Background

Chemical modifications of RNA have emerged as a new mechanism for the post-transcriptional regulation of gene expression (Zhao et al., 2017). In contrast to a handful of well-studied modified bases that constitute the DNA epigenome, over a hundred modified ribonucleosides are present in all types of RNA. This diversity offers new possibilities to modulate gene expression, significantly expanding the metabolic and regulatory functions of RNA (Zhao et al., 2017). Mainly studied in abundant transfer RNA (tRNA) and ribosomal RNA (rRNA), ribonucleoside modifications have more recently been described in messenger RNAs (mRNAs), where they form the basis of the epitranscriptome (Zhao et al., 2017). These epitranscriptomic modifications have the potential to control all steps of mRNA metabolism including structure, stability, location and translation.

Several modifications, including N6-methyladenosine (m6A), N6,2’-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), pseudouridine (Ψ), inosine (I), 5-methylcytidine (m5C), 5-hydroxylmethylcytidine (hm⁵C), 7-methylguanosine (m7G) and 2’-O-methylnucleosides (Nm) are found in eukaryotic mRNA and can influence the metabolism and function of mRNA (Li et al., 2016). While the majority of mRNA modifications are methylation events, only a single acetylated ribonucleoside has been described in eukaryotes, occurring at the N4-position of cytidine (N4-acetylcytidine, ac4C, Figure 1A). ac4C is one of the universally conserved RNA modifications present in all domains of life and had previously been observed in rRNA and tRNA (Ito et al., 2014a and 2014b; Sharma et al., 2015). More recently, ac4C was observed in RNA viruses and in polyadenylated RNA (poly(A)-RNA) of yeast and human cancer cells (Dong et al., 2016; McIntyre et al., 2018; Tardu et al., 2018), raising the possibility that ac4C is present in mRNA. In all cases, ac4C production was attributed to the enzyme NAT10 (Figure 1A), or its homologs (Ito et al., 2014a and 2014b; Sharma et al., 2015), suggesting a non-redundant activity.

To investigate the occurrence of ac4C in human mRNA, we generated tools for antibody-based enrichment and transcriptome-wide mapping, a method we named acRIP-seq (Arango et al., 2018). Additionally, to assess ac4C regulation and function, we deleted NAT10 in HeLa cells. The sum of these studies confirmed NAT10-dependent acetylation of mRNAs (Arango et al., 2018). Overall, acetylation was enriched within coding sequences and loss of ac4C through NAT10 ablation led to a specific decrease in the expression of defined ac4C targets. More specifically, we found that ac4C increases mRNA stability and enhances translation efficiency of mRNAs when the modification is present in coding sequences (Arango et al., 2018).

Here, we report a detailed protocol for acetylated RNA immunoprecipitation and sequencing, acRIP-seq (Figures 1B-1C). This method is used to identify acetylated mRNA regions transcriptome-wide.


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^® Ultra^TM 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.