We developed a general method to detect cellular small molecule-RNA conjugates that does not rely on the reactivity of the small molecule, revealing NAD-linked RNA in E. coli and S. venezuelae. Subsequent characterization shows NAD is a 5’ modification of RNA, cannot be installed in vitro through aberrant transcriptional initiation, is only found among smaller cellular RNAs, and is present at a surprisingly high abundance of ~3000 copies per cell.

Compared with the rapidly expanding set of known biological roles for RNA, the known chemical diversity of cellular RNA has remained limited primarily to canonical RNA, 3 -aminoacylated tRNAs, nucleobase-modified RNAs, and 5 -capped mRNAs in eukaryotes. We developed two methods to detect in a broad manner chemically labile cellular small molecule-RNA conjugates. The methods were validated by the detection of known tRNA and rRNA modifications. The first method analyzes small molecules cleaved from RNA by base or nucleophile treatment. Application to Escherichia coli and Streptomyces venezuelae RNA revealed an RNAlinked hydroxyfuranone or succinyl ester group, in addition to a number of other putative small molecule-RNA conjugates not previously reported. The second method analyzes nuclease-generated mononucleotides before and after treatment with base or nucleophile and also revealed a number of new putative small molecule-RNA conjugates, including 3 -dephospho-CoA and its succinyl-, acetyl-, and methylmalonyl-thioester derivatives. Subsequent experiments established that these CoA species are attached to E. coli and S. venezuelae RNA at the 5 terminus. CoA-linked RNA cannot be generated through aberrant transcriptional initiation by E. coli RNA polymerase in vitro, and CoA-linked RNA in E. coli is only found among smaller (Շ200 nucleotide) RNAs that have yet to be identified. These results provide examples of small molecule-RNA conjugates and suggest that the chemical diversity of cellular RNA may be greater than previously understood. mass spectrometry ͉ RNA modifications ͉ coenzyme A O ver the past few decades, RNA has emerged as much more than an intermediary in biology's central dogma. Ribozymes (1), riboswitches (2), microRNAs (miRNAs) (3), small interfering RNAs (siRNAs) (4), Piwi-interacting RNAs (piRNAs) (5), small nuclear RNAs (snRNAs) (6), CRISPR sRNAs (7), RNA transcriptional regulators (8), and long noncoding RNAs (9, 10) are all examples of RNAs that are thought to play a wide range of catalytic, regulatory, or defensive roles in the cell. Models of early biotic systems have proposed even broader roles for RNA, including the possibility that RNA-tethered molecules participated in RNA-templated chemical reactions as an early form of metabolism (11)(12)(13)(14)(15)(16).In contrast with these newer insights into its functional diversity, the known chemical diversity of natural RNA has remained limited primarily to canonical polyribonucleotides, 3Ј-aminoacylated tRNAs (17), modified nucleobases in a variety of RNAs (18), and 5Ј-capped mRNAs in eukaryotes (19)(20). This disparity between functional and chemical diversity, coupled with the powerful functional properties of synthetic small molecule-nucleic acid conjugates (21-24) led us to speculate that small molecule-RNA conjugates beyond those previously described may exist in modern cells as evolutionary fossils or even as novel RNAs with functions enabled by their modifications.To begin to explore this possibility, we have developed and implemented a ...

Coronary heart disease (CHD) accounts for one in every six deaths in US individuals. Great advances have been made in identifying important risk factors for CHD, such as hypertension, diabetes mellitus, smoking and hypercholesterolaemia, which have led to major developments in therapy. In particular, statins represent one of the greatest successes in the prevention of CHD. While these standard risk factors are important, an obvious opportunity exists to take advantage of ongoing scientific research to better risk-stratify individuals and to identify new treatment targets. In this Review, we summarize ongoing scientific research in a number of metabolic molecules or features, including lipoproteins, homocysteine, calcium metabolism and glycaemic markers. We evaluate the current state of the research and the strength of evidence supporting each emerging biomarker. We also discuss whether the associations with CHD are strong and consistent enough to improve current risk stratification metrics, and whether these markers enhance our understanding of the underlying biology of CHD and thus point towards new treatment options.