Technical problems with YTHDF1 knockdown accounts for the putative translation-promoting effect of YTHDF1. (A) The YTHDF paralogs exhibit similar disordered regions, prion-like regions, net charge, and hydrophobicity along the length of each protein. The disordered region score (green) was calculated using D2P2. This algorithm utilizes multiple different disordered protein prediction tools to generate scores for disordered regions (Oates et al. 2013). PLAAC (Lancaster et al. 2014) was used to calculate the prion-like likelihood ratio. A sliding window of 10 amino acids was used to calculate net charge per residue, probability of charged residues, and hydrophobicity. The plots were generated by CIDER (Holehouse et al. 2017) and ProtScale (ExPASy). All physiochemical parameters are similar along the length of all YTHDF paralogs, consistent with the idea that the low-complexity domains have similar properties and functions. (Image taken from Zaccara and Jaffrey 2020.) (B) We downloaded the data sets listing the ribosome-protected fragment number for each gene in each YTHDF1 replicate from Wang et al. (2015). We also downloaded the data for the matched control siRNA. In the case of Replicate 2, YTHDF1 expression is very low, consistent with knockdown. However, in Replicate 1, the expression of YTHDF1 is much higher than the control expression, which is inconsistent with a knockdown. Instead, this is more consistent with an overexpression or some other type of artifact. Because of this problem with Replicate 1, it was analyzed separately below. (C) Reexamination of the effect of YTHDF1 knockdown for each replicate separately. Because we could not confirm that Replicate 1 was a knockdown, we analyzed Replicate 1 and Replicate 2 separately. In the case of Replicate 1, we could see a clear decrease in translation, as measured by the number of ribosome-protected fragments for each gene which contained high levels of m6A. However, this replicate does not appear to be a knockdown because it has high levels of YTHDF1. If this is an overexpression, the reduced translation may represent YTHDF1 aggregates which trap m6A-containing mRNA. In the case of Replicate 2, which is a clear knockdown as seen above, there is no effect on the number of ribosome-protected fragments for highly methylated RNAs compared to nonmethylated RNAs. Thus, the original data set does not support the idea that YTHDF1 promotes the translation of m6A-containing mRNA based on Replicate 2, which is the only valid datable replicate from this study.
