Sequence- and structure-specific cytosine-5 mRNA methylation by NSUN6.

The highly abundant N6-methyladenosine (m6A) RNA modification affects most aspects of mRNA function, yet the precise function of the rarer 5-methylcytidine (m5C) remains largely unknown. Here, we map m5C in the human transcriptome using methylation-dependent individual-nucleotide resolution cross-linking and immunoprecipitation (miCLIP) combined with RNA bisulfite sequencing. We identify NSUN6 as a methyltransferase with strong substrate specificity towards mRNA. NSUN6 primarily targeted three prime untranslated regions (3'UTR) at the consensus sequence motif CTCCA, located in loops of hairpin structures. Knockout and rescue experiments revealed enhanced mRNA and translation levels when NSUN6-targeted mRNAs were methylated. Ribosome profiling further demonstrated that NSUN6-specific methylation correlated with translation termination. While NSUN6 was dispensable for mouse embryonic development, it was down-regulated in human tumours and high expression of NSUN6 indicated better patient outcome of certain cancer types. In summary, our study identifies NSUN6 as a methyltransferase targeting mRNA, potentially as part of a quality control mechanism involved in translation termination fidelity.

Codon usage optimization in pluripotent embryonic stem cells.

BACKGROUND: The uneven use of synonymous codons in the transcriptome regulates the efficiency and fidelity of protein translation rates. Yet, the importance of this codon bias in regulating cell state-specific expression programmes is currently debated. Here, we ask whether different codon usage controls gene expression programmes in self-renewing and differentiating embryonic stem cells. RESULTS: Using ribosome and transcriptome profiling, we identify distinct codon signatures during human embryonic stem cell differentiation. We find that cell state-specific codon bias is determined by the guanine-cytosine (GC) content of differentially expressed genes. By measuring the codon frequencies at the ribosome active sites interacting with transfer RNAs (tRNA), we further discover that self-renewing cells optimize translation of codons that depend on the inosine tRNA modification in the anticodon wobble position. Accordingly, inosine levels are highest in human pluripotent embryonic stem cells. This effect is conserved in mice and is independent of the differentiation stimulus. CONCLUSIONS: We show that GC content influences cell state-specific mRNA levels, and we reveal how translational mechanisms based on tRNA modifications change codon usage in embryonic stem cells.