ALBA proteins facilitate cytoplasmic YTHDF-mediated reading of m<sup>6</sup>A in plants

Reichel, Marlene; Tankmar, Mathias Due; Rennie, Sarah; Arribas-Hernández, Laura; Lewinski, Martin; Köster, Tino; Wang, Naiqi; Millar, Anthony A.; Staiger, Dorothee; Brodersen, Peter

doi:10.1101/2024.06.06.597704

2024

DOI: 10.1101/2024.06.06.597704

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ALBA proteins facilitate cytoplasmic YTHDF-mediated reading of m⁶A in plants

Marlene Reichel,

Mathias Due Tankmar,

Sarah Rennie

et al.

Abstract: N6-methyladenosine (m6A) exerts many of its regulatory effects on eukaryotic mRNAs by recruiting cytoplasmic YT521-B homology domain family (YTHDF) proteins. Here, we show that in Arabidopsis, the interaction between m6A and the major YTHDF protein ECT2 also involves the mRNA-binding ALBA protein family. ALBA and YTHDF proteins physically associate via a deeply conserved short linear motif in the intrinsically disordered region of YTHDF proteins, their mRNA target sets overlap, and ALBA4 binding sites are juxt… Show more

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ECT2 peptide sequences outside the YTH domain regulate its m ⁶ A-RNA binding

Seigneurin-Berny,

Karczewski,

Delaforge

et al. 2024

RNA Biology

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The m 6 A epitranscriptomic mark is the most abundant and widespread internal RNA chemical modification, which through the control of RNA acts as an important factor of eukaryote reproduction, growth, morphogenesis and stress response. The main m 6 A readers constitute a super family of proteins with hundreds of members that share a so-called YTH RNA binding domain. The majority of YTH proteins carry no obvious additional domain except for an Intrinsically Disordered Region (IDR). In Arabidopsis thaliana IDRs are important for the functional specialization among the different YTH proteins, known as Evolutionarily Conserved C -Terminal region, ECT 1 to 12. Here by studying the ECT2 protein and using an in vitro biochemical characterization, we show that full-length ECT2 and its YTH domain alone have a distinct ability to bind m 6 A, conversely to previously characterized YTH readers. We identify peptide regions outside of ECT2 YTH domain, in the N-terminal IDR, that regulate its binding to m 6 A-methylated RNA. Furthermore, we show that the selectivity of ECT2 binding for m 6 A is enhanced by a high uridine content within its neighbouring sequence, where ECT2 N-terminal IDR is believed to contact the target RNA in vivo . Finally, we also identify small structural elements, located next to ECT2 YTH domain and conserved in a large set of YTH proteins, that enhance its binding to m 6 A-methylated RNA. We propose from these findings that some of these regulatory regions are not limited to ECT2 or YTH readers of flowering plants but may be widespread among eukaryotic YTH readers.

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ECT2 peptide sequences outside the YTH domain regulate its m ⁶ A-RNA binding

Seigneurin-Berny,

Karczewski,

Delaforge

et al. 2024

RNA Biology

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Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture

Reichel,

Schmidt,

Rettel

et al. 2024

BMC Plant Biol

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Background The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA- binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oligo(dT) beads and the RNA-binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce. Results Here, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seedlings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5’untranslated region (UTR). The efficiency of RNA capture was greatly improved by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5’UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction. Conclusion We adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up scaling the protocol should be applicable for less abundant transcripts.

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ALBA proteins facilitate cytoplasmic YTHDF-mediated reading of m⁶A in plants

Cited by 2 publications

References 86 publications

ECT2 peptide sequences outside the YTH domain regulate its m ⁶ A-RNA binding

ECT2 peptide sequences outside the YTH domain regulate its m ⁶ A-RNA binding

Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture

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ALBA proteins facilitate cytoplasmic YTHDF-mediated reading of m6A in plants

Cited by 2 publications

References 86 publications

ECT2 peptide sequences outside the YTH domain regulate its m 6 A-RNA binding

ECT2 peptide sequences outside the YTH domain regulate its m 6 A-RNA binding

Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture

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ALBA proteins facilitate cytoplasmic YTHDF-mediated reading of m⁶A in plants

ECT2 peptide sequences outside the YTH domain regulate its m ⁶ A-RNA binding

ECT2 peptide sequences outside the YTH domain regulate its m ⁶ A-RNA binding