Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2
- University of Copenhagen, Denmark
- University of Bielefeld, Germany
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 cross-linking 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.
Data availability
All sequencing data (iCLIP-seq, HyperTRIBE, mRNA-seq, small RNA-seq) have been deposited in the European Nucleotide Archive under accession code PRJEB44359.All code is available at GitHubhttps://github.com/sarah-ku/targets_arabidopsis
-
Principles of mRNA targeting and regulation via Arabidopsis YTHDF proteinsEuropean Nucleotide Archive, PRJEB44359.
Article and author information
Author details
Funding
H2020 European Research Council (ERC-2016-COG 726417)
- Peter Brodersen
Independent Research Fund Denmark (9040-00409B)
- Peter Brodersen
European Molecular Biology Organization (STF 7614)
- Laura Arribas-Hernández
Deutsche Forschungsgemeinschaft (STA653/14-1)
- Prof. Dr. Dorothee Staiger
H2020 European Research Council (638173)
- Robin Andersson
Independent Research Fund Denmark (6108-00038B)
- Robin Andersson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Arribas-Hernández et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,156
- views
-
- 571
- downloads
-
- 56
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2
eLife 10:e72375.
https://doi.org/10.7554/eLife.72375
Further reading
-
- Genetics and Genomics
Advances in single-cell sequencing technologies have provided novel insights into the dynamics of gene expression and cellular heterogeneity within tissues and have enabled the construction of transcriptomic cell atlases. However, linking anatomical information to transcriptomic data and positively identifying the cell types that correspond to gene expression clusters in single-cell sequencing data sets remains a challenge. We describe a straightforward genetic barcoding approach that takes advantage of the powerful genetic tools in Drosophila to allow in vivo tagging of defined cell populations. This method, called Targeted Genetically-Encoded Multiplexing (TaG-EM), involves inserting a DNA barcode just upstream of the polyadenylation site in a Gal4-inducible UAS-GFP construct so that the barcode sequence can be read out during single-cell sequencing, labeling a cell population of interest. By creating many such independently barcoded fly strains, TaG-EM enables positive identification of cell types in cell atlas projects, identification of multiplet droplets, and barcoding of experimental timepoints, conditions, and replicates. Furthermore, we demonstrate that TaG-EM barcodes can be read out using next-generation sequencing to facilitate population-scale behavioral measurements. Thus, TaG-EM has the potential to enable large-scale behavioral screens in addition to improving the ability to multiplex and reliably annotate single-cell transcriptomic experiments.
-
- Genetics and Genomics
RNAs in circulation carry sequence-specific regulatory information between cells in plant, animal, and host-pathogen systems. Such RNA can cross generational boundaries, as evidenced by somatic double-stranded RNA (dsRNA) in the nematode Caenorhabditis elegans silencing genes of matching sequence in progeny. Here we dissect the intergenerational path taken by dsRNA from parental circulation and discover that cytosolic import through the dsRNA importer SID-1 in the parental germline and/or developing progeny varies with developmental time and dsRNA substrates. Loss of SID-1 enhances initiation of heritable RNA silencing within the germline and causes changes in the expression of the sid-1-dependent gene sdg-1 that last for more than 100 generations after restoration of SID-1. The SDG-1 protein is enriched in perinuclear germ granules required for heritable RNA silencing but is expressed from a retrotransposon targeted by such silencing. This auto-inhibitory loop suggests how retrotransposons could persist by hosting genes that regulate their own silencing.
