The human 18S rRNA base methyltransferases DIMT1L and WBSCR22-TRMT112 but not rRNA modification are required for ribosome biogenesis

Abstract

At the heart of the ribosome lie rRNAs, whose catalytic function in translation is subtly modulated by posttranscriptional modifications. In the small ribosomal subunit of budding yeast, on the 18S rRNA, two adjacent adenosines (A1781/A1782) are N(6)-dimethylated by Dim1 near the decoding site, and one guanosine (G1575) is N(7)-methylated by Bud23-Trm112 at a ridge between the P- and E-site tRNAs. Here we establish human DIMT1L and WBSCR22-TRMT112 as the functional homologues of yeast Dim1 and Bud23-Trm112. We report that these enzymes are required for distinct pre-rRNA processing reactions leading to synthesis of 18S rRNA, and we demonstrate that in human cells, as in budding yeast, ribosome biogenesis requires the presence of the modification enzyme rather than its RNA-modifying catalytic activity. We conclude that a quality control mechanism has been conserved from yeast to human by which binding of a methyltransferase to nascent pre-rRNAs is a prerequisite to processing, so that all cleaved RNAs are committed to faithful modification. We further report that 18S rRNA dimethylation is nuclear in human cells, in contrast to yeast, where it is cytoplasmic. Yeast and human ribosome biogenesis thus have both conserved and distinctive features.

FIGURE 1:

The 18S rRNA m⁷G and modifications are conserved in S. cerevisiae and Homo sapiens. (A) Secondary structure of the 18S rRNA. The insets illustrate conservation of rRNA sequence and secondary structure near the N⁷-methylguanosine (m⁷G) modification introduced by Bud23-Trm112 in yeast and WBSCR22-TRMT112 in humans and in the vicinity of the two N⁶,N⁶-dimethyladenosines synthesized by Dim1 in yeast and DIMT1L in humans. Modified nucleotide positions are highlighted in red. The 5′, central (C), 3′ major (3′ M), and 3′ minor (3′ m) domains are indicated. (B) Three-dimensional representation of the yeast small subunit (model based on Protein Data Bank entry 3U5B) with posttranscriptional modifications and functional sites highlighted. The decoding site (DCS, in cyan) at the base of helix 44 (in anthracite) and the mRNA entry (green circle) and exit (red circle) sites are indicated. Residues shown as green and red spheres are 2′-O methylated or pseudouridylated, respectively. Bd, body; Bk, beak; H, head; Lf, left foot; Nk, neck; Pt; platform, Rf, right foot; Sh, shoulder.

FIGURE 2:

Subcellular distribution of DIMT1L, WBSCR22, and TRMT112. (A–C) DIMT1L is a nucleolar protein. WBSCR22 and TRMT112 localize to the nucleoplasm, with nucleolar exclusion for TRMT112, and to a polarized perinuclear structure (white arrows), overlapping partially with the Golgi and lysosomes. Cells were counterstained with DAPI for DNA labeling. Image sections were captured in confocal mode with a Yokogawa spinning disk on a Zeiss Axiovert microscope at 40× magnification. Scale bar, 10 μm. (A) HeLa cells stably expressing the nucleolar protein fibrillarin fused to GFP (FIB-GFP) were processed for immuno­fluorescence with a primary antibody specific to DIMT1L, WBSCR22, or TRMT112 and a red fluorescent secondary antibody. The intrinsic GFP fluorescence was used to visualize fibrillarin. (B) HeLa cells decorated with a WBSCR22- or TRMT112-specific primary antibody, revealed with a green fluorescent secondary antibody. (C) HeLa cells incubated with either Lyso- or Golgi-tracker and decorated with a TRMT112-specific primary antibody, revealed with a red fluorescent secondary antibody.

