Passive shaping of intra- and intercellular m6A dynamics via mRNA metabolism

Reviewer #1 (Public review):

Summary:

Here, the authors propose that changes in m6A levels may be predictable via a simple model that is based exclusively on mRNA metabolic events. Under this model, m6A mRNAs are "passive" victims of RNA metabolic events with no "active" regulatory events needed to modulate their levels by m6A writers, readers, or erasers; looking at changes in RNA transcription, RNA export, and RNA degradation dynamics is enough to explain how m6A levels change over time.

The relevance of this study is extremely high at this stage of the epi transcriptome field. This compelling paper is in line with more and more recent studies showing how m6A is a constitutive mark reflecting overall RNA redistribution events. At the same time, it reminds every reader to carefully evaluate changes in m6A levels if observed in their experimental setup. It highlights the importance of performing extensive evaluations on how much RNA metabolic events could explain an observed m6A change.

Weaknesses:

It is essential to notice that m6ADyn does not exactly recapitulate the observed m6A changes. First, this can be due to m6ADyn's limitations. The authors do a great job in the Discussion highlighting these limitations. Indeed, they mention how m6ADyn cannot interpret m6A's implications on nuclear degradation or splicing and cannot model more complex scenario predictions (i.e., a scenario in which m6A both impacts export and degradation) or the contribution of single sites within a gene.

Secondly, since predictions do not exactly recapitulate the observed m6A changes, "active" regulatory events may still play a partial role in regulating m6A changes. The authors themselves highlight situations in which data do not support m6ADyn predictions. Active mechanisms to control m6A degradation levels or mRNA export levels could exist and may still play an essential role.

(1) "We next sought to assess whether alternative models could readily predict the positive correlation between m6A and nuclear localization and the negative correlations between
m6A and mRNA stability. We assessed how nuclear decay might impact these associations by introducing nuclear decay as an additional rate, δ. We found that both associations were robust to this additional rate (Supplementary Figure 2a-c)."
Based on the data, I would say that model 2 (m6A-dep + nuclear degradation) is better than model 1. The discussion of these findings in the Discussion could help clarify how to interpret this prediction. Is nuclear degradation playing a significant role, more than expected by previous studies?

(2) The authors classify m6A levels as "low" or "high," and it is unclear how "low" differs from unmethylated mRNAs.

(3) The authors explore whether m6A changes could be linked with differences in mRNA subcellular localization. They tested this hypothesis by looking at mRNA changes during heat stress, a complex scenario to predict with m6ADyn. According to the collected data, heat shock is not associated with dramatic changes in m6A levels. However, the authors observe a redistribution of m6A mRNAs during the treatment and recovery time, with highly methylated mRNAs getting retained in the nucleus being associated with a shorter half-life, and being transcriptional induced by HSF1. Based on this observation, the authors use m6Adyn to predict the contribution of RNA export, RNA degradation, and RNA transcription to the observed m6A changes. However:

(a) Do the authors have a comparison of m6ADyn predictions based on the assumption that RNA export and RNA transcription may change at the same time?

(b) They arbitrarily set the global reduction of export to 10%, but I'm not sure we can completely rule out whether m6A mRNAs have an export rate during heat shock similar to the non-methylated mRNAs. What happens if the authors simulate that the block in export could be preferential for m6A mRNAs only?

(c) The dramatic increase in the nucleus: cytoplasmic ratio of mRNA upon heat stress may not reflect the overall m6A mRNA distribution upon heat stress. It would be interesting to repeat the same experiment in METTL3 KO cells. Of note, m6A mRNA granules have been observed within 30 minutes of heat shock. Thus, some m6A mRNAs may still be preferentially enriched in these granules for storage rather than being directly degraded. Overall, it would be interesting to understand the authors' position relative to previous studies of m6A during heat stress.

(d) Gene Ontology analysis based on the top 1000 PC1 genes shows an enrichment of GOs involved in post-translational protein modification more than GOs involved in cellular response to stress, which is highlighted by the authors and used as justification to study RNA transcriptional events upon heat shock. How do the authors think that GOs involved in post-translational protein modification may contribute to the observed data?

(e) Additionally, the authors first mention that there is no dramatic change in m6A levels upon heat shock, "subtle quantitative differences were apparent," but then mention a "systematic increase in m6A levels observed in heat stress". It is unclear to which systematic increase they are referring to. Are the authors referring to previous studies? It is confusing in the field what exactly is going on after heat stress. For instance, in some papers, a preferential increase of 5'UTR m6A has been proposed rather than a systematic and general increase.

Reviewer #2 (Public review):

Dierks et al. investigate the impact of m6A RNA modifications on the mRNA life cycle, exploring the links between transcription, cytoplasmic RNA degradation, and subcellular RNA localization. Using transcriptome-wide data and mechanistic modelling of RNA metabolism, the authors demonstrate that a simplified model of m6A primarily affecting cytoplasmic RNA stability is sufficient to explain the nuclear-cytoplasmic distribution of methylated RNAs and the dynamic changes in m6A levels upon perturbation. Based on multiple lines of evidence, they propose that passive mechanisms based on the restricted decay of methylated transcripts in the cytoplasm play a primary role in shaping condition-specific m6A patterns and m6A dynamics. The authors support their hypothesis with multiple large-scale datasets and targeted perturbation experiments. Overall, the authors present compelling evidence for their model which has the potential to explain and consolidate previous observations on different m6A functions, including m6A-mediated RNA export.

Reviewer #3 (Public review):

Summary:

This manuscript works with a hypothesis where the overall m6A methylation levels in cells are influenced by mRNA metabolism (sub-cellular localization and decay). The basic assumption is that m6A causes mRNA decay and this happens in the cytoplasm. They go on to experimentally test their model to confirm its predictions. This is confirmed by sub-cellular fractionation experiments which show high m6A levels in the nuclear RNA. Nuclear localized RNAs have higher methylation. Using a heat shock model, they demonstrate that RNAs with increased nuclear localization or transcription, are methylated at higher levels. Their overall argument is that changes in m6A levels are rather determined by passive processes that are influenced by RNA processing/metabolism. However, it should be considered that erasers have their roles under specific environments (early embryos or germline) and are not modelled by the cell culture systems used here.

Strengths:

This is a thought-provoking series of experiments that challenge the idea that active mechanisms of recruitment or erasure are major determinants for m6A distribution and levels.