The modified base 5-methylcytosine (m ⁵ C) is well studied in DNA, but investigations of its prevalence in cellular RNA have been largely confined to tRNA and rRNA. In animals, the two m ⁵ C methyltransferases NSUN2 and TRDMT1 are known to modify specific tRNAs and have roles in the control of cell growth and differentiation. To map modified cytosine sites across a human transcriptome, we coupled bisulfite conversion of cellular RNA with next-generation sequencing. We confirmed 21 of the 28 previously known m ⁵ C sites in human tRNAs and identified 234 novel tRNA candidate sites, mostly in anticipated structural positions. Surprisingly, we discovered 10 275 sites in mRNAs and other non-coding RNAs. We observed that distribution of modified cytosines between RNA types was not random; within mRNAs they were enriched in the untranslated regions and near Argonaute binding regions. We also identified five new sites modified by NSUN2, broadening its known substrate range to another tRNA, the RPPH1 subunit of RNase P and two mRNAs. Our data demonstrates the widespread presence of modified cytosines throughout coding and non-coding sequences in a transcriptome, suggesting a broader role of this modification in the post-transcriptional control of cellular RNA function.

MicroRNAs (miRNAs) repress translation of target mRNAs by interaction with partially mismatched sequences in their 3′ UTR. The mechanism by which they act on translation has remained largely obscure. We examined the translation of mRNAs containing four partially mismatched miRNA-binding sites in the 3′ UTR in HeLa cells cotransfected with a cognate miRNA. The mRNAs were prepared by in vitro transcription and were engineered to employ different modes of translation initiation. We find that the 5′ cap structure and the 3′ poly(A) tail are each necessary but not sufficient for full miRNA-mediated repression of mRNA translation. Replacing the cap structure with an internal ribosome entry site from either the cricket paralysis virus or the encephalomyocarditis virus impairs miRNA-mediated repression. Collectively, these results demonstrate that miRNAs interfere with the initiation step of translation and implicate the cap-binding protein eukaryotic initiation factor 4E as a molecular target.

Circular (circ)RNAs are to transcriptomics what the famous hidden musical theme is to Elgar's Enigma Variations : woven into the entire composition but not unambiguously identified or understood by scholars to this day. Now, two studies (Hansen et al , 2013; Memczak et al , 2013) have made substantial progress in both, identifying thousands of circRNAs and beginning to crack the enigma of their cellular function, by demonstrating that the circRNAs CiRS‐7/CDR1as and SRY function as natural and highly stable sponges for specific microRNAs. Much work over decades has documented the complex ways in which genetic information is transcribed and processed into functional RNAs to ultimately shape phenotypes. Well‐appreciated phenomena include alternative promoter choice, splicing, editing and 3′ end formation to generate surprisingly diverse mRNA populations, as well as the widespread, regulated and complex production of non‐coding RNAs from genic as well as intergenic regions of the genome, reviewed in Mercer et al (2012). Occasional evidence for the formation of circular RNAs (circRNAs) had also been presented over the years and examples such as the circular testis‐determining RNA SRY have achieved some prominence (Capel et al , 1993). Nevertheless, circRNAs have mostly been disregarded as rare, some form of transcriptional noise or RT–PCR artefacts. Thus, demonstrations of their widespread and substantial presence within transcriptomes have only recently come to the fore (Salzman et …

In the past century, few areas of biology advanced as much as our understanding of the pathways of intermediary metabolism. Initially considered unimportant in terms of gene regulation, crucial cellular fate changes, cell differentiation, or malignant transformation are now known to involve 'metabolic remodeling' with profound changes in the expression of many metabolic enzyme genes. This review focuses on the recent identification of RNA-binding activity of numerous metabolic enzymes. We discuss possible roles of this unexpected second activity in feedback gene regulation ('moonlighting') and/or in the control of enzymatic function. We also consider how metabolism-driven post-translational modifications could regulate enzyme-RNA interactions. Thus, RNA emerges as a new partner of metabolic enzymes with far-reaching possible consequences to be unraveled in the future.