FIGURE 3:

WBSCR22 and TRMT112 form a complex, and TRMT112 is required for the metabolic stability of WBSCR22. (A–C) WBSCR22 and TRMT112 interact directly. Constructs encoding polyhistidine-tagged WBSCR22 and nontagged TRMT112 were overexpressed in bacterial cells, total extracts were purified over a nickel column, and eluates were tested by Coomassie blue staining (A) and Western blotting with antibodies against WBSCR22 (B) or TRMT112 (C). Lane 1, molecular weight marker; 2–5, cells coexpressing His₆-WBSCR22 and TRMT112; 6–9, cells expressing only untagged TRMT112. Lanes 2 and 6, total extract from noninduced cells; 3 and 7, total extract from IPTG-induced cells; 4 and 8, supernatant loaded on Ni-NTA column; 5 and 9, 50 mM imidazole eluates, revealing copurification of WBSCR22 and TRMT112 in extracts prepared from bacterial cells coexpressing His₆-WBSCR22 and TRMT112, or the absence of any purification from cells expressing only the construct encoding untagged TRMT112. As a control, lane 10 shows the purified Hemk2-TRMT112 complex (Figaro et al., 2008). (D) Depletion of WBSCR22 is efficient at the protein level. HeLa cells were treated for 3 d with one of five different siRNAs (#1–#5) targeting WBSCR22. Total protein was analyzed by Western blotting with antibodies against TRMT112 and WBSCR22. β-Actin was used as loading control. (E) TRMT112 is required for WBSCR22 metabolic stability. HeLa cells were treated for 3 d with an siRNA (#1, #2, or #3) targeting TRMT112. Total protein extracts were processed as in C. The residual level of TRMT112 was estimated by densitometry to be between 50 and 70%.

FIGURE 4:

DIMT1L, WBSCR22, and TRMT112 are required for distinct pre-rRNA processing steps, and the pre-rRNA processing defects are conserved in different cell types and do not depend on p53. WI-38, RKO, and HCT116 cells were treated for 3 d with a specific siRNA targeting DIMT1L, WBSCR22, or TRMT112. Paired HCT116 cell lines expressing p53 or not were used (+/+ and –/–). Total RNA was extracted, resolved on denaturing agarose gel, transferred to a nylon membrane, and hybridized with probes. The probes used were as follows: (I, II) LD1844, (III) LD1827, and (IV) LD1828. The detected pre-rRNA species are indicated to the right and schematized. (V, VI) The mature 28S and 18S rRNAs stained with ethidium bromide. For each sample, the 28S/18S ratio was calculated from Agilent bioanalyzer electropherograms. All RNA species were quantified with a Phosphorimager normalized with respect to the nontargeting control (SCR), and their abundances represented as a heatmap using the color code indicated to the right. The sequences of the siRNAs used are listed in Supplemental Table S3 (WBSCR22#1, TRMT112#2, and DIMT1L#2). Note that in the heatmap for RKO cells, the signal for lane 8 was corrected for loading.

FIGURE 5:

Dynamic analysis of pre-rRNA processing defects in cells depleted of DIMT1L, WBSCR22, or TRMT112. (I) HeLa cells were depleted of WBSCR22, TRMT112, or DIMT1L for 3 d by means of a specific siRNA, pulse labeled for 30 min with l-(methyl-³H)-methionine, chased with an excess of cold methionine, and collected at different time points over a 4-h period. Total RNA was extracted, resolved on an agarose gel, transferred to a GeneScreen membrane, and then exposed by fluorography. (II, III) The ethidium bromide–stained mature 28S and 18S rRNAs. The sequences of the siRNAs used are listed in Supplemental Table S3 (WBSCR22#1, TRMT112#2, and DIMT1L#2).

