Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively

Abstract

Yeast 25S rRNA was reported to contain a single cytosine methylation (m(5)C). In the present study using a combination of RP-HPLC, mung bean nuclease assay and rRNA mutagenesis, we discovered that instead of one, yeast contains two m(5)C residues at position 2278 and 2870. Furthermore, we identified and characterized two putative methyltransferases, Rcm1 and Nop2 to be responsible for these two cytosine methylations, respectively. Both proteins are highly conserved, which correlates with the presence of two m(5)C residues at identical positions in higher eukaryotes, including humans. The human homolog of yeast Nop2, p120 has been discovered to be upregulated in various cancer tissues, whereas the human homolog of Rcm1, NSUN5 is completely deleted in the William's-Beuren Syndrome. The substrates and function of both human homologs remained unknown. In the present study, we also provide insights into the significance of these two m(5)C residues. The loss of m(5)C2278 results in anisomycin hypersensitivity, whereas the loss of m(5)C2870 affects ribosome synthesis and processing. Establishing the locations and enzymes in yeast will not only help identifying the function of their homologs in higher organisms, but will also enable understanding the role of these modifications in ribosome function and architecture.

Figure 1.

RP-HPLC analysis of the 25S rRNA of Δrcm1 and Δtrm4. Nucleosides compositions of the 25S rRNAs from the wild type, Δrcm1 and Δtrm4 mutant were analyzed by RP-HPLC. (A) RP-HPLC chromatogram from the wild type, (B) Δrcm1 and (C) Δtrm4 mutant. The peak corresponding to the m⁵C with a retention time of approximately 12 min reduces to half in the Δrcm1 mutant compared with wild type and Δtrm4.

Figure 2.

Mung bean nuclease protection assay for the identification of new m⁵C residue in the 25S rRNA of S. cerevisiae. To identify the second m⁵C modification, whole 25S rRNA was subjected to mung bean nuclease protection assay. Synthetic oligonucleotides complementary to the different regions of 25S rRNA were designed and hybridized to the 25S rRNA of wild type. Two fragments corresponding to domain IV of 25S rRNA (from base 2253 to 2300) marked red (A) and region corresponding to domain V of 25S rRNA (from base 2840 to 2889) also marked in red (B) were discovered to contain m⁵C residues. (C) RP-HPLC chromatogram of the fragments derived from region displayed in red (A) corresponding to already predicted m⁵C 2278 residue. (D) RP-HPLC chromatogram of the fragments derived from region marked in red (B). Both fragments contain single m⁵C residue with the retention time of ∼12 min. To identify the m⁵C residue missing in Δrcm1, the statuses of these two m⁵C residues were analyzed in the 25S rRNA of Δrcm1 strain. (E) RP-HPLC chromatogram of fragments corresponding to region 2253–2300 of 25S rRNA marked in red (A). (F) RP-HPLC chromatogram of fragments corresponding to region 2840–2889 of 25S rRNA marked in red (B). Interestingly, m⁵C residue from the fragments corresponding to m⁵C2278 was absent, highlighting the involvement of Rcm1 in catalyzing the m⁵C2278 modification of 25S rRNA.

Figure 3.

Mapping of the exact position of m⁵C residue in the domain V of S. cerevisiae. To identify the precise location of the m⁵C residue within the fragment corresponding to the PTC region of 25S rRNA, shown in panel (A), fragments marked in panel (B) were isolated using mung bean nuclease protection assay. The nucleosides composition of these fragments was next analyzed using RP-HPLC. (C) RP-HPLC chromatogram of oligo-2867, oligo-2870 (D) and oligo-2884 (E). As evident from the chromatograms, the 25S rRNA fragments corresponding to oligo-2870 contains m⁵C residues, whereas other two fragments lack m⁵C residues. Together, this distinctly confirmed that the m⁵C residue in the PTC region (2839 to 2890) is located at position 2870.

Figure 4.

Rcm1 catalyzes methylation of m⁵C2278 of 25S rRNA in vivo. (A) In silico 3D structure model of Rcm1. The 3D structure prediction of Rcm1 was performed using a recently described protocol of Kelley and Sternberg, (2009), explained in detail in Supplementary Methods. Rcm1 is predicted to be a Rossmann-like fold methyltransferase. Two highly conserved cysteine residue, Cys330 in motif IV and Cys404 in motif VI, are highlighted as red and green spheres in the cartoon. To substantiate, Rcm1 to be involved in performing m⁵C modification, plasmid pSH31 carrying C-terminally heptahistidine tagged Rcm1 was transformed into Δrcm1 mutant strain. (B) RP-HPLC chromatogram of 25S rRNA from Δrcm1 strain carrying pSH31 plasmid. As a control, the status of m⁵C modification of the Δrcm1 strain with mutant rcm1-C404A (C). Like wild-type Rcm1, mutant protein was expressed as C-terminally heptahistidine tagged from plasmid pSH31-b. It became apparent from the chromatograms that the recombinant Rcm1 expressed from pSH31 plasmid was able to methylate the C-5 of C2278 of 25S rRNA, whereas the substitution of catalytically vital cysteine404 to alanine abolished the catalytic potential of Rcm1. (D) Western blot using anti-His antibodies was made to analyze the expression of heptahistidine tagged Rcm1 and mutant rcm1-C404A.

