m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5’ splice site
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eLife assessment
This is an important paper reporting that an adenosine methyltransferase in the model plant Arabidopsis functions to target a key RNA component of the spliceosome, as in fission yeast, and thereby contributes to intron recognition. By contrast, the authors report no major role for the methyltransferase in targeting mRNAs, as reported in previous studies in Arabidopsis. While some of the evidence is convincing, other evidence is incomplete. The conclusions that mRNAs are not a significant target and that specific intronic sequences define sensitivity to the methyltransferase require additional support.
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Abstract
Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5’ splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the AC A GA box is modified by N 6 methylation, but the role of this m 6 A modification is poorly understood. Here, we show that m 6 A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIONA1, is required for Arabidopsis U6 snRNA m 6 A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5’ splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m 6 A influences 3’ splice site usage. We generalise these findings to reveal two major classes of 5’ splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA AC A GA box. We conclude that U6 snRNA m 6 A modification contributes to the selection of degenerate 5’ splice sites crucial to alternative splicing.
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eLife assessment
This is an important paper reporting that an adenosine methyltransferase in the model plant Arabidopsis functions to target a key RNA component of the spliceosome, as in fission yeast, and thereby contributes to intron recognition. By contrast, the authors report no major role for the methyltransferase in targeting mRNAs, as reported in previous studies in Arabidopsis. While some of the evidence is convincing, other evidence is incomplete. The conclusions that mRNAs are not a significant target and that specific intronic sequences define sensitivity to the methyltransferase require additional support.
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Reviewer #1 (Public Review):
This manuscript reports the function of FIO1, a mammalian METTL16 homolog, in Arabidopsis. The authors found FIO1 affects early flower phenotype through regulating splicing via U6 m6A modification. This paper confirmed FIO1-mediated m6A methylation on U6 RNA, consistent with two recently published reports. The manuscript contains quite a thorough splicing analysis on how splicing is affected in the fio1 mutant where U6 m6A is absent, and a detailed explanation of how m6A could affect base pairing and secondary structure involving U6 at different temperatures.
1. FLC mRNA can be m6A methylated. The authors appear to suggest the effect is secondary. More analysis and explanation are required. For instance the authors could measure m6A level on FLC in fio1 mutant, mta mutant, and compare it with that of wt.
2. …
Reviewer #1 (Public Review):
This manuscript reports the function of FIO1, a mammalian METTL16 homolog, in Arabidopsis. The authors found FIO1 affects early flower phenotype through regulating splicing via U6 m6A modification. This paper confirmed FIO1-mediated m6A methylation on U6 RNA, consistent with two recently published reports. The manuscript contains quite a thorough splicing analysis on how splicing is affected in the fio1 mutant where U6 m6A is absent, and a detailed explanation of how m6A could affect base pairing and secondary structure involving U6 at different temperatures.
1. FLC mRNA can be m6A methylated. The authors appear to suggest the effect is secondary. More analysis and explanation are required. For instance the authors could measure m6A level on FLC in fio1 mutant, mta mutant, and compare it with that of wt.
2. The authors used nanopore m6A sequencing to map m6A in mRNA from wt and fio1 mutant strains. I would suggest either RIP-seq or mass spectrometry measurement to confirm the loss of fio1 leads to limited mRNA m6A changes.
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Reviewer #2 (Public Review):
Overall, I think that the screen or mutants in the Arabidopsis flowering pathway and its outcome are biologically interesting and important. The authors show that FIO1 methylates U6 snRNA and not (or rarely) mRNA. However, subsequent to this, the results are entirely from bioinformatics of RNAseq data from the derived mutants; there are no further experiments performed, either to confirm or test newly-derived hypotheses. Furthermore, the main hypothesis, that 5'SS pos.+4 identity is critical for sensitivity to U6 N6-methylation, was already described in yeast S. pombe, based on data from mutants in the pombe ortholog Mtl16. Minimally, the conclusions based on bioinformatics should be confirmed with experimental data. In addition, there are examples throughout the manuscript where the authors state results or …
Reviewer #2 (Public Review):
Overall, I think that the screen or mutants in the Arabidopsis flowering pathway and its outcome are biologically interesting and important. The authors show that FIO1 methylates U6 snRNA and not (or rarely) mRNA. However, subsequent to this, the results are entirely from bioinformatics of RNAseq data from the derived mutants; there are no further experiments performed, either to confirm or test newly-derived hypotheses. Furthermore, the main hypothesis, that 5'SS pos.+4 identity is critical for sensitivity to U6 N6-methylation, was already described in yeast S. pombe, based on data from mutants in the pombe ortholog Mtl16. Minimally, the conclusions based on bioinformatics should be confirmed with experimental data. In addition, there are examples throughout the manuscript where the authors state results or conclusions without providing any data; this is not acceptable and data supporting these assertions must be included.
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