Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2
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Evaluation Summary:
Gene regulation triggered by recognising N6-methyladenosine (m6A) by RNA-binding proteins emerged as a fundamental process for development and adaptation in most eukaryotes. This paper is of interest to scientists within the field of post-transcriptional gene regulation and RNA biology. In a deep and thorough analysis, the authors resolved previously contradictory reports of sequence motifs associated with methylation sites in messenger RNAs and shed light on the proteins that recognise this modification.
(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/10.1101/2021.08.01.454660v1
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Abstract
Specific recognition of N6 -methyladenosine (m 6 A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH domain protein ECT2 is thought to bind to mRNA at URU(m 6 A)Y sites, yet RR(m 6 A)CH is the canonical m 6 A consensus site in all eukaryotes and ECT2 functions require m 6 A-binding activity. Here, we apply iCLIP ( i ndividual nucleotide resolution c ross l inking and i mmuno p recipitation) and HyperTRIBE ( t argets of R NA-binding proteins i dentified b y e diting) to define high-quality target sets of ECT2 and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m 6 A)CH. Pyrimidine-rich motifs are enriched around, but not at m 6 A sites, reflecting a preference for N6 -adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m 6 A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m 6 A-YTH function in plants and reveals new insight into the mode of RNA recognition by YTH domain-containing proteins.
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Evaluation Summary:
Gene regulation triggered by recognising N6-methyladenosine (m6A) by RNA-binding proteins emerged as a fundamental process for development and adaptation in most eukaryotes. This paper is of interest to scientists within the field of post-transcriptional gene regulation and RNA biology. In a deep and thorough analysis, the authors resolved previously contradictory reports of sequence motifs associated with methylation sites in messenger RNAs and shed light on the proteins that recognise this modification.
(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.or…
Evaluation Summary:
Gene regulation triggered by recognising N6-methyladenosine (m6A) by RNA-binding proteins emerged as a fundamental process for development and adaptation in most eukaryotes. This paper is of interest to scientists within the field of post-transcriptional gene regulation and RNA biology. In a deep and thorough analysis, the authors resolved previously contradictory reports of sequence motifs associated with methylation sites in messenger RNAs and shed light on the proteins that recognise this modification.
(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/10.1101/2021.08.01.454660v1
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Reviewer #1 (Public Review):
In plants, as in many eukaryotic organisms, the m6A modification of RNA molecules plays critical roles in development, stress response and adaptation. This modification alters the RNA molecule properties changing their function, folding, and how they are post-transcriptionally processed and modified. RNA-binding proteins containing YTH domains recognize these modified nucleotides. In this manuscript, the authors used a combination of iCLIP (individual-nucleotide resolution crosslinking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to identify mRNA targeted by the YTH protein ECT2. In addition, the same data was used to define the sequence motifs bound and around ECT2 crosslink sites.
The authors concluded that RRACH sites are the most enriched motifs at m6A …
Reviewer #1 (Public Review):
In plants, as in many eukaryotic organisms, the m6A modification of RNA molecules plays critical roles in development, stress response and adaptation. This modification alters the RNA molecule properties changing their function, folding, and how they are post-transcriptionally processed and modified. RNA-binding proteins containing YTH domains recognize these modified nucleotides. In this manuscript, the authors used a combination of iCLIP (individual-nucleotide resolution crosslinking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to identify mRNA targeted by the YTH protein ECT2. In addition, the same data was used to define the sequence motifs bound and around ECT2 crosslink sites.
The authors concluded that RRACH sites are the most enriched motifs at m6A sites in Arabidopsis and that ECT2 binds to these sites. It also reports that the intrinsically disordered region of ECT2 participates in RNA binding through U-rich elements abundant upstream of m6A-sites. Finally, they reported that URUAY motifs, previously reported as targets of ECT2 are not the preferential target sites and even disfavors ECT2 binding.
The conclusion achieved in the paper is of general interest to the community studying post-transcriptional gene regulation as the datasets generated and analyzed here. The experimental data provided is solid and fully support the conclusions. A companion paper provides the missing information regarding specificity by comparing ECT2 and ECT3.
