Identification and quantification of modified nucleosides inSaccharomyces cerevisiaemRNAs

Abstract

Shortened title: mRNA modification quantification and discovery by UHPLC-MS/MSABSTRACT Post-transcriptional nucleoside modifications have long been recognized as key modulators of non-coding RNA structure and function. There is an emerging appreciation that the chemical modification of protein-coding messenger RNAs (mRNAs) also plays critical roles in the cell. Although there are over 100 known RNA modifications found in biology only a handful have been identified in mRNAs. We sought to identify and quantify modifications present in the mRNAs of yeast cells using a high throughput ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method that measures the levels of 36 types of RNA nucleosides in parallel. We detected the presence of six modified nucleosides in mRNAs at relatively high abundances: N7methylguanosine, N6-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytosine, N4acetylcytidine and 5-formylcytidine. Additionally, we investigated how the levels of mRNA modifications vary in response to cellular stress. We find that the concentrations of mRNA modifications including N6-methyladenosine and N4-acetylcytidine change in response to heat stress, glucose starvation and/or oxidative stress. This work expands the repertoire of potential chemical modifications in mRNAs, and utilizes a high-throughput approach to search for modifications that highlights the value of integrating massspectrometry tools in the mRNA modification discovery and characterization pipeline.

Translational offsetting as a mode of estrogen receptor α‐dependent regulation of gene expression

“…A volume of 4 μl (96 to 487 ng/μl) total RNA was used to evaluate levels of cm 5 U, mcm 5 U, and mcm 5 s 2 U, by LC‐MS/MS using a similar method as described (Basanta‐Sanchez et al , ; Tardu et al , ). Briefly, prior to UHPLC‐MS analysis, each sample was mixed with 0.4 pg/μl of internal standard (IS), isotopically labeled guanosine, [ 13 C][ 15 N]‐G.…”

A Chemical Signature for Cytidine Acetylation in RNA

“…The ability to query ac4C sensitively and site-specifically will be an essential component in genetic screens aimed at identifying snoRNA adapters for cytidine acetyltransferases, and potentially also help define rules for enzymatic ac4C-targeting . Finally, this method may be useful for identifying putative sites of ac4C in coding RNAs, which have recently been proposed to exist in yeast . One limitation of our method as currently constituted is an inability to analyze ac4C in densely modified targets such as tRNAs, whose sequencing is challenged by the presence of nucleobases that disrupt Watson–Crick base-pairing and limit RT procession .…”

N4-acetylation of Cytidine in mRNA by NAT10 Regulates Stability and Translation

“…Here enters the study by Arango et al (2018) with a new modified base in human mRNA, N 4 -acetylcytidine (ac 4 C), and joins a recent preprint report that used mass spectrometry to detect ac 4 C in mRNA of the yeast Saccharomyces cerevisiae (Tardu et al, 2018). The new field of epitranscriptomics is experiencing growing pains, a natural result of the demanding experimental and bioinformatic need to identify a relatively weak signal in rare molecules that is more often than not invisible to reverse transcription.…”