between nucleotide modification and antibiotic resistance.

It is well established that protein addition during the ribosomal assembly is thermodynamically corporative.[4-5] We are currently investigating the presence of similar binding corporativity between the ribosomal proteins and three RNA modification enzymes (RsuA, RsmG and RsmC) that bind to bacterial ribosomes during ribosome biogenesis. Our laboratory has developed several fluorescence- and gel-based assays to detect binding of these enzymes to 16S RNA. These assays will be used to determine the binding cooperativity between modification enzymes and ribosomal proteins.[5-6] In addition we are also probing structural changes in ribosomal RNA associated with modification enzyme binding using RNase and hydroxyl radical footprinting experiments. These analyses will reveal the structural basis for thermodynamic cooperativity in protein additions.

Interestingly, there are more than 100 nucleotide modifications found in various different RNAs. These modified nucleotides are known to be involved in localized structural and thermodynamic perturbations.[7-8] At the same time, these modifications also can perturb protein binding, and hence influence ribosome biogenesis. It has been shown that disrupting the pseudouridylation and methylation machinery in yeast cells leads to temperature sensitive growth defects. Similarly, bacterial ribosomes reconstituted with in vitro transcribed 23S rRNAs that the lack of nucleotide modifications show a five-fold reduction in peptidyl-transferase activity. The second goal of our lab is to investigate how modified nucleotides (pseudouridine, m2G and m7G) perturb local RNA structure, RNA folding thermodynamics and RNA-protein interactions using various biophysical methods.

References

1. Shajani, Z., Sykes, M. T., Williamson, J. R., Assembly of bacterial ribosomes, Annu. Rev. Biochem. 80, 501-526 (2011).
2. Motorin, Y., Helm M., RNA nucleotide methylation. Wiley Interdiscip. Rev. RNA. 2, 611-631 (2011).
3. Nishimura, K., Hosaka, T., Tokuyama, S., Okamoto, S., Ochi, K. Mutations in rsmG, encoding a 16S rRNA methyltransferase, result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3, J. Bacteriol. 189, 3876-3883 (2008).
4. Recht, M. I., Williamson, J. R., Central domain assembly: thermodynamics and kinetics of S6 and S18 binding to an S15-RNA complex, J. Mol. Biol. 313, 35-48 (2001).
5. Abeysirigunawardena, S. C., Woodson, S. A., Differential effects of ribosomal proteins and Mg2+ ions on a conformational switch during 30S ribosome 5'-domain assembly, RNA. 21, 1859-1865 (2015).
6. Kim, H., Abeysirigunawardena, S.C., Chen, K., Mayerle, M., Ragunathan, K., Luthey-Schulten, Z., Ha, T., Woodson, S.A., Protein-guided RNA dynamics during early ribosome assembly. Nature. 506, 334-338 (2014).
7. Abeysirigunawardena, S. C., Chow, C. S., pH-dependent structural changes of helix 69 from Escherichia coli 23S ribosomal RNA, RNA. 14, 782-792 (2008).
8. Chow, C. S., Lamichhane, T. N., Mahto, S. K., Expanding the nucleotide repertoire of the ribosome with post-transcriptional modifications. ACS Chem Biol. 2, 610-609 (2007).