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Reviewer #2 (Public Review):
Most eukaryotes can contain N6-methyladenosine (m6A) in a significant proportion of their mRNAs, and in both plants and animals m6A is required for normal developmental programmes. The modification is usually found towards the 3' end of transcripts within the sequence RR(m6A)CH (R=G/A, H=A/C/U). YTH-domain proteins of plants and animals have a binding pocket that accepts the m6A, and binding to methylated transcripts by such proteins is required to bring about many of the developmental programmes associated with the requirement for m6A. However, previous reports have suggested that one of the major Arabidopsis YTH proteins (ECT2) actually binds the sequence URU(m6A)Y. The work reported here uses UV cross linking of a tagged ECT2 as well as A to inosine editing imparted by an ECT2-ADAR fusion, to identify …
Reviewer #2 (Public Review):
Most eukaryotes can contain N6-methyladenosine (m6A) in a significant proportion of their mRNAs, and in both plants and animals m6A is required for normal developmental programmes. The modification is usually found towards the 3' end of transcripts within the sequence RR(m6A)CH (R=G/A, H=A/C/U). YTH-domain proteins of plants and animals have a binding pocket that accepts the m6A, and binding to methylated transcripts by such proteins is required to bring about many of the developmental programmes associated with the requirement for m6A. However, previous reports have suggested that one of the major Arabidopsis YTH proteins (ECT2) actually binds the sequence URU(m6A)Y. The work reported here uses UV cross linking of a tagged ECT2 as well as A to inosine editing imparted by an ECT2-ADAR fusion, to identify transcripts that are bound by ECT2 and shows that these correlate strongly with sites mRNAs previously identified as containing m6A. Thus supporting ECT2 as a bona fide m6A reader. In addition, the use of UV crosslinking (iCLIP) suggests that ECT2 binds to m6A predominantly in the DR(m6A)CH sequence context in vivo, consistent with major methylation consensus of eukaryotes. The authors suggest that the earlier claim for URUAY as the target site might have been due to the use of formaldehyde for crosslinking (which will promote protein-protein as well as protein-RNA crosslinks). As well as providing a detailed list of ECT2 target transcripts, and showing that this largely overlaps with previously identified m6A containing mRNAs, the authors also undertook a detailed study of other sequence motifs associated with m6A and ECT2 binding. The key conclusions from this are that pyrimidine-rich motifs are enriched around, but not at m6A-sites, which likely reflects a preference for such motifs in the broader recognition of mRNAs by components of the enzyme complex that carries out the methylation. In particular, oligo-U and UNUNU motifs are frequently found upstream of the DR(m6A)CH site and evidence is presented that these can become UV crosslinked to intrinsically disordered region (IDR) N-terminal of ECT2. The relevance and importance of this binding by the IDR remains to be determined, but the suggestion that it helps stabilise binding to m6A containing transcripts is a reasonable hypothesis.
While the authors conclude that URUAY is not the main binding site for ECT2, where it is present close to a DR(m6A)CH site, it could become cross linked to ECT2. However, URUAY was more enriched proximal to previously identified DR(m6A)CH that were not bound by ECT2, which the authors interpret as indicating that another RNA binding protein is competing for such sites.
Questions raised
The current report does a thorough job of identifying transcripts that are bound by ECT2. However, plants have a large family of YTH proteins, indeed, Arabidopsis has 11 of the YTHDF type of which ECT2 is a member, and these fall into four sub-groups. It remains an open question whether all of these family members are binding the same set of transcripts, either competing with each other or acting redundantly. If the different ECT proteins are competing with each other, it will be important to determine whether the binding of different ECTs brings about different outcomes and if so whether they have different affinities for the methylated targets (or whether their binding can be moderated eg by environmental conditions). Answering these questions will likely shed light on the complexity of m6A post-transcriptional regulation.
The suggestion that there is a protein that binds to URUAY sites and competes or displaces ECT2 is intriguing and will require further experiments to verify and hopefully identify. Whether such a protein is recognising just the URUAY motif or is binding this motif in the context of a nearby DR(m6A)CH site is also an open question. However, in the latter case, it might be that such a competitor is one of the other ECT family members.
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