A YTHDF-PABP axis is required for m⁶A-mediated organogenesis in plants

Abstract

N6-methyladenosine (m6A) in mRNA is key to eukaryotic gene regulation. Many m6A functions involve specialized RNA-binding proteins that recognize m6A via a YT521-B Homology (YTH) domain. YTH domain proteins contain long intrinsically disordered regions (IDRs) that may mediate phase separation and interaction with protein partners, but whose precise biochemical functions remain largely unknown. The Arabidopsis thaliana YTH domain proteins ECT2, ECT3 and ECT4 accelerate organogenesis through stimulation of cell division in organ primordia. Here, we focus on ECT2 to reveal molecular underpinnings of this function of ECT2/3/4. We show that stimulation of leaf formation requires the long N-terminal IDR, and we identify two short IDR-elements required for ECT2-mediated organogenesis. Of these two, a tyrosine-rich 19-amino acid region is necessary for binding to a small subset of proteins that includes the major cytoplasmic poly(A)-binding proteins PAB2, PAB4 and PAB8. Remarkably, overexpression of PAB4 in leaf primordia partially rescues the delayed leaf formation in ect2 ect3 ect4 mutants, suggesting that the ECT2-PAB2/4/8 interaction on target mRNAs of organogenesis-related genes may overcome limiting PAB concentrations in primordial cells.

Insights into the conservation and diversification of the molecular functions of YTHDF proteins

“…Second, the complementation analyses of the reverse chimeras show that the IDRs of Ath ECTs with divergent functions contribute substantially to their different molecular properties. This finding implies a key role of IDRs in defining the molecular activities of YTHDF proteins, and is in line with the conclusions of a recent study of mammalian YTHDFs [ 48 ], with the functional importance of the IDR of Hs YTHDF2 inferred from early tethering studies [ 9 ], and with the isolation of IDR-dependent interactors proposed to be important for Hs YTHDF2 and Ath ECT2 function [ 28 – 30 , 96 , 98 ]. Third, the ability to direct correct leaf formation correlates with a combination of two biophysical properties of the IDRs of plant YTHDFs: a clear propensity to phase separate, and an organization of charged residues that creates an electric dipole.…”

“…We were able to identify two biophysical properties of the IDR of ECT1—its stronger predicted propensity to phase separate and its charge distribution—that are distinct from the ECT2/3/4/5/6/7/8/10 group, but note that additional changes, e.g. in SLiMs mediating interaction with regulatory factors, may also contribute to the different functionality [ 98 ].…”

Plant YTHDF proteins are direct effectors of antiviral immunity against an N6‐methyladenosine‐containing RNA virus

“…These deletions were originally designed to identify regions of importance for the developmental function of ECT2. Of six ~50–80 aa deletions, four had no effect on leaf formation (preprint: Tankmar et al, 2023 ). Since Tyr residues may promote IDR‐driven phase separation (Wang et al , 2018 ; Martin et al , 2020 ; Bremer et al , 2022 ), we selected the ECT2 ΔN5 mutant carrying a deletion in a Tyr‐rich region present in many ECTs for analysis of AMV resistance (Figs 6A , and EV5A and B ).…”

“…Cloning and line selection of ECT2 ΔN5 ‐mCherry was performed by USER cloning and agrobacterium‐mediated transformation as described in Tankmar (preprint: Tankmar et al , 2023 ). In brief, USER‐compatible primers (LA336, MT5, MT6, LA337) were used to amplify fragments from ECT2Pro:ECT2gDNA‐mCherry:ECT2Ter ; a construct previously generated by Arribas‐Hernández et al ( 2018 ).…”

“…Tankmar et al, 2023). Since Tyr residues may promote IDR-driven phase separation(Wang et al, 2018;Martin et al, 2020;Bremer et al, 2022), we selected the ECT2 DN5 mutant carrying a deletion in a Tyr-rich region present in many ECTs for analysis of AMV resistance(Figs 6A, and EV5A and B).…”