The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation
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Evaluation Summary:
Post-transcriptional gene regulation mediated by YTH proteins that bind N6-methyladenosine (m6A) in mRNA is a pathway that has emerged of importance in eukaryotic biology. This paper focuses on two evolutionarily close proteins of this family, ETC2 and ETC3 to find redundant, specific and divergent functions. The authors addressed previously contradictory reports regarding the subcellular location of these proteins. The paper is of general interest to scientists within the field of post-transcriptional gene regulation and RNA biology and is not limited to the plant research community. This will advance our understanding of mRNA methylation and its role in plant biology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
This manuscript was co-submitted with: https://www.biorxiv.org/content/10.1101/2021.04.18.440342v2
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Abstract
Gene regulation via N6 -methyladenosine (m 6 A) in mRNA involves RNA-binding proteins that recognize m 6 A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only a few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant, ectopic, and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2 , ECT3 , and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady-state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.
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Evaluation Summary:
Post-transcriptional gene regulation mediated by YTH proteins that bind N6-methyladenosine (m6A) in mRNA is a pathway that has emerged of importance in eukaryotic biology. This paper focuses on two evolutionarily close proteins of this family, ETC2 and ETC3 to find redundant, specific and divergent functions. The authors addressed previously contradictory reports regarding the subcellular location of these proteins. The paper is of general interest to scientists within the field of post-transcriptional gene regulation and RNA biology and is not limited to the plant research community. This will advance our understanding of mRNA methylation and its role in plant biology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with …
Evaluation Summary:
Post-transcriptional gene regulation mediated by YTH proteins that bind N6-methyladenosine (m6A) in mRNA is a pathway that has emerged of importance in eukaryotic biology. This paper focuses on two evolutionarily close proteins of this family, ETC2 and ETC3 to find redundant, specific and divergent functions. The authors addressed previously contradictory reports regarding the subcellular location of these proteins. The paper is of general interest to scientists within the field of post-transcriptional gene regulation and RNA biology and is not limited to the plant research community. This will advance our understanding of mRNA methylation and its role in plant biology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
This manuscript was co-submitted with: https://www.biorxiv.org/content/10.1101/2021.04.18.440342v2
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Reviewer #1 (Public Review):
In metazoan systems, YTH-domain proteins recognize N6-methyladenosine (m6A) in mRNA to affect these molecules half-life or their translation efficiency. What remains unknown is whether mammalian YTHDF proteins act redundantly or not. Conversely, in plants EVOLUTIONARILY CONSERVED C-TERMINAL REGION (ECT) 2-4 were reported to work redundantly to promote proliferation of primed stem cells in organ primordial. However, it is largely unknown how these functions are achieved. In this manuscript, the authors rely on the data obtained in a companion manuscript to define ECT2 and ECT3 targeted mRNAs, redundant functions, and specific targets. In addition, the manuscript also describes divergent functions that make plant YTHDF proteins unique compared to mammals.
The authors also used super-resolution microscopy to …
Reviewer #1 (Public Review):
In metazoan systems, YTH-domain proteins recognize N6-methyladenosine (m6A) in mRNA to affect these molecules half-life or their translation efficiency. What remains unknown is whether mammalian YTHDF proteins act redundantly or not. Conversely, in plants EVOLUTIONARILY CONSERVED C-TERMINAL REGION (ECT) 2-4 were reported to work redundantly to promote proliferation of primed stem cells in organ primordial. However, it is largely unknown how these functions are achieved. In this manuscript, the authors rely on the data obtained in a companion manuscript to define ECT2 and ECT3 targeted mRNAs, redundant functions, and specific targets. In addition, the manuscript also describes divergent functions that make plant YTHDF proteins unique compared to mammals.
The authors also used super-resolution microscopy to precisely define ECT2 sub-cellular distribution, demonstrated that ECT2/3/4 do not influence poly(A)-site choice in their targets as previously suggested, and showed that ECT2/3-targets are repressed in the absence of these proteins.
