Profiling of 2′-O-Me in human rRNA reveals a subset of fractionally modified positions and provides evidence for ribosome heterogeneity

Abstract

Ribose methylation is one of the two most abundant modifications in human ribosomal RNA and is believed to be important for ribosome biogenesis, mRNA selectivity and translational fidelity. We have applied RiboMeth-seq to rRNA from HeLa cells for ribosome-wide, quantitative mapping of 2′-O-Me sites and obtained a comprehensive set of 106 sites, including two novel sites, and with plausible box C/D guide RNAs assigned to all but three sites. We find approximately two-thirds of the sites to be fully methylated and the remainder to be fractionally modified in support of ribosome heterogeneity at the level of RNA modifications. A comparison to HCT116 cells reveals similar 2′-O-Me profiles with distinct differences at several sites. This study constitutes the first comprehensive mapping of 2′-O-Me sites in human rRNA using a high throughput sequencing approach. It establishes the existence of a core of constitutively methylated positions and a subset of variable, potentially regulatory positions, and paves the way for experimental analyses of the role of variations in rRNA methylation under different physiological or pathological settings.

High-throughput and site-specific identification of 2′-O-methylation sites using ribose oxidation sequencing (RibOxi-seq)

“…The most probable explanation for our results is that G4227 is only partially methylated, with the unmethylated population allowing the exposure of U4226 (Supplemental Table S1). This result is consistent with the data obtained using Ribometh-seq as well as observations of fractional methylation from primer extension experiments (Maden 1986;Krogh et al 2016). Such patterns prompted us to consider the possibility that annotated 2 ′ -O-methylation sites not detected by our method may be the result of a complete lack of methylation.…”

“…4). It is interesting to note that this site was also not detected using Ribometh-seq in HeLa cells (Krogh et al 2016). Further evidence from more cell lines will be required to confirm whether this site is actually modified in other cells or tissues.…”

“…All site-annotations and numbering correspond to the hg19 reference genome. The lists of known sites we used were curated as previously described (Krogh et al 2016). By applying a cutoff value of log 2 fold change of >7 and adjusted P-value of <0.0001, 39 out of 40 known 18S sites and 60 out of 66 known 28S sites were detected with high confidence ( Fig.…”

“…S2). The number of high confidence sites consisted of 93.3% known sites, which include sites newly found and MS validated by Krogh et al (2016) using Ribometh-seq. Using such cutoffs, only three novel sites (18S: U354, 28S A1322, and A3717) were found.…”

“…This method overall has much better specificity and accuracy, as it has successfully detected about the same number of sites in rRNAs as have been annotated. Several novel sites were also validated by mass spectrometry (Krogh et al 2016). However, the method relies heavily on negative rather than positive signals.…”