Poster abstracts

Poster number 114 submitted by Zezhong Wan

mRNA modifications impact stop codon recognition

Zezhong Wan (Department of Chemistry, University of Michigan), Tyler J. Smith (Department of Chemistry, University of Michigan), Kristin S. Koutmou (Department of Chemistry, University of Michigan)

As templates for protein synthesis by the ribosome, mRNAs use the four standard nucleosides to record the sequence of amino acids. In addition to A, C, G and U, they can also possess modified nucleobases that impact protein expression. Present in all phylogenetic domains, there is significant chemical diversity among modified RNA nucleosides 1, including the addition of atoms (e.g. H), functional groups (e.g. methyl-, acetyl-, or carboxyl-)2 or nucleoside isomers (e.g. pseudouridine)3. At the molecular level, mRNA modifications have the potential to affect the lifespan and activity of transcripts by altering post-transcriptional splicing, translation, and decay4. Among the entire process, translation is central to protein synthesis. The initiation and elongation steps of translation are dependent on mRNA/tRNA interactions, while termination requires recognition of stop codon by release factors (RF)5. However, how and when these steps in the protein synthesis pathway can be affected by chemical modifications to single nucleobases still remain unclear. Here we explored the impact of modified bases on translation termination. We utilized a fully-reconstituted E.coli in vitro translation system to examine how the inclusion of a series of guanosine modifications in stop codons (UGA and UAG) influences the kinetics of peptide release. Our results suggest that the rate of peptide release is highly dependent on H-bond donor/acceptor positions around the purine ring, and indicate that the H-bond between N1 site on the purine ring and residues in RF domains is potentially crucial for RF recognition of stop codons.

1.Roundtree, Ian A., et al. "Dynamic RNA modifications in gene expression regulation." Cell 169.7 (2017): 1187-1200.
2.Cantara, William A., et al. "The RNA modification database, RNAMDB: 2011 update." Nucleic acids research 39.suppl_1 (2010): D195-D201.
3.Limbach, Patrick A., Pamela F. Crain, and James A. McCloskey. "Summary: the modified nucleosides of RNA." Nucleic acids research 22.12 (1994): 2183-2196.
4.Li, Sheng, and Christopher E. Mason. "The pivotal regulatory landscape of RNA modifications." Annu Rev Genomics Hum Genet 15.1 (2014): 127-150.
5.Korkmaz, Gürkan, et al. "Comprehensive analysis of stop codon usage in bacteria and its correlation with release factor abundance." Journal of Biological Chemistry 289.44 (2014): 30334-30342.

Keywords: mRNA modification, translation, stop codon