Translational modulation as a consequence of codon choice and miRNA regulation in caenorhabditis elegans [electronic resource]
Keywords: Genetics

The conversion of genetic information from the language of nucleic acids into that of proteins is fundamental to nearly every biological process. The regulation of this process of gene expression—when, where, and how much product is produced from each gene—is a crucial determinant of cell state and identity. Gene expression is tightly regulated at every metabolic step, including transcription of DNA into mRNA, splicing of introns, export from the nucleus, polypeptide synthesis, and post-translational modification and stability. Regulation of protein synthesis acts directly or indirectly through the ribosome, as it is the interaction of ribosomes with mRNAs and dozens of protein and RNA translation factors that determine the efficiency and fidelity of translation. Emerging technologies allow the analysis of translation in contexts that were previously inaccessible. In my thesis work, I employed a technique known as ribosome profiling to examine snapshots of the positions of millions of ribosomes on cellular mRNAs in vivo. In the first part of my thesis, I used ribosome profiling data from Caenorhabditis elegans and cultured human tumor cells to investigate the sequence determinants of the rate of ribosome elongation. The genetic code is redundant, meaning that a single amino acid can usually encoded by more than one trinucleotide sequence, or codon. The choice from among these "synonymous" codons has long been suspected to influence local ribosomal elongation rates, with possible consequences on the folding of nascent polypeptides. By examining "snapshots" of the in vivo positions of ribosomes on thousands of mRNAs, we were able to determine that mRNA positions featuring "wobble" codons in the ribosomal decoding center were associated with greater ribosome occupancy, and thus most likely with slower translation elongation. These wobble codons pair with cognate tRNA anticodons using alternative base-pairing strategies that are less energetically stable than canonical Watson-Crick interactions. The observed wobble retention was strong for codons that employ G:U pairing at the wobble position, and weaker for those using I:U pairs. Importantly, this property of translation elongation was observed in both C. elegans samples and human cells, indicating that wobble-related slowing is a conserved feature of metazoan translation. In the second part of my thesis, I used ribosome profiling, together with traditional, low-throughput methods to investigate the molecular consequences of regulation by C. elegans miRNAs. miRNAs are post-transcriptional regulators of gene expression that have been proposed to act by directing mRNA degradation, deadenylation, translation initiation, by interfering with a post-initiation stage of translation or by triggering proteolysis. C. elegans features several well-validated, natural miRNA::mRNA interactions that serve to reduce target protein levels at specific developmental time points as part of the so-called heterochronic pathway. mRNA-seq measurements confirmed earlier observations from other groups that the abundance of target mRNAs decreases only slightly upon the onset of miRNA regulation. Analysis of ribosome footprint abundance showed that miRNA regulation is often associated with decreased ribosome loading in later larval stages, but changes in ribosome loading are negligible at time points where miRNA inhibition becomes active. Additionally, profiles of ribosome footprints did not appreciably change, rendering several models of post-initiation translational regulation unlikely. These data suggest a model in which processes beyond mRNA degradation and translational repression contribute to regulation by this set of miRNAs, including a possible role for post- or peri-translational destruction of peptide products.