A tRNA modification with aminovaleramide facilitates AUA decoding in protein synthesis.

Modified tRNA anticodons are critical for proper mRNA translation during protein synthesis. It is generally thought that almost all bacterial tRNAsIle use a modified cytidine-lysidine (L)-at the first position (34) of the anticodon to decipher the AUA codon as isoleucine (Ile). Here we report that tRNAsIle from plant organelles and a subset of bacteria contain a new cytidine derivative, designated 2-aminovaleramididine (ava2C). Like L34, ava2C34 governs both Ile-charging ability and AUA decoding. Cryo-electron microscopy structural analyses revealed molecular details of codon recognition by ava2C34 with a specific interaction between its terminal amide group and an mRNA residue 3'-adjacent to the AUA codon. These findings reveal the evolutionary variation of an essential tRNA modification and demonstrate the molecular basis of AUA decoding mediated by a unique tRNA modification.

Base-resolution analysis of 5-hydroxymethylcytidine by selective oxidation and reverse transcription arrest.

While 5-hydroxymethylcytidine in RNA (hm5C) is associated with cellular development and differentiation, its distribution and biological function remain largely unexplored because suitable detection methods are lacking. Here, we report a base-resolution sequencing method for hm5C in RNA by applying peroxotungstate-mediated chemical conversion of hm5C to trihydroxylated thymine (thT). Reverse transcription by SuperScript III terminated at the thT site, probably because of its unnatural nucleobase structure producing truncated cDNA. Consequently, base-resolution analysis of the hm5C sites in RNA was achieved with both Sanger sequencing and Illumina sequencing analysis by comparing sequencing data before and after peroxotungstate treatment.

Chemistry-Driven Epigenetic Investigation of Histone and DNA Modifications.

In the regulation processes of gene expression, genomic DNA and nuclear proteins, including histone proteins, cooperate with each other, leading to the distinctive functions of eukaryotic cells such as pluripotency and differentiation. Chemical modification of histone proteins and DNA has been revealed as one of the major driving forces in the complicated epigenetic regulation system. However, understanding of the precise molecular mechanisms is still limited. To address this issue, researchers have proposed both biological and chemical strategies for the preparation and detection of modified proteins and nucleic acids. In this review, we focus on chemical methods around the field of epigenetics. Chemical protein synthesis has enabled the preparation of site-specifically modified histones and their successful application to various in vitro assays, which have emphasized the significance of posttranslational modifications of interest. We also review the modification-specific chemical reactions against synthetic and genomic DNA, which enabled discrimination of several modified bases at single-base resolution.

Base-Resolution Analysis of 5-Hydroxymethylcytosine by One-Pot Bisulfite-Free Chemical Conversion with Peroxotungstate.

5-Hydroxymethylcytosine (hmC) is an essential intermediate in the active DNA demethylation pathway. Here we report a new base-resolution method for measuring hmC by combining peroxotungstate-mediated oxidation and sequencing analysis. We reveal that an oxidized product of hmC, trihydroxylated thymine (thT), tolerated the incorporation of dATP as a substrate in the process of DNA polymerase elongation. By comparing the results of Sanger sequencing before and after the oxidation, we observed that hmC sites on single-stranded DNAs could be discriminated from unmethylated cytosines. We found that a thermal cycle condition during peroxotungstate treatment enhanced the oxidation reaction of hmC in double-stranded DNA. Furthermore, Illumina sequencing analysis of hmC-containing synthetic genome fragments enabled us to identify simultaneously the positions of hmC in base resolution. This bisulfite-free simple hmC detection technique could facilitate the acquisition of epigenomic information.