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

N6-methyladenosine (m 6 A) 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 thaliana , the interaction between m 6 A 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 and their mRNA target sets overlap, with ALBA4 binding sites being juxtaposed to m 6 A sites. These binding sites correspond to pyrimidine-rich elements previously found to be important for m 6 A binding to ECT2. Accordingly, both the biological functions of ECT2, and its binding to m 6 A targets in vivo, require ALBA association. Our results introduce the YTHDF-ALBA complex as the functional cytoplasmic m 6 A-reader in Arabidopsis , and define a molecular foundation for the concept of facilitated m 6 A reading, which increases the potential for combinatorial control of biological m 6 A effects. Synopsis YTHDF proteins read the m 6 A-code on eukaryotic mRNA in the cytoplasm. This report shows that in Arabidopsis , m 6 A is read not simply by YTHDF proteins, but by their complexes with the ancient RNA-binding ALBA proteins. A short linear motif (SLiM), deeply conserved in the intrinsically disordered region of plant m 6 A-binding YTHDF proteins, is required for their RNA-binding in vivo. This SLiM is necessary for physical association with the ALBA family of RNA-binding proteins. The ALBA protein ALBA4 binds to pyrimidine-rich elements juxtaposed to m 6 A in mRNA targets of the major YTHDF proteins ECT2 and ECT3. ALBA proteins are required for biological effects of m 6 A and for ECT2-binding to m 6 A targets in vivo. The plant m 6 A reader consists of a complex between ECT2/3 and the RNA-binding protein ALBA.