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. 2016 Sep 19;44(16):7884-95.
doi: 10.1093/nar/gkw482. Epub 2016 Jun 1.

"V体育ios版" Profiling of 2'-O-Me in human rRNA reveals a subset of fractionally modified positions and provides evidence for ribosome heterogeneity

Nicolai Krogh  1 , Martin D Jansson  2 , Sophia J Häfner  2 , Disa Tehler  2 , Ulf Birkedal  1 , Mikkel Christensen-Dalsgaard  1 , Anders H Lund  3 , Henrik Nielsen  4
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"VSports在线直播" Profiling of 2'-O-Me in human rRNA reveals a subset of fractionally modified positions and provides evidence for ribosome heterogeneity

Nicolai Krogh et al. Nucleic Acids Res. .

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 VSports手机版. 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. .

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"VSports手机版" Figures

Figure 1.
Figure 1.
Outline of experimental strategies. (A) In RiboMeth-seq (RMS) analysis, library fragments obtained by alkaline degradation were ligated to adapters (blue), sequenced and 5′ and 3′ read-ends recorded. Ends at cleavable bonds will be highly represented whereas ends at bonds protected by 2′-O-methylation will be under-represented proportionally to the fraction of molecules modified at the position. (B) For mass spectrometry (MS) validation, a fragment encompassing the queried positions was isolated by annealing of a complementary oligo followed by digestion of unprotected RNA with RNases. The protected fragment was subjected to RNase A or T1 degradation followed by MALDI-TOF MS. (C) Distribution of RMS scores in HeLa rRNA. The score expresses the fraction of molecules methylated at the queried position. The figure excludes the two methylations in 5.8S rRNA.
Figure 2.
Figure 2.
A new methylation at LSU-C1868. (A) Base pairing of snoRD48 and target. (B and C) MS analysis. Both methylated and unmethylated fragments are present in the RNase A and T1 spectra, as expected from the RMS score. The sequence of the fragment is shown above the spectra, with an indication of the expected cleavage sites by RNases. All identified fragments are labeled in red. Arrows mark C1868 positions and spectra peaks with indications of observed and theoretical (in brackets) masses. (D) LSU-C1868 is located at the base of H39 that harbors two 2′-O-Me (M) and one pseudouridine (Ψ) and close to H38, the ‘A-site finger’.
Figure 3.
Figure 3.
A new methylation at LSU-G3771. (A) Base pairing of snoRD15A and target. (B) MS analysis. Only the methylated fragment is observed, as expected from the high RMS score. The sequence of the fragment is shown above the spectrum, with an indication of the expected cleavage sites by RNase A. All identified fragments are labeled in red. Arrows mark the G3771 position and spectrum peak for the methylated fragment with indications of observed and theoretical (in brackets) masses as well as the expected position of the unmethylated fragment that was not observed. (C) LSU-G3771 is located at the base of H71 and base paired to the 2′-O -methylated residue LSU-C3787.
Figure 4.
Figure 4.
Sequence analysis of snoRDs guiding 2′-O-methylation of rRNA. The analysis was applied to the minimal set of snoRDs defined in this study. (A) Distribution of box D types and consensus sequence represented as a sequence logo for box D generated by WebLogo (65). (B) Same for box D’. (C) Distribution of snoRDs using box D, box D’ and both. (D) Analysis of base pair type in relation to the distance from box D. The numbers show the type of base pair (or mismatch) at the indicated distance from box D cumulated for all proposed interactions between the selected set of snoRDs in Supplementary Table S6 and their target. (E and F) Distributions of duplex length and free energy of the interaction between snoRD and target, respectively.
Figure 5.
Figure 5.
RMS and snoRD expression analyses of HeLa and HCT116 cells. (A and B) RMS analysis of SSU and LSU rRNA, respectively. RMS score data plotted as the average ±s.d. for biological triplicates (C) RMS score for individual HeLa 2′-O-Me rRNA positions plotted against the corresponding summed average expression from two experiments of all snoRDs targeting the site. Bars indicate standard deviation in RMS score for triplicate experiments. (D) RMS score as a function of the number of genes encoding snoRDs targeting the position. (E) Log2 fold-change of RMS score for individual 2′-O-Me rRNA positions between HeLa and HCT116 cells plotted against the log2 expression fold change for the respective snoRD guides.
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V体育安卓版 - References

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