C Coverage and P -values obtained for the Human Let-7e precursor sequence. Position 18 is indicated. D Analysis of the power to detect m7G modification at position 18 in the human Let-7e. The curves show the power for 4 different detection levels of m7G modifications with the sequencing depth observed for Let-7e. Kouzarides and colleagues recently reported m 7 G modifications within a subset of human miRNAs, including Let-7e, for which the m 7 G modification was validated and localized to position 18 using mass spectroscopy of a RNaseA fragment of the miRNA Figure 4A For Let-7e, we have very high sequencing depth Figure 4B but we do not observe any mutations in the treated samples for this position.
In this specific experiment, tRNAs m 7 G positions are detected with mutational rates in the range of 0. The fact that we do not observe any mutations for Let-7e position 18 demonstrates that for the HeLa cells used in our study, this position is not m 7 G modified at a biologically relevant level. In our analysis, E. However, in neither of the two analyses, we find any evidence of internal mRNA positions being m 7 G modified.
In contrast, we find the known ribosomal m 7 G modifications at E. The two known E.
The known yeast ribosomal m7G modification at is indicated. Mutational profiling has previously been applied to obtain nucleotide resolution mapping of RNA-protein interactions, m 1 A modifications and for Selective 2-hydroxyl acylation analysed by primer extension SHAPE based probing of RNA structure, and here we demonstrate that this strategy also can be successfully applied to detect internal m 7 G modifications. Abasic sites in RNA are relatively stable, and during the subsequent reverse transcription m 7 G positions are permanently recorded as mutations in the cDNA sequence.
Compared to methods based on the mapping of reverse transcription stops, mutational profiling makes better use of the sequencing reads and has improved sensitivity because bias from RNA fragmentation, adapter ligation and secondary structures is reduced. In global RNA modification mapping experiments, it becomes very important to establish the background detection rates and account for these when modifications are mapped We developed a pipeline that incorporates re-estimated base qualities, which are used to provide estimated mutation frequencies using an EM-algorithm.
Dissecting the role of RNA modification regulatory proteins in melanoma
In addition, our tool applies log likelihood ratio tests to obtain P -values for differences in mutation rates both within replicates and between the control and NaBH 4 treated samples, making m 7 G-MaP-seq highly suitable for global analyses. This is in agreement with a recent publication using NaBH 4 treatment for detection of ac 4 C 34 and the known susceptibility of D for cleavage of the dihydrouridine ring upon reduction by NaBH 4 In contrast, for m 1 A positions NaBH 4 treatment was not expected to lead to increased mutation rates.
Rather, the alkaline conditions during reduction could potentially lead to Dimroth rearrangement to m 6 A and thereby decrease mutation rates, as previously used to improve m 1 A signal in global sequencing-based mapping experiments Typically, the Dimroth rearrangement is performed at elevated temperatures 35 , whereas we here perform NaBH 4 treatment on ice, which likely explains that we do not observe widespread rearrangement to m 6 A and decreased mutation rates. NaBH 4 treatment of m 1 A results in the formation of 1-methylhydroadenosine, which is fairly stable, but can be re-oxidized to m 6 A The reduction to 1-methylhydroadenosine results in the loss of planar conformation and aromatic character of the six-membered ring.
Our results suggest that 1-methylhydroadenosine is relatively stable under our conditions and that A is relatively efficiently incorporated into cDNA during reverse transcription at 1-methylhydroadenosine positions Supplementary Figure S5B. We find that different m 7 G positions have variation in their m 7 G-MaP-seq mutational rates, which may be the result of differences in the level of m 7 G modification, but probably also reflects that the local sequence influences the efficiency of abasic sites formation during reduction and of reverse transcriptase abasic site read-through.
Compared to our method m 7 G-seq includes an additional step to biotinylate the abasic sites, which allows subsequent enrichment of the RNA fragments. For this reason, m 7 G-seq will probably be better to identify low frequency m 7 G modification than m 7 G-MaP-seq. In future applications of m 7 G-MaP-seq, we plan to combine our protocol with m 7 G specific antibody immunoprecipitation of RNA fragment 12 , 13 , 23 before the NaBH 4 treatment, which potentially would facilitate the detection of positions with low frequency m 7 G modifications.
In contrast, we did not observe any signal at position , and in human LSU rRNA, which were recently reported to be m 7 G modified based on a high resolution cryoEM structure of the human 80S ribosome As in our study, the cryoEM study is based on rRNA purified from HeLa cells, suggesting that the inconsistent results are not cell type related.
Our data, together with mass spectroscopy based analysis of human LSU rRNA from the Isobe group 39 , therefore strongly suggests that m 7 G modifications are not reliably detected from current cryoEM data, which is furthermore supported by the fact that the cryoEM study did not detect the well-known m 7 G modification on SSU position In these experiments, we observe m 7 G modifications on tRNAs only, for which a subset is known to be m 7 G modified at position 46 in the variable loop, whereas no other sRNA was found to be modified Supplementary Figure S7.
Our results are in agreement with existing mapping of yeast m 7 G modifications, including tRNAs previously found not to be m 7 G modiifed Supplementary Table S3 Their study implemented AlkB treatment to increase sequencing depth of tRNA carrying modifications that terminate reverse transcription and convincingly identified the entire subset of mouse tRNA that are m 7 G modified. Alternatively, the A cells used in their study may have more abundant miRNA m 7 G modifications than the HeLa cells used in our study. For both species, our analysis has high sequencing depth on a substantial fraction of Gs in the transcriptome, 20 and 16 percent, for E.
Future application of m 7 G-seq or m 7 G-MaP-seq with higher sequencing depth and potentially combined with antibody mediated m 7 G pull-down will allow detection of potential low frequency m 7 G modifications in highly expressed mRNA and high frequency m 7 G modifications in lowly expressed mRNA.
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The method can be implemented with existing RNA-sequencing protocols and we provide an efficient computational pipeline for data analysis. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide.
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Article Contents. Oxford Academic. Google Scholar. Line Dahl Poulsen. Finn Kirpekar. Anders Albrechtsen. Jeppe Vinther. To whom correspondence should be addressed.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Cite Citation.
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Glen Report - Reversible m6A RNA modification
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