Selective Recognition of PTRE1 Transcripts Mediated by Protein-protein Interaction between the RNA m6A Reader ECT2 and PTRE1.

ABSTRACT

N6-methyladenosine (m6A) is a prevalent internal post-transcriptional modification in eukaryotic RNAs, and its function is executed by m6A-binding proteins known as "readers". Our previous research revealed that the Arabidopsis m6A reader ECT2 positively regulates transcript levels of proteasome regulator PTRE1 and several 20S proteasome subunits, enhancing 26S proteasome activity. However, the mechanism of selective recognition of m6A targets by these readers like ECT2 remains unclear. In this study, we further demonstrate that ECT2 physically interacts with PTRE1 and several 20S proteasome subunits. This interaction occurs on the ribosome and involves the N-terminus of PTRE1, suggesting that ECT2 might bind to the nascent PTRE1 polypeptide. Deletion of ECT2's protein interaction domain impairs its ability to bind mRNA, while mutations in the m6A RNA binding site do not affect such protein-protein interaction. Furthermore, introducing a novel protein-binding domain into ECT2 elevates transcript levels of the proteins interacting with this domain. Our findings suggest that interaction with PTRE1 protein enhances ECT2's binding to PTRE1 m6A mRNAs during translation, thereby regulating PTRE1 mRNA levels.