Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2.

Laura Arribas-Hernández,Sarah Rennie,Dorothee Staiger,Martin Lewinski,Carlotta Porcelli,Robin Andersson,Tino Köster,Peter Brodersen

doi:10.7554/elife.72375

Laura Arribas-Hernández, Sarah Rennie + Show 6 more

Open Access

https://doi.org/10.7554/elife.72375

Copy DOI

Journal: eLife	Publication Date: Sep 30, 2021
Citations: 42	License type: CC BY 4.0

Affiliation: Bielefeld University, University of Copenhagen

Abstract

Specific recognition of N6-methyladenosine (m6A) 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(m6A)Y sites, yet RR(m6A)CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A-binding activity. Here, we apply iCLIP (individual nucleotide resolution crosslinking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) 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(m6A)CH. Pyrimidine-rich motifs are enriched around, but not at m6A 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 m6A 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 m6A-YTH function in plants and reveals new insight into the mode of RNA recognition by YTH domain-containing proteins.

Highlights

N6-methyladenosine (m6A) is the most abundant modified nucleotide in eukaryotic mRNA bodies
To identify positions with significantly higher editing rates in ECT2-FLAG-ADAR lines compared to controls, we developed a new approach to detect differential editing (Figure 1B) that will be described in detail in a subsequent report
The ADAR expression level (TPMs) correlated strongly with editing proportions among ECT2-FLAG-ADAR lines (Figure 1G, Figure 1—figure supplement 1H), and editing proportions were higher for target mRNAs that are co-expressed with ECT2 in a large percentage of cells according to single-cell RNA-seq (Denyer et al 2019) (Figure 1H), lending further support to the conclusion that the observed editing is ADAR-specific and driven to target mRNAs by ECT2

Summary

Introduction

N6-methyladenosine (m6A) is the most abundant modified nucleotide in eukaryotic mRNA bodies. It is required for embryonic development and stem cell differentiation in several animals and plants (Zhong et al 2008; Batista et al 2014; Ping et al 2014; Geula et al 2015; Zhang et al 2017) and for the control of the meiotic program in yeast (Shah and Clancy 1992; Clancy et al 2002; Agarwala et al 2012). The characteristic motif and gene-body location is not detected in organisms that lack METTL3/METTL14 homologs, such as the nematode

Results

Discussion

Conclusion