Identification of sites of 2'-O-methylation vulnerability in human ribosomal RNAs by systematic mapping

Abstract

Ribosomal RNA modifications are important in optimizing ribosome function. Sugar 2'-O-methylation performed by fibrillarin-associated box C/D antisense guide snoRNAs impacts all steps of translation, playing a role in disease etiology (cancer). As it renders adjacent phosphodiester bonds resistant to alkaline treatment, 2'-O-methylation can be monitored qualitatively and quantitatively by applying next-generation sequencing to fragments of randomly cleaved RNA. We remapped all sites of 2'-O-methylation in human rRNAs in two isogenic diploid cell lines, one producing and one not producing the antitumor protein p53. We identified sites naturally modified only partially (confirming the existence in cells of compositionally distinct ribosomes with potentially specialized functions) and sites whose 2'-O-methylation is sensitive to p53. We mapped sites particularly vulnerable to a reduced level of the methyltransferase fibrillarin. The remarkable fact that these are largely sites of natural hypomodification provides initial insights into the mechanism of partial RNA modification. Sites where methylation appeared vulnerable lie peripherally on the 3-D structure of the ribosomal subunits, whereas the numerous modifications present at the core of the subunits, where the functional centers lie, appeared robustly made. We suggest that vulnerable sites of 2'-O-methylation are highly likely to undergo specific regulation during normal and pathological processes.

Figure 1

Distribution of the 106 ribose-methylated residues on 3-D models of human ribosomal subunits. (A) Entire ribosome: solvent view. The models of the subunits are based on PDB entry 4UG0. The four rRNAs are shown as ribbons (18S, dark grey; 28S, light gray; 5S, red; 5.8S, purple) with the modifications as spheres. The color code used for the modifications is described in panels B and C. SSU, small subunit; LSU, large subunit. (B) Small subunit (40S): interface view. Left, 18S rRNA shown as a dark grey ribbon with the modifications as spheres. Right, simplified view showing only the modifications and the E-site tRNA for orientation. The decoding site (DCS) is highlighted in black. Small ribosomal subunit morphological features are indicated: Bk, beak; H, head; Sh, shoulder; Bd, body; Lf, left foot; Rf, right foot; and h45, helix 45. Each modification is color-coded according to the categories identified in this work. (C) Large subunit (60S): interface view. Legend as in panel B. The peptidyl transferase center (PTC) residues are highlighted in black. Two PTC residues are 2′-O-methylated (shown in grey). CP, central protuberance.

Figure 2

Effects of progressive fibrillarin depletion on ribosome biogenesis and p53 steady-state accumulation. (A) Fibrillarin depletion elicits an antitumoral p53-dependent nucleolar stress response. Total protein extracted from HCT116 p53 +/+ and HCT116 p53 −/− cells treated with an siRNA against fibrillarin (#528 or #529) for 1, 2, or 3 days, or with a non-targeting control siRNA (SCR, scramble) analyzed by western blotting with antibodies against fibrillarin (FBL), p53, or β-actin (loading control). Uncropped blots are shown in Supplementary Figure 8. (B) Fibrillarin expression is influenced by p53. Total protein extracted from HCT116 p53 +/+cells, from HCT116 p53 −/− cells, and from HCT116 p53 +/+cells treated with an siRNA against p53 (#210) for 3 days, analyzed by western blotting with antibodies against the indicated proteins (FBL and β-actin). Inset, p53-siRNA-mediated depletion in HCT116 p53 +/+cells is highly efficient. Total protein extracted from HCT116 p53 +/+cells treated for 3 days with an siRNA targeting p53 (#210) or with a non-targeting control siRNA (SCR). The blot was probed with antibodies against the indicated proteins (p53 and β-actin). Uncropped blots are shown in Supplementary Figure 8. (C) Fibrillarin is required for mature rRNA accumulation. HCT116 p53 +/+ and HCT116 p53 −/− cells were depleted of fibrillarin for 1, 2, or 3 days with a specific siRNA (#528 or #529) or with a non-targeting control siRNA (SCR, scramble). Total RNA was extracted and resolved on denaturing gels and mature rRNAs were visualized by ethidium bromide staining. Densitometric quantification of the signals is shown. rRNA levels in cells depleted of fibrillarin are normalized with respect to the levels observed in cells treated with a non-targeting silencer (SCR). The uncropped gel is shown in Supplementary Figure 9. Note that the effects of Fibrillarin depletion on pre-rRNA processing and p53 steady-state accumulation were observed independently at least three times, confirming for the processing data our previous observations (see Www.RibosomeSynthesis.Com).

Figure 3

Clustering of 2′-O-methylation on human 18S and 5.8S rRNAs according to the sensitivity of methylation to fibrillarin depletion in the presence or absence of p53 in cells. A methylation score was computed for each position at the different time points of fibrillarin depletion (1, 2, and 3 days) in both HCT116 p53 +/+ and HCT116 p53 −/− cells. The fibrillarin depletion was performed twice independently with two different siRNAs (#528, and #529). The heatmap displays normalized mean methylation scores. All methylation scores were clustered with the software ‘R’ (hclust). The modified positions are indicated on the right. All positions are in 18S rRNA, except two which are in 5.8S rRNA (highlighted as 5.8S-Um14 and 5.8S-Gm75). They are highlighted as follows: asterisk, a position particularly sensitive to fibrillarin depletion; red, hypomodification in HCT116 p53 +/+ and in HCT116 p53 −/− cells; green, hypomodification only in HCT116 p53 −/− cells. The inset shows the color key and histogram.

Figure 4

Clustering of human rRNA 2′-O-methylation on 28S rRNA sites according to their sensitivity to fibrillarin depletion in the presence or absence of p53 in cells. Legend as in Fig. 3 for the 2′-O-methylated residues detected on the 28S rRNA. Asterisks, positions particularly sensitive to fibrillarin depletion; red, hypomodification in HCT116 p53 +/+ and in HCT116 p53 −/− cells; green, hypomodification only in HCT116 p53 −/− cells.

Figure 5

Clustering of snoRNA levels according to their sensitivity to fibrillarin depletion. SnoRNA abundances were inferred from numbers of sequencing reads in the RiboMethSeq dataset and clustered with ‘R’ (hclust). All detected box C/D snoRNAs (SNORD) are represented (categories 1 to 6). Representative H/ACA snoRNAs (SNORA) are shown (category 7). The inset shows the color key and histogram. snoRNAs labelled in orange are involved in the modification of the variable positions identified in this work.