YTHDF proteins show similar binding to all m6A sites. (A) Shown is a Venn diagram depicting the highly selective nature of YTHDF paralog binding to different transcripts. This image, using data taken from Shi et al. (2017), shows the previous view that some transcripts are uniquely bound by YTHDF1, YTHDF2, or YTHDF3, while others are bound to two or three of the paralogs. This model implies that the function of each m6A site will depend on the YTHDF protein that binds to it. (B) Shown is a quantification of read length size from the PAR-CLIP data that identified YTHDF-binding sites in the HIV genome (Tirumuru et al. 2016). The PAR-CLIP data were comprised of extremely short reads, which cannot be reliably mapped to the transcriptome, and which explained why this study initially reported highly different binding patterns of the YTHDF proteins. (C) Schematic representation of the problem with using cutoff thresholds to call peaks. Shown are two examples of CLIP data sets in which three peaks are clearly seen. When a threshold is applied, some peaks fall below the threshold while others are above the threshold. However, qualitatively the binding properties appear essentially identical. If the threshold is lowered slightly, different peaks are called resulting in a different conclusion, that is, that the patterns of binding in the two data sets are identical. This highlights how threshold cutoffs can give the misleading conclusion that specific m6A sites are only bound by YTHDF1 (the middle site), and certain sites are only bound by YTHDF (right site). (D) Reexamination of YTHDF1 and YTHDF2 binding properties using a correlation analysis. In this approach, every m6A site in the transcriptome was analyzed, and the read counts from a YTHDF1 CLIP data set and a YTHDF2 CLIP data set were used to determine the position on the scatterplot. As can be seen, a strongly linear correlation is seen, demonstrating that every m6A site exhibits highly correlated levels of YTHDF1 and YTHDF2 binding. The dashed circle shows the region in which m6A sites would be located if they selectively bound YTHDF2 or YTHDF1. These sites were not found, demonstrating that there are no YTHDF1-unique or YTHDF2-unique sites. (E) Shown is CLIP data from HEK293 cells for YTHDF1, YTHDF2, and YTHDF3 on two transcripts proposed to be a YTHDF1-unique (top) and YTHDF2-unique transcript (bottom). As can be seen, the overall pattern of binding is essentially the same. Shown in red is the miCLIP data set which shows the location of m6A peaks, with sites indicated as red dots (taken from Zaccara and Jaffrey 2020).