Experimentally this is a solid paper that re-evaluates several aspects of YTHDF proteins disproving some functions wrongly attributed to these proteins. The conclusions achieved in the paper are of general interest to the community studying post-transcriptional gene regulation and especially to RNA biology. The experimental data provided is solid and support the conclusions. This paper is built on a companion paper that provides the foundations for the study.
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Reviewer #2 (Public Review):
Many plant mRNAs contain N6-methyladenosine (m6A) towards the 3' end of the transcript. YTH-domain proteins of plants and animals have a binding pocket that accepts the m6A. These YTH proteins fall into two broad classes, YTHDF type and YTHDC type. Arabidopsis has two YTHDC types and one of these is a fusion with the CPSF30 polyadenylation factor and has been implicated in 3' processing of transcripts in the nucleus. In contrast, Arabidopsis, like many plants, has an expanded family of 11 YTHDF type proteins which can be divided into 3 or possibly 4 subgroups based on sequence similarity and evolutionary trees. ECT2, ECT3 and ECT4 fall into one of these subgroups. Clear developmental phenotypes are only seen when both ECT2 and ECT3 are knocked out and these phenotypes are stronger when ECT4 is also absent - …
Reviewer #2 (Public Review):
Many plant mRNAs contain N6-methyladenosine (m6A) towards the 3' end of the transcript. YTH-domain proteins of plants and animals have a binding pocket that accepts the m6A. These YTH proteins fall into two broad classes, YTHDF type and YTHDC type. Arabidopsis has two YTHDC types and one of these is a fusion with the CPSF30 polyadenylation factor and has been implicated in 3' processing of transcripts in the nucleus. In contrast, Arabidopsis, like many plants, has an expanded family of 11 YTHDF type proteins which can be divided into 3 or possibly 4 subgroups based on sequence similarity and evolutionary trees. ECT2, ECT3 and ECT4 fall into one of these subgroups. Clear developmental phenotypes are only seen when both ECT2 and ECT3 are knocked out and these phenotypes are stronger when ECT4 is also absent - suggesting that the four proteins act redundantly (at least in part). Previous publications have suggested that ECT2 can move to/be found in the nucleus and that it may promote alternative polyadenylation. The work reported here shows that ECT2 is cytoplasmic and that the nuclear location previously reported is likely artifactual.
The authors identify a high confidence set of transcripts that are bound by ECT3, and they demonstrate that there is a very high level of overlap between ECT3 and ECT2 targets. Through a series of experiments, they show that binding of these target mRNAs is likely occurring in the same cells (rather than different tissue/cell types), and thus the two proteins are likely to be truly acting redundantly.
A key finding is that in the ECT2, ECT3, ECT4 triple mutant, the high confidence target mRNAs are reduced in abundance - thus suggesting a role in stabilising m6A containing transcripts.
Questions raised
The authors have previously shown that there are subtle differences between ECT2 and ECT3 mutant plants, most notably in the direction of root growth. It will be interesting to determine if this can be explained by the small number of differentially bound target mRNAs.
It remains formally possible that, whilst ECT2 and ECT3 bind the same mRNA targets and behave redundantly under most conditions, under certain treatments or environmental stimuli, they might be differentially post-translationally modified or may interact with different protein partners to bring about different consequences for bound transcripts. A larger question relates to the remaining YTHDF type proteins belonging to the other three subgroups. It will be interesting to determine whether these too act redundantly with ECT2, carry out a similar function in different cell types, or act to bring about different or even antagonistic outcomes.
Perhaps the most exciting finding is that transcripts that are targets of ECT2 and ECT3 are preferentially reduced in abundance in the triple mutant. This suggests a role for these proteins in cytoplasmic stabilisation of their mRNA targets, which would be consistent with observations from low m6A plants. The authors point out that it is also formally possible that this increase in transcript abundance could result from a feedback effect on transcription, and mRNA half-life measurements would be required to exclude this. However, a further possibility is that the stabilising effect could be indirect, many mRNAs are degraded co-translationally; if ECT2 were to act as a translational repressor, then increased translation of target transcripts in the mutant could actually result in their reduced steady-state levels.
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