Principles of mRNA targeting and regulation via Arabidopsis YTHDF proteins

Arribas-Hernández, Laura; Rennie, Sarah; Schon, Michael; Porcelli, Carlotta; Enugutti, Balaji; Andersson, Robin; Nodine, Michael; Brodersen, Peter


Post-transcriptional gene regulation dependent on N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A through a YTH domain. Here, we determine targets of the Arabidopsis YTH-domain protein ECT2, using a combination of two orthogonal methodologies: UV crosslinking and immunoprecipitation (CLIP) and the proximity-labeling method HyperTRIBE (targets of RNA binding proteins identified by editing), the latter of which was also used to determine targets for the YTH-domain protein ECT3. We further constructed Smart-seq2 libraries from isolated ECT2 mCherry-expressing protoplasted root cell populations with or without ECT2/3/4 function, comparing them to standard RNA-seq for DAG4 in roots and analysing the abundance effects and polyadenylation site usage on ECT2/3 mRNA targets. Overall, our data help to clarify the principles underlying mRNA recognition and regulation by ECT2 and other plant YTHDF proteins.