Epitranscriptome Analysis
The epitranscriptome encompasses RNA modifications that influence RNA structure and function and regulate gene expression. More than a hundred and fifty distinct RNA modifications have been discovered in nature. Post-transcriptional regulatory markers occur in messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), circular RNAs (circRNAs), micro RNAs (miRNA), and long non-coding RNAs (lncRNAs). Liquid Chromatography Mass Spectrometry (LC-MS) is utilized to quantify the abundance of m5C, hm5C, f5C, ca5C, m4C, m6A, pseudouridine (Ψ), and many other modified RNA nucleotides. Next generation sequencing (NGS) can identify some of the modified RNA nucleotides in context of epitranscriptome-wide analyses. Epitranscriptomics is expanding with new profiling methods and discoveries of promising biomarkers and therapeutic targets.
Profiling RNA modifications with sequencing or mass spectrometry
NEB offers enzymes, reagents, and kits that are ideal for NGS or LC-MS epitranscriptome analysis:
- Our suite of Ribonucleases (RNases) have utilities for mapping RNA modifications with next generation sequencing or LC-MS.
- RNase 4 (NEB #M1284) tolerates common RNA modifications and cleaves at uridine-purine (U/R) dinucleotide sites to generate a larger population of uniquely mappable oligonucleotides to improve RNA species characterization by LC-MS/MS.
- Nucleoside Digestion Mix (NEB #M0649) digests RNA with common epitranscriptome modifications to single nucleosides in one step for quantitative analysis by LC-MS/MS.
- XRN-1 (NEB # M0338) can be used to distinguish between capped and uncapped mRNA.
- Sce Pseudouridine Synthase I (Sce PUS I) (NEB # M0526) isomerizes uridine to pseudouridine in predominantly single-stranded RNA in vitro.
- EpiMark® N6-Methyladenosine Enrichment Kit (NEB #E1610) enriches m6A modified RNA in immunoprecipitation protocols for downstream next-generation sequencing or RT-qPCR.
- Additionally, our NEBNext® next generation sequencing portfolio includes useful solutions for mapping the epitranscriptome.
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- What types of epigenetic modifications can be identified using the Nucleoside Digestion Mix?
- Does the Nucleoside Digestion Mix digest RNA?
- How much RNase 4 (NEB #M1284) should I use to digest RNA for LC-MS/MS nucleotide sequence mapping?
- How much of the control RNAs should I use if I want to spike them into my sample?
- Will any NEB methyltransferases (methylases) work on RNA?
- Grünberg, S., Doyle, L. A., Wolf, E. J., Dai, N., Ivan R. Corrêa, J., Yigit, E., Stoddard, B. L. (2023) The structural basis of mRNA recognition and binding by eukaryotic pseudouridine synthase PUS1 bioRxiv; 2021.2012.2008.471817, PubMedID: 37939088, DOI: https://doi.org/10.1101/2021.12.08.471817
- Wolf, E.J., Dai, N., Chan, S.H., Corrêa, I.R. Jr. (2023) Selective Characterization of mRNA 5′ End-Capping by DNA Probe-Directed Enrichment with Site-Specific Endoribonucleases. ACS Pharmacol. Transl. Sci; 6(11), 1692-1702. DOI: 10.1021/acsptsci.3c00157
- Yan B. et al. (2021) ReCappable Seq: Comprehensive Determination of Transcription Start Sites derived from all RNA polymerases. Genome Research; DOI: 10.1101/gr.275784.121
- Wolf, E.J., Grünberg, S., Dai, N., Chen, T.H., Roy, B., Yigit, E., Corrêa, I.R. (2022) Human RNase 4 improves mRNA sequence characterization by LC-MSMS. Nucl. Acids Res; 50(18), e106. PubMedID: 35871301, DOI: 10.1093/nar/gkac632
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