Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jun 20;9(12):e3278.
doi: 10.21769/BioProtoc.3278.

Immunoprecipitation and Sequencing of Acetylated RNA

Daniel Arango  1 , David Sturgill  1 , Shalini Oberdoerffer  1
Affiliations

Immunoprecipitation and Sequencing of Acetylated RNA

Daniel Arango et al. Bio Protoc. .

Abstract

Generation of the epitranscriptome through chemical modifications of protein-coding messenger RNAs (mRNAs) has emerged as a new mechanism of post-transcriptional gene regulation. While most mRNA modifications are methylation events, a single acetylated ribonucleoside has been described in eukaryotes, occurring at the N4-position of cytidine (N4-acetylcytidine or ac4C). Using a combination of antibody-based enrichment of acetylated regions and deep sequencing, we recently reported ac4C as a novel mRNA modification that is catalyzed by the N-acetyltransferase enzyme NAT10. In this protocol, we describe in detail the procedures to identify acetylated mRNA regions transcriptome-wide using acetylated RNA immunoprecipitation and sequencing (acRIP-seq).

Keywords: Epitranscriptome; N4-acetylcytidine; ac4C; acRIP-seq; mRNA modifications.

PubMed Disclaimer

Conflict of interest statement

Competing interestsThe authors declare no competing interests.

Figures

Figure 1.
Figure 1.. acRIP-seq identifies acetylated regions within mRNA transcriptome-wide.
A. NAT10 catalyzes cytidine acetylation. B-C. Schematics of acRIP-seq. Fragmented poly(A) RNA is immunoprecipitated (IP) with anti-ac4C antibodies or Isotypic IgG control (B). RNA from acRIP, IgG-IP and Inputs are used to construct Illumina sequences using the NEBNext® UltraTM II Directional RNA Library protocol followed by high-throughput sequencing and bioinformatical analysis to identify putative acetylated regions transcriptome-wide (C). USER: Uracil-Specific Excision Reagent.
Figure 2.
Figure 2.. Bioanalyzer profiles.
A. Representative profiles of unfragmented (top) and fragmented (bottom) poly(A) RNA. B. Representative profiles of RNA isolated from immunoprecipitation with IgG control (top) or anti-ac4C antibodies (bottom). C. Representative profiles of DNA libraries from Input (top), IgG (middle) or acRIP (bottom).
See this image and copyright information in PMC

References

    1. AbouHaidar M. G. and Ivanov I. G.(1999). Non-enzymatic RNA hydrolysis promoted by the combined catalytic activity of buffers and magnesium ions. Z Naturforsch C 54(7-8): 542-548. - PubMed
    1. Arango D., Sturgill D., Alhusaini N., Dillman A. A., Sweet T. J., Hanson G., Hosogane M., Sinclair W. R., Nanan K. K., Mandler M. D., Fox S. D., Zengeya T. T., Andresson T., Meier J. L., Coller J. and Oberdoerffer S.(2018). Acetylation of cytidine in mRNA promotes translation efficiency. Cell 175(7): 1872-1886 e1824. - PMC - PubMed
    1. Bolger A. M., Lohse M. and Usadel B.(2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics(Oxford, England) 30: 2114-2120. - PMC - PubMed
    1. Dong C., Niu L., Song W., Xiong X., Zhang X., Zhang Z., Yang Y., Yi F., Zhan J., Zhang H., Yang Z., Zhang L. H., Zhai S., Li H., Ye M. and Du Q.(2016). tRNA modification profiles of the fast-proliferating cancer cells. Biochem Biophys Res Commun 476(4): 340-345. - PubMed
    1. Ito S., Akamatsu Y., Noma A., Kimura S., Miyauchi K., Ikeuchi Y., Suzuki T. and Suzuki T.(2014). A single acetylation of 18 S rRNA is essential for biogenesis of the small ribosomal subunit in Saccharomyces cerevisiae. J Biol Chem 289(38): 26201-26212. - PMC - PubMed

LinkOut - more resources