Holistic Optimization of Bioinformatic Analysis Pipeline for Detection and Quantification of 2′-O-Methylations in RNA by RiboMethSeq

Pichot, Florian; Marchand, Virginie; Ayadi, Lilia; Bourguignon‐Igel, Valérie; Helm, Mark; Motorin, Yuri

doi:10.3389/fgene.2020.00038

Cited by 23 publications

(18 citation statements)

References 23 publications

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“…RiboMeth-seq was performed using the Illumina sequencing technology and raw data were processed as previously described ( 14 , 25 ). The median number of total reads reaches 7.2 millions after trimming, these reads being aligned on the 7.2 kb-long rRNA sequences, that corresponds to the optimal sequencing depth ( 14 , 26 ).…”

Section: Methodsmentioning

confidence: 99%

Ribosomal RNA 2′O-methylation as a novel layer of inter-tumour heterogeneity in breast cancer

et al. 2020

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Recent epitranscriptomics studies unravelled that ribosomal RNA (rRNA) 2′O-methylation is an additional layer of gene expression regulation highlighting the ribosome as a novel actor of translation control. However, this major finding lies on evidences coming mainly, if not exclusively, from cellular models. Using the innovative next-generation RiboMeth-seq technology, we established the first rRNA 2′O-methylation landscape in 195 primary human breast tumours. We uncovered the existence of compulsory/stable sites, which show limited inter-patient variability in their 2′O-methylation level, which map on functionally important sites of the human ribosome structure and which are surrounded by variable sites found from the second nucleotide layers. Our data demonstrate that some positions within the rRNA molecules can tolerate absence of 2′O-methylation in tumoral and healthy tissues. We also reveal that rRNA 2′O-methylation exhibits intra- and inter-patient variability in breast tumours. Its level is indeed differentially associated with breast cancer subtype and tumour grade. Altogether, our rRNA 2′O-methylation profiling of a large-scale human sample collection provides the first compelling evidence that ribosome variability occurs in humans and suggests that rRNA 2′O-methylation might represent a relevant element of tumour biology useful in clinic. This novel variability at molecular level offers an additional layer to capture the cancer heterogeneity and associates with specific features of tumour biology thus offering a novel targetable molecular signature in cancer.

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Section: Methodsmentioning

confidence: 99%

Ribosomal RNA 2′O-methylation as a novel layer of inter-tumour heterogeneity in breast cancer

et al. 2020

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“…These counts for A, C and G are used for local normalization of the U cleavages to background (NormUcount, see Materials and Methods), to account for local accessibility of different RNA regions. Further bioinformatic steps of analysis are similar to the well-established RiboMethSeq procedure ( 54 ), and allow calculation of scores termed ScoreMEAN, ScoreA and ScoreC for Ucleavage profiles, where ψ residues appear as protected positions (similarly to Nm residues in RiboMethSeq).…”

Section: Resultsmentioning

confidence: 99%

“…Resulting U profiles were used for calculation of ’RiboMethSeq-like’ scores (ScoreMEAN, A, B and PsiScore, equivalent to ScoreC in RiboMethSeq). Window of four neighboring nucleotides (±2 nt) was used for score calculation ( 54 ).…”

Section: Methodsmentioning

confidence: 99%

HydraPsiSeq: a method for systematic and quantitative mapping of pseudouridines in RNA

Marchand

Pichot

Neybecker

et al. 2020

Nucleic Acids Research

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Developing methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed ‘HydraPsiSeq’, a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10–50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at ∼20–25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.

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“…These rRNA sequences were thus corrected to fit to the observed sequencing data (Supplementary Information and Methods). Then, in order to map all possible candidate sites, we performed the same approach as reported in [ 30 ]. We used threshold values for ScoreMEAN and Score A2, respectively 0.93 and 0.5; although in some cases, less strict ScoreMEAN limit (0.92 or lower) was applied.…”

Section: Resultsmentioning

confidence: 99%

Mapping rRNA 2’-O-methylations and identification of C/D snoRNAs in Arabidopsis thaliana plants

et al. 2021

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In all eukaryotic cells, the most abundant modification of ribosomal RNA (rRNA) is methylation at the ribose moiety (2ʹ-O-methylation). Ribose methylation at specific rRNA sites is guided by small nucleolar RNAs (snoRNAs) of C/D-box type (C/D snoRNA) and achieved by the methyltransferase Fibrillarin (FIB).Here we used the Illumina-based RiboMethSeq approach for mapping rRNA 2ʹ-O-methylation sites in A. thaliana Col-0 (WT) plants. This analysis detected novel C/D snoRNA-guided rRNA 2ʹ-O-methylation positions and also some orphan sites without a matching C/D snoRNA. Furthermore, immunoprecipitation of Arabidopsis FIB2 identified and demonstrated expression of C/D snoRNAs corresponding to majority of mapped rRNA sites. On the other hand, we show that disruption of Arabidopsis Nucleolin 1 gene (NUC1), encoding a major nucleolar protein, decreases 2ʹ-O-methylation at specific rRNA sites suggesting functional/structural interconnections of 2ʹ-O-methylation with nucleolus organization and plant development. Finally, based on our findings and existent database sets, we introduce a new nomenclature system for C/D snoRNA in Arabidopsis plants.

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Holistic Optimization of Bioinformatic Analysis Pipeline for Detection and Quantification of 2′-O-Methylations in RNA by RiboMethSeq

Cited by 23 publications

References 23 publications

Ribosomal RNA 2′O-methylation as a novel layer of inter-tumour heterogeneity in breast cancer

Ribosomal RNA 2′O-methylation as a novel layer of inter-tumour heterogeneity in breast cancer

HydraPsiSeq: a method for systematic and quantitative mapping of pseudouridines in RNA

Mapping rRNA 2’-O-methylations and identification of C/D snoRNAs in Arabidopsis thaliana plants

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