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. 2016 Oct 14;44(18):8951-8961.
doi: 10.1093/nar/gkw564. Epub 2016 Jun 20.

VSports最新版本 - The complete chemical structure of Saccharomyces cerevisiae rRNA: partial pseudouridylation of U2345 in 25S rRNA by snoRNA snR9

Masato Taoka  1 , Yuko Nobe  2 , Yuka Yamaki  2 , Yoshio Yamauchi  2 , Hideaki Ishikawa  3 , Nobuhiro Takahashi  3 , Hiroshi Nakayama  4 , Toshiaki Isobe  5
Affiliations

The complete chemical structure of Saccharomyces cerevisiae rRNA: partial pseudouridylation of U2345 in 25S rRNA by snoRNA snR9

"VSports最新版本" Masato Taoka et al. Nucleic Acids Res. .

Abstract

We present the complete chemical structures of the rRNAs from the eukaryotic model organism, Saccharomyces cerevisiae The final structures, as determined with mass spectrometry-based methodology that includes a stable isotope-labelled, non-modified reference RNA, contain 112 sites with 12 different post-transcriptional modifications, including a previously unidentified pseudouridine at position 2345 in 25S rRNA. Quantitative mass spectrometry-based stoichiometric analysis of the different modifications at each site indicated that 94 sites were almost fully modified, whereas the remaining 18 sites were modified to a lesser extent. Superimposed three-dimensional modification maps for S. cerevisiae and Schizosaccharomyces pombe rRNAs confirmed that most of the modified nucleotides are located in functionally important interior regions of the ribosomes. We identified snR9 as the snoRNA responsible for pseudouridylation of U2345 and showed that this pseudouridylation occurs co-transcriptionally and competitively with 2'-O-methylation of U2345 VSports手机版. This study ends the uncertainty concerning whether all modified nucleotides in S. cerevisiae rRNAs have been identified and provides a resource for future structural, functional and biogenesis studies of the eukaryotic ribosome. .

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Figures (V体育安卓版)

Figure 1.
Figure 1.
Nucleotide heterogeneity at position 2345 in Saccharomyces cerevisiae 25S rRNA. LC-MS and MS2 of the RNase T1 digest of U/C-5D-labelled and reverse-phase LC-purified 25S rRNA (100 fmol). (A) Extracted ion monitoring of fragments containing a nt at position 2345 shows that nt2345 is a mixture containing 9% U, 74% Um, 4% Ψ and 13% Ψm (Table 2). The extracted ion masses have m/z values of 1421.688 (upper panel) and 1418.184 (lower panel) within a mass window of ±10 ppm. U, 2334UCmCCΨAUCUACUAΨCΨA2351Gp; Ψ, 2334UCmCCΨAUCUACΨAΨCΨA2351Gp; Um, 2334UCmCCΨAUCUACUmAΨCΨA2351Gp; Ψm, 2334UCmCCΨAUCUACΨmAΨCΨA2351Gp. (B) Mass spectra for the oligonucleotides containing U2345. The most abundant isotopomers are marked by asterisks. The m/z values of the most abundant signals coincide with the theoretical values within 5 ppm. (C) Tandem mass spectrum of 2334UCmCCΨAUCUACΨmAΨCΨA2351Gp. The blue arrows in the spectrum identify the major a, c, w and y ions. The cleavage positions of the assigned ions are mapped on the RNA sequence in the inset. Errors determined by Ariadne in the MS2 signals are plotted under the spectrum.
Figure 2.
Figure 2.
3D modification map of the Sc ribosome. The modified nt found in this study are mapped on the 3D structures of the Sc rRNAs (3U5B.pdb and 3U5D.pdb). The RNA backbones are shown as white wires and the colour codes for the modified nt (shown as balls) are: yellow, Ψ; red, 2′-O-methylated nt; blue, base modified nt found in Sc and Sp rRNAs; purple, modified nt unique to Sc rRNA; and white, modified nt unique to Sp rRNA. (A) Sc rRNAs. (B–D) Superimposed maps of modified nt found in Sc and Sp rRNAs; (B) SSU and LSU, (C) SSU and (D) LSU. Insets in C and D show the surface structures of the subunits. Domain names are in white and their positions are indicated by arrows.
Figure 3.
Figure 3.
Deletion of snR9 prevents pseudouridylation of U2345. (A) Potential base-pairing interactions between snR9 and Sc 25S rRNA. The upper sequence is that of the snoRNA snR9 with its hairpin shown as a solid line. The positions of A33, G67 and G69, which were mutated in our study, are indicted by the arrows. The lower sequence is that of the 25S rRNA around Ψ2345. The lower arrow points to Ψ2345. (B) Extracted ion chromatogram (EIC) of the fragments containing 2345ΨmA2347Ψp produced by the RNase A digestion of the 25S rRNA from wild-type (upper panel), ΔsnR9 (middle panel) and ΔsnR33 (lower panel) Sc strains. The strains were cultured in the U/C-5-D labelling medium at 30°C and purified 25S rRNA was digested with RNase A. Each digest (50 fmol) was subjected to LC-MS. The sequence and m/z value of ΨmAΨp are shown in the figure. The most intense signal in the ΔsnR33 spectrum was set to a relative intensity of 100%, and the peaks in the wild-type spectrum were scaled accordingly. The arrows in the left panels indicate the position of the MS signal for the Ψm2345-containing fragment, which was not detected in the ΔsnR9 chromatogram.
Figure 4.
Figure 4.
Episomal expression of snR9 in ΔsnR9_2 restores U2345 pseudouridylation. EICs of fragments from the RNase A digestion of 25S rRNA containing U2345 are shown. 25S rRNA was purified from ΔsnR9_2 that had been transformed with pSEC, pSECR9-WT, pSECR9-A33G, pSECR9-G67A or pSECR9-G69A. Each strain was cultured at 30°C in U/C-5-D labelling medium. The extracted 25S rRNAs were individually digested with RNase A, and the resulting digests (50 fmol) were subjected to LC-MS. The sequence and m/z value of ΨmAΨp are indicated. A mass window of ±10 ppm was used the chromatograms. The most intense peak in the pSECR-WT spectrum was set to 100%. The MS signal of ΨmAΨp was detected only in the spectrum of ΔsnR9 that had been transformed with pSECR9-WT, which enabled expression of wild-type snR9.
Figure 5.
Figure 5.
Loss of U2345 pseudouridylation causes no changes in cell growth and ribosome synthesis. (A) Cell growth study. Serial 10-fold dilutions of the isogenic wild-type BY5208 and ΔsnR9 strains grown at 19°C for 48 h or 30°C and 37°C for 24 h were spotted onto YPD-containing agar plates. (B) Ribosome profiles of BY5208 (upper panel) and ΔsnR9 (lower panel) strains. Ribosomal and polysomal fractions prepared from the wild-type and ΔsnR9 strains (each cultured at 30°C for 24 h in YPD medium) were subjected to sucrose density gradient centrifugation. A254 was monitored.
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