tRNA Metabolism and Neurodevelopmental Disorders.

tRNAs are short noncoding RNAs required for protein translation. The human genome includes more than 600 putative tRNA genes, many of which are considered redundant. tRNA transcripts are subject to tightly controlled, multistep maturation processes that lead to the removal of flanking sequences and the addition of nontemplated nucleotides. Furthermore, tRNAs are highly structured and posttranscriptionally modified. Together, these unique features have impeded the adoption of modern genomics and transcriptomics technologies for tRNA studies. Nevertheless, it has become apparent from human neurogenetic research that many tRNA biogenesis proteins cause brain abnormalities and other neurological disorders when mutated. The cerebral cortex, cerebellum, and peripheral nervous system show defects, impairment, and degeneration upon tRNA misregulation, suggesting that they are particularly sensitive to changes in tRNA expression or function. An integrated approach to identify tRNA species and contextually characterize tRNA function will be imperative to drive future tool development and novel therapeutic design for tRNA-associated disorders.

Acetylation of Cytidine in mRNA Promotes Translation Efficiency.

Generation of the "epitranscriptome" through post-transcriptional ribonucleoside modification embeds a layer of regulatory complexity into RNA structure and function. Here, we describe N4-acetylcytidine (ac4C) as an mRNA modification that is catalyzed by the acetyltransferase NAT10. Transcriptome-wide mapping of ac4C revealed discretely acetylated regions that were enriched within coding sequences. Ablation of NAT10 reduced ac4C detection at the mapped mRNA sites and was globally associated with target mRNA downregulation. Analysis of mRNA half-lives revealed a NAT10-dependent increase in stability in the cohort of acetylated mRNAs. mRNA acetylation was further demonstrated to enhance substrate translation in vitro and in vivo. Codon content analysis within ac4C peaks uncovered a biased representation of cytidine within wobble sites that was empirically determined to influence mRNA decoding efficiency. These findings expand the repertoire of mRNA modifications to include an acetylated residue and establish a role for ac4C in the regulation of mRNA translation.

Tethered function assays using 3' untranslated regions.

Proteins that regulate mRNA metabolism are often bipartite: an RNA binding activity confers substrate specificity, and a "functional" domain elicits the biological outcome. In some cases, these two activities reside on different polypeptides that form a complex on the mRNA. Regardless, experimental separation of RNA binding from function facilitates analysis of both properties, liberating each from the constraints of the other. "Tethered function" assays bring a protein to a reporter RNA through a designed RNA-protein interaction. The function of the tethered protein-whether that be stability, translation, localization, or transport, or otherwise-is then assessed. We refer to this approach as a "tethered function" assay, since it can be examined. The approach does not require knowledge of the natural RNA binding sites, or of the composition of the naturally occurring protein complexes. It can be useful in dissecting how proteins that act on RNAs work, and in identifying active components of multiprotein complexes. RNA-binding proteins previously have been linked to foreign RNA-binding specificities, for a wide variety of purposes. We emphasize here the particular value of tethering to the 3' untranslated region of eukaryotic mRNAs, and the opportunities it presents for the analysis of how those mRNAs are regulated. We discuss experimental design, as well as published and potential applications.