Poster abstracts
Poster number 153 submitted by Rebecca Wegman
Dissecting the role of protein arginine methylation on translation initiation factor eIF1A
Rebecca L. Wegman (Department of Biological Sciences, University at Buffalo ), Skyler Kelly (Department of Biological Sciences, University at Buffalo ), Michael Langberg (Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center), Min-kui Luo (Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center), Sarah E. Walker (Department of Biological Sciences, University at Buffalo ), Michael C. Yu (Department of Biological Sciences, University at Buffalo )
Abstract:
Eukaryotic translation initiation is a vital process for proper expression of proteins within a cell. At the start of initiation, a pre-initiation complex, (PIC) binds to the capped 5’-end of the mRNA, and moves along the untranslated region using the anti-codon of the initiator tRNA to identify a proper AUG start codon. Previous work has shown that both N- and C- terminal tails of one eukaryotic initiation factor, eIF1A, provide critical interactions that stabilize the PIC in either an open conformation (conducive to scanning) or a closed conformation (inhibitory of scanning and supportive of initiation) (1,2). The balance between the open and closed conformations of the PIC is critical to proper start codon recognition and thus it is important to gain a better understanding of how this conformational change is regulated.
It is known that translation factors can be regulated through post-translation modifications, such as phosphorylation and methylation. Recently, we carried out bioorthogonal profiling to comprehensively identify substrates of yeast protein arginine methyltransferase Hmt1 in vivo, and this approach yielded eIF1A as a candidate. To validate our proteomic identification, we carried out an in vitro methylation assay to show that eIF1A can act as an Hmt1 substrate. Structural analysis of eIF1A suggests two potential sites of arginine methylation – amino acids 13 and 14 in the N-terminal tail. These two arginines reside within or adjacent to the signature RG/RGG box that is commonly found in Hmt1 substrates. To better understand how methylation of eIF1A can potentially affect its function, we generated a combination of substitution mutants of eIF1A (R to K or R to A) at these positions and determined their effects on fidelity of translation start codon usage relative to wild-type. Our data demonstrate that methylation of eIF1A decreases fidelity of start codon recognition as these mutations severely decrease usage of near-cognate start codons. Overall, our data suggest that eIF1A methylation stabilizes interactions of the N-terminal tail in a closed conformation of the PIC that is not conducive to scanning allowing arrest at near-cognate codons. This allows Hmt1 to fine tune the fidelity of start codon recognition for proper translation initiation.
References:
1) Fekete et al. (2007). N- and C-terminal residues of eIF1A have opposing effect on the fidelity of start codon selection. The EMBO Journal, 26(6), 106-1614.
2) Saini et al. (2010). Regulatory elements in eIF1A control the fidelity of start codon selection by modulating tRNAiMet binding to the ribosome. Genes & Development, 24, 97-110.
Keywords: eIF1A, Methylation , Translation