RNA-RNA and RNA-Protein Interactions in Translational Regulation

Across Bacteria, Archaea, and Eukarya, genetic information contained within mRNA is translated to protein by large ribonucleoprotein complexes called ribosomes. Eukaryotic Ribosomes are comprised of the large subunit (60S) and the small subunit (40S) which together total to over 70 proteins that are scaffolded by ribosomal RNA (rRNA). Translation is a tightly regulated process partitioned into initiation, elongation, and termination steps, each with their own set of protein factors, to achieve coordination for proper synthesis of proteins. For example, translation initiation requires initiation factors (eIFs) to facilitate 40S and 60S assembly into the 80S complex at the mRNA start codon. For eukaryotic elongation, coordination relies on aminoacyl-tRNAs and elongation factors (eEFs), and termination relies on release factors (eRFs) to facilitate the dissociation of the 40S and 60S and the release of the nascent peptide. These examples all involve protein factors but protein-based methods are not the only type of signals the cell employs for translational regulation. Our understanding of translational regulation through non-coding RNAs (ncRNAs) such as transfer RNA (tRNA) and small nucleolar RNA (snoRNA) has been expanding.

tRNAs bridge the gap between codon and cognate amino acid during translation as well-known adaptor molecules. tRNAs adopt a cloverleaf-like secondary structure and a L-shaped tertiary structure which allows the tRNA to dock into the ribosome and interface with the mRNA at the same time. Importantly, tRNAs are also heavily decorated RNA molecules with an average of 13 modifications per cytoplasmic tRNA and 5 per mitochondrial tRNA. There are over 100 tRNA modifications and certain modifications have been shown to affect tRNA stability, charging, and tRNA biogenesis. Importantly, modifications at the anticodon can both expand and restrict decoding during translation.

snoRNAs generally exist as ribonucleoprotein (snoRNPs) complexes and are classified primarily into two families, C/D box and H/ACA box snoRNAs, based on conserved sequence motifs. Both families have been shown to play an indirect role in translation as snoRNAs are primarily known for guiding chemical modifications of rRNAs that are important for proper ribosome biogenesis and function. C/D box snoRNAs guide 2’-O-methylation and H/ACA box snoRNAs guide psuedouridylation (Ψ).

In Chapter 2 we investigate cell-type and tRNA queuosine (Q) modification-dependencies on cell proliferation. Among the six cell lines tested, proliferation only decreased in MCF7 cells when tRNAs were Q-modified, but the proliferation can be restored by the transfection of Q-modification-dependent tRF^His. tRNA-seq showed that in MCF7 cells, tRNA Q-modification reduced m¹A58 and m³C32 modification levels, consistent with reduced translation. Additionally, polysome profiling showed a Q-modification associated codon usage pattern that corresponded with altered translation efficiency of ribosomal proteins required for faster proliferation. Protein pull-down using tRFHis identified Musashi RNA binding protein 2 (Msi2) as a tRFHis binding protein. Msi2 is known to enhance mitochondrial activities, and Msi2 knockdown reduced the tRF^His dependent cell proliferation effect. This study highlights a way tRNAs can affect cell proliferation by impacting translation via modifications through a proposed mechanism of Msi2 binding.

In Chapter 3, we expand on a previous study that describes a H/ACA snoRNA, SNORA73 as a “ternary-glue” (TAG) snoRNA which binds a target mRNA and the signal recognition particle (SRP) 7SL RNA to promote protein secretion, indicating a role of snoRNA that links mRNAs to the SRP pathway to enable the translation of endoplasmic reticulum (ER) translocated proteins. We identify 30 candidate TAG-snoRNAs, of which 15 were expressed across five human cell lines, and develop an RNAplex-based pipeline (TAGSNOplex) to predict TAG-snoRNA targeting mRNAs that contain signal peptide coding sequences and belong to secretory, cell surface, and intracellular membrane proteins. We validate our predicted targeting of the PTK7 protein by SNORA21 whose knockdown reduces the surface display of PTK7 without changing total PTK7 protein level. These results further explore the direct role snoRNAs have in translational regulation and provide a resource for finding potential mRNA targets of TAG-snoRNAs that regulate the localization of secretory, cell surface, and membrane proteins.

Finally in Chapter 4, we investigate the spatial dynamics of cytoplasmic tRNA modification in human cells to characterize what fraction- cytoplasmic, nucleoplasm, or chromatin – do modifications appear in. Together, this work provides insights on how tRNAs and snoRNAs can regulate translation in alternative ways through RNA-protein or RNA-RNA interactions.