Single-molecule analysis of RNA:protein interactions reveals sequential binding of different YTHDF proteins to the same mRNA molecules [TRIBE_STAMP]

N6-methyladenosine (m6A) plays critical roles in gene expression control by recruiting the cytoplasmic reader proteins YTHDF1,2 and 3. Recently, the function of YTHDF proteins and whether they bind to shared or distinct sets of methylated mRNAs in cells has been the subject of debate. Here, we developed TRIBE-STAMP, an approach for single-molecule detection of the target RNAs of two RNA binding proteins simultaneously in cells. Applying TRIBE-STAMP to the YTHDF proteins revealed that most target mRNAs are shared among the three YTHDF proteins. Surprisingly, we also found that individual mRNA molecules can be bound by more than one YTHDF protein throughout their lifetime. Furthermore, we show that YTHDF proteins bind sequentially to target methylated mRNAs, with YTHDF1 binding before YTHDF2 or YTHDF3. Our data reveal shared molecular interactions among the YTHDF proteins and support a model in which YTHDF1 and YTHDF3 are unlikely to promote rapid mRNA decay. YTHDF1, YTHDF2 and YTHDF3 TRIBE-STAMP in HEK293T cells. YTHDF-ADAR expressing plasmids were transfected in cells stably expressing YTHDF-APOBEC1 proteins for 24h. Total RNA was extracted, and libraries prepared in triplicates for cells transfected with YTHDF1- or YTHDF2-ADAR or in duplicates for cells transfected with YTHDF3-ADAR. cDNA was sequenced in paired end mode using read lengths of 150bp and 250bp. For identification of editing sites by each protein, cells transfected with plasmids expressing APOBEC1 or ADAR alone were used as controls.