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. 2013 Dec;19(12):1684-92.
doi: 10.1261/rna.036806.112. Epub 2013 Oct 22.

HAMR: High-Throughput Annotation of Modified Ribonucleotides

Free PMC article

HAMR: High-Throughput Annotation of Modified Ribonucleotides

Paul Ryvkin et al. RNA. .
Free PMC article

Abstract

RNA is often altered post-transcriptionally by the covalent modification of particular nucleotides; these modifications are known to modulate the structure and activity of their host RNAs. The recent discovery that an RNA methyl-6 adenosine demethylase (FTO) is a risk gene in obesity has brought to light the significance of RNA modifications to human biology. These noncanonical nucleotides, when converted to cDNA in the course of RNA sequencing, can produce sequence patterns that are distinguishable from simple base-calling errors. To determine whether these modifications can be detected in RNA sequencing data, we developed a method that can not only locate these modifications transcriptome-wide with single nucleotide resolution, but can also differentiate between different classes of modifications. Using small RNA-seq data we were able to detect 92% of all known human tRNA modification sites that are predicted to affect RT activity. We also found that different modifications produce distinct patterns of cDNA sequence, allowing us to differentiate between two classes of adenosine and two classes of guanine modifications with 98% and 79% accuracy, respectively. To show the robustness of this method to sample preparation and sequencing methods, as well as to organismal diversity, we applied it to a publicly available yeast data set and achieved similar levels of accuracy. We also experimentally validated two novel and one known 3-methylcytosine (3mC) sites predicted by HAMR in human tRNAs. Researchers can now use our method to identify and characterize RNA modifications using only RNA-seq data, both retrospectively and when asking questions specifically about modified RNA.

Keywords: RNA modification; RNA sequencing; tRNA.

Figures

FIGURE 1.
FIGURE 1.
Mismatch rates along small RNA reads (<44 nt) mapping to three types of RNAs.
FIGURE 2.
FIGURE 2.
(Left) Locations of known tRNA modifications predicted to affect RT incorporation and (right) modification sites predicted by HAMR mapped onto a tRNA (RFAM) consensus structure. Values indicate the percentage of tRNA families where the site is present.
FIGURE 3.
FIGURE 3.
Proportion of sites with known modifications that we predicted to harbor modifications in human tRNA using HAMR. (Black) Modifications are known to affect RT incorporation; (dark gray) modifications predicted to affect RT incorporation based on their structure at the Watson–Crick edge of the nucleoside; (light gray) modifications known to have no effect on RT, or there is no evidence showing that they do under the conditions in this experiment.
FIGURE 4.
FIGURE 4.
Frequencies of observed nonreference nucleotides at sites with known modifications of (A) adenosines and (B) guanosines in human tRNAs. Each point represents one observation in one sample. The axes are labeled by template-strand cDNA sequence and are the complement of the nucleotide that was incorporated by RT into the cDNA. (C,D) Results of this same analysis in S. cerevisiae.
FIGURE 5.
FIGURE 5.
HAMR identifies known and novel modification sites in human tRNAs. Random-primed RT-qPCR analysis of three human tRNAs (as specified) after RNA immunoprecipitation using either an antibody specific for 3mC or an IgG control (see Materials and Methods for additional details). qPCR loading was normalized to a nonmodified tRNA (Lys [UUU]). The tRNA models demonstrate the specific site of the HAMR identified 3mC modification sites (cytosine 3 for Met [CAU], as well as cytosine 32 for mtMet [CAU] and mtThr [UGU]). Gray and black labels denote tRNAs where the HAMR-identified 3mC site is novel or known, respectively. (mt) Mitochondrial tRNAs. Error bars, ±SD. (**) P-value < 0.01.

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