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

Abstract

Gene regulation dependent on N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A through a YTH domain. The Arabidopsis YTH-domain protein ECT2 is thought to influence mRNA 3′-end formation via binding to URU(m6A)Y sites, an unexpected conclusion given that ECT2 functions require its m6A binding activity, and that RR(m6A)CH is the m6A consensus site in all eukaryotes. Here, we apply the orthogonal techniques individual nucleotide-resolution UV-crosslinking and immunoprecipitation (iCLIP) and HyperTRIBE to define high-quality target sets of the YTH-domain proteins ECT2 and ECT3. The results show that in vivo, ECT2 does in fact bind to RR(m6A)CH. URUAY and other pyrimidine-rich motifs are enriched around, but not at m6A-sites, reflecting a preference for N6-adenosine methylation of RRACH islands in pyrimidine-rich regions. Such regions may also be implicated in ECT2-binding. In particular, a series of properties unique to the URUAY motif suggest that URUAY-type sequences act as sites of competition between unknown RNA-binding proteins and the intrinsically disordered region of ECT2. We also show that the abundance of many ECT2/3 mRNA targets is decreased in meristematic cells devoid of ECT2/3/4-activity. In contrast, loss of ECT2/3/4 activity has no effect on polyadenylation site usage in ECT2/3 targets, consistent with the exclusive cytoplasmic localization of ECT2 observed by super-resolution confocal microscopy. Our study reconciles conflicting results between genetic observations on N6-adenosine methylation and ECT2/3/4 function on the one side, and ECT2 target identification on the other, and point to regulation of cytoplasmic mRNA function, including abundance, as a mechanism of plant YTHDF action.

Comparison of TRIBE and STAMP for identifying targets of RNA binding proteins in human andDrosophilacells

“…Importantly, this allows target identification in very small numbers of cells and neurons without requiring biochemistry. TRIBE has been useful to a number of other labs working in different fields and model organisms ranging from humans to malaria parasites (Liu et al 2019;Alizzi et al 2020;Nguyen et al 2020;Arribas-Hernández et al 2021;Cheng et al 2021;Singh et al 2021;van Leeuwen et al 2022).…”

Detection, regulation, and functions of RNA N6-methyladenosine modification in plants

“…Therefore, these proteins containing the YTH domain in Arabidopsis all have the potential to bind m 6 A ( Wang et al., 2014 , 2015 ). ECT2 has been demonstrated to bind m 6 A-containing RNA directly in vitro and in vivo ( Scutenaire et al., 2018 ; Wei et al., 2018 ), and the binding sites of ECT2 to mRNA contain the plant-specific motif URUAY ( Wei et al., 2018 ) and the canonical site RRACH ( Arribas-Hernández et al., 2021a , 2021b ). Further genetic experiments showed that the functions of ECT2 are redundant with those of ECT3 and ECT4, suggesting that ECT3 and ECT4 are also m 6 A readers ( Arribas-Hernandez et al., 2018 , 2020 ).…”

m6A‐mediated regulation of crop development and stress responses

“…However, we currently do not know how m 6 A machineries achieve selectivity toward certain motif sequences to accomplish m 6 A installation, removal and recognition. One likely scenario is that they may be localized to diverse sequence contexts through Regulation of m 6 A on mRNA metabolism in crops m 6 A possesses multiple regulatory effects on mRNA metabolism in Arabidopsis, such as mRNA stability (Anderson et al, 2018;Arribas-Hern andez et al, 2021b;Duan et al, 2017;Kramer et al, 2020;Shen et al, 2016;Wei et al, 2018), transcriptome integrity (Pontier et al, 2019), and alternative polyadenylation (Hou et al, 2021;Hu et al, 2021a;Parker et al, 2020;Song et al, 2021). Currently, substantial progresses have been made in deciphering the influence of m 6 A methylation on crop mRNA metabolism, mainly mRNA degradation and translation (Shao et al, 2021).…”