Figure 5.

Growth analysis and antibiotic sensitivity of the Δrcm1. (A) Ten-fold serial dilutions of the wild-type and Δrcm1 strains were spotted onto solid YPD plates and were incubated at different temperatures. (B) Anisomycin and Paromomycin sensitivity tests were performed by spotting 5 μl of anisomycin (5 µg/ml) and paromomycin solution (200 mg/ml) on filter discs, which were then applied on YPD plates containing the strains indicated. Polysome profile analysis was performed to detect any defects in 60S biogenesis and translation of Δrcm1 compared with isogenic wild type. (C) Polysome profile of the wild type and (D) Polysome profile of Δrcm1. The 59 kDa band corresponds to Rcm1 protein. M stands for protein marker.

Figure 6.

Nop2 catalyzes the C-5 methylation of C2870 in the 25S rRNA of yeast. (A) In silico 3D structure model of Nop2. Like Rcm1, Nop2 is also predicted to be a Rossmann-like fold methyltransferase. Two highly conserved cysteine residues; Cys424 in motif IV and Cys478 in motif VI are highlighted as red and blue spheres in the cartoon. To demonstrate Nop2 to be a RNA cytosine methyltransferase, we generated methyltransferase-dead mutant of nop2, where the catalytically significant cysteine residues Cys424 and Cys478 were exchanged with alanine. All these mutant proteins were expressed from plasmid pSH17-a (C424A) and pSH17-b (C478A) in a Δnop2 strain. Surprisingly, the exchange of Cys424 to alanine failed to complement the deletion of Nop2. (B) RP-HPLC chromatogram of 25S rRNA from Δnop2 carrying wild-type Nop2 expressed from plasmid pSH17. (C) RP-HPLC chromatogram of 25S rRNA from Δnop2 with mutant nop2-C478A expressed from plasmid pSH17-b. Approximately 50% reduction as observed in Δrcm1, in the chromatogram of 25S rRNA of strain carrying mutant nop2-C478A authenticated Nop2 to be an m⁵C methyltransferase. To further validate that Nop2 is responsible for m⁵C 2870, we constructed a strain where rcm1 was deleted from Δnop2 + nop2-C478A strain. (D) Overlaid RP-HPLC chromatogram of the nucleosides derived from the 25S rRNA of double mutant Δrcm1Δnop2 + nop2-C478A (red) and isogenic wild type (black). The Δrcm1Δnop2 + nop2-C478A contains no m⁵C nucleosides, validating the specific involvement of Rcm1 and Nop2 in the m⁵C modification of 25S rRNA at position 2278 and 2870. (E) To examine the expression of mutant nop2-C478A protein, a western blot using monoclonal antibodies against Nop2 was made. The Nop2 protein was detected at 90 kDa. The lower 70 kDa band corresponds to degradation product.

Figure 7.

Loss of m⁵C2870 affects ribosome biogenesis and polysome assembly. (A) Ten-fold serial dilutions of the strains were spotted onto solid YPG plates and were incubated at different temperatures. (B) Illustration for the 35S primary transcript. 35S rRNA contains 18S, 5.8S and 25S rRNA sequences separated by ITS1 and ITS2. The processing of the 35S precursor to mature rRNA involves endonucleolytic and exonucleolytic steps at specific sites (C) Polysome profile of Δnop2 + Nop2 and (D) of Δnop2 + nop2-C478A strain. Halfmers formations are indicated by asterisk. (E) Northern blot analysis of the Δnop2 + nop2-C478A mutant. The membrane was hybridized with radioactive labelled probes for ITS1 (i), ITS2 (ii) and to oligonucleotides specific to 18S and 25S rRNA (iii), shown as f, i, d and j in the panel (B), respectively. (F) Quantitative analysis of northern blot. The northern blots were quantified using Image Quant v 5.2 (GE life sciences) and the fold change for various precursors in the mutant strain nop2-C478A were calculated using isogenic wild type as a control.

Figure 8.

Investigation of any role of Trm112 in m⁵C modifications of 25S rRNA. To analyze any role of Trm112 in assisting Nop2 and Rcm1 in performing the m⁵C modification, the status of m⁵C modifications in Δtrm112 mutant were analyzed. RP-HPLC chromatogram of 25S rRNA isolated from (A) wild type and (B) Δtrm112. No significant change in the amount of m⁵C residues was observed in Δtrm112 mutant compared with the isogenic wild type.

Figure 9.

Localization of m⁵C residues and summary of the present study. S. cerevisiae contains two m⁵C residues, m⁵C2278 in the helix 70 (shown as blue spheres) and m⁵C2870 in the helix 89 (shown as red spheres) of 25S rRNA. Nop2 and Rcm1 perform the methylation of these cytosines, individually and the two highly conserved cysteine residues (shown in red and green spheres) are indispensible for the catalytic reaction. The PDB files 3U5B and 3U5E were used for the representation of ribosomal RNA. The cartoon was made by PyMol software (PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.). The pdb files generated by Phyre were used for the surface model of Nop2 and Rcm1.