FIGURE 6:

DIMT1L and WBSCR22 are required for 18S rRNA and m⁷G₁₆₃₉ methylation, respectively. (A–D) Primer extension mapping of the substrate nucleotides of DIMT1L and WBSCR222 on human 18S rRNA. Total RNA was extracted from HeLa cells depleted for 3 d with a DIMT1L-targeting (A) or WBSCR22-targeting (B) siRNA and analyzed by primer extension with oligonucleotide LD2120, LD2122, or LD2141. (C, D) The positions of the oligonucleotides and of the rRNA species. (E) PNO1 is required for efficient DIMT1L-mediated dimethylation. Total RNA was extracted from HeLa cells depleted for 3 d with a siRNA specific to DIMT1L or PNO1 and processed by primer extension with oligonucleotide LD2141. The level of residual dimethylation estimated with a Phosphorimager is indicated to the right. The results shown are means of three independent experiments. The sequences of the siRNAs used are listed in Supplemental Table S3 (WBSCR22#1, DIMT1L#2). (F) PNO1 is not required for the metabolic stability of DIMT1L. Total protein extract from HeLa cells depleted for 3 d with a siRNA specific to DIMT1L or PNO1 and tested by Western blotting with the antibodies indicated. Right, residual level of DIMT1L and PNO1 mRNAs assessed by qRT-PCR.

FIGURE 7:

The methyltransferase function of DIMT1L is not required for pre-rRNA processing. Protein levels and pre-rRNA processing in HEK293 cells stably expressing, via an siRNA-resistant mRNA (siRNAr), a construct encoding the wild-type or a catalytically deficient (Y131G) Flag-tagged version of DIMT1L in the presence or absence of endogenous DIMT1L. The allele encoding catalytically deficient DIMT1L and, as controls, the wild-type construct and the empty plasmid were integrated at the same genomic locus. Expression of the recombinant constructs was under the control of an inducible Tet promoter (bent arrow) and was induced by incubating the cells in 0.2 μg/ml tetracycline. Expression of the endogenous DIMT1L gene was suppressed by incubating the cells for 3 d with siRNA DIMT1L#2 (see Supplemental Table S3). As control, a nontargeting siRNA (SCR, scramble) was used. (A) Western blot analysis with an antibody against DIMT1L. The antibody detects the endogenous DIMT1L protein, displaying the expected molecular weight of 35 kDa, and the Flag-tagged recombinant variants, detected at 38 kDa. β-Actin was used as loading control. The anti-DIMT1L antibody detects a faint nonspecific band (asterisk) above Flag-tagged DIMT1L. Hybridizing the membrane with an anti-Flag antibody revealed only recombinant proteins of the expected size (unpublished data). (B) Northern blot analysis. Total RNA treated as described in Figure 4 was probed with oligonucleotide LD1827. The abundances of the various RNAs detected were established by Phosphorimager quantification and normalized with respect to the nontargeting SCR control and are presented as a heatmap with the color code indicated to the right.

FIGURE 8:

The methyltransferase function of WBSCR22 is not required for pre-rRNA processing. Protein levels and pre-rRNA processing in HEK293 cells stably expressing, via an siRNA-resistant mRNA (siRNAr), a construct encoding the wild-type or a catalytically deficient (G63E, D82K, and D63E/D82K) Flag-tagged version of WBSCR22 in the presence or absence of endogenous WBSCR22. The constructs encoding catalytically deficient WBSCR22 and, as controls, the wild-type construct and the empty plasmid were integrated at the same genomic locus. Expression of the recombinant constructs was under the control of an inducible Tet promoter (bent arrow) and was induced by incubating the cells in 0.2 μg/ml tetracycline. Expression of the endogenous WBSCR22 gene was suppressed by incubating the cells for 3 d with siRNA WBSCR22#1 (see Supplemental Table S3). As control, a nontargeting siRNA (SCR, scramble) was used. (A) Western blot analysis with an antibody against WBSCR22. The antibody detects the endogenous WBSCR22 protein, displaying the expected molecular weight of 32 kDa, and the Flag-tagged recombinant variants, detected at 35 kDa. β-Actin was used as loading control. Hybridizing the membrane with an anti-Flag antibody revealed only the recombinant variants of the expected size (unpublished data). (B) Northern blot analysis. Total RNA treated as described in Figure 4 was probed with oligonucleotide LD1827. The abundances of the various RNAs detected were established by Phosphorimager quantification and normalized with respect to the nontargeting SCR control and are presented as a heatmap with the color code indicated to the right.