The mechanism underlying redundant functions of the YTHDF proteins

Abstract

The YTH N⁶-methyladenosine RNA binding proteins (YTHDFs) mediate the functional effects of N⁶-methyladenosine (m⁶A) on RNA. Recently, a report proposed that all YTHDFs work redundantly to facilitate RNA decay, raising questions about the exact functions of individual YTHDFs, especially YTHDF1 and YTHDF2. We show that YTHDF1 and YTHDF2 differ in their low-complexity domains (LCDs) and exhibit different behaviors in condensate formation and subsequent physiological functions. Biologically, we also find that the global stabilization of RNA after depletion of all YTHDFs is driven by increased P-body formation and is not strictly m⁶A dependent.

Keywords: P-body; Translation control; YTHDF1; YTHDF2; YTHDF3; m6A.

Fig. 1

YTHDF1 and YTHDF2 have different molecular functions. a UpSet plots comparing transcripts analyzed with m⁶A-centric and RBP target-centric approaches. b Venn diagram showing RNA analyzed by Zaccara et al. and RNA analyzed in this work. c Cumulative plots showing changes in RNA translation efficiency (TE, left) and abundance (right) after YTHDF1 knockdown using data from Zaccara et al. P values were determined by a Mann-Whitney-Wilcoxon test. d Cumulative plots showing changes in RNA translation efficiency (TE, left) and abundance (right) after YTHDF2 knockdown using data from Zaccara et al. P values were determined by a Mann-Whitney-Wilcoxon test. e Sequence alignment of YTHDF proteins showing differences in the low complexity domain (LCD) region. Conservation scores were calculated by Jalview [22]. f Heatmaps showing the similarity scores calculated by the BLOSUM62 algorithm of YTHDF LCDs (left) and full-length proteins (right). g Higher-order structures formed by LCDs of YTHDFs under an electron microscopy (EM). Scale bar: 200 nm. h Cytoplasmic RNP granule protein localization map of the cell generated by t-distributed stochastic neighbor embedding (t-SNE) from the CELL MAP project [23]. YTHDF1 and eIFs are delineated in red, YTHDF2 and CNOTs are delineated in blue, and YTHDF3 is delineated in yellow. For boxplots, the center line represents the median, the box limits show the upper and lower quartiles, and whiskers represent 1–99%. P values were determined by a Mann-Whitney-Wilcoxon test

Fig. 2

Increased P-body formation accounts for the global RNA stabilization after YTHDF1-3 triple knockdown. a Boxplots showing changes in RNA abundance after single knockdown or triple knockdown of YTHDFs in HeLa cells. Gray boxes, non-targets (NT) in each analysis (for siDF1, non-DF1 bound transcripts; for siDF2, non-DF2 bound transcripts; for siDF3, non-DF3 bound transcripts; for siDF1-3, non-methylated transcripts). Colored boxes, YTHDF1 targets (brown), YTHDF2 targets (red), YTHDF3 targets (blue), and m⁶A modified transcripts (purple). b Fluorescence microscopy analysis of P-body numbers after single knockdown or triple knockdown of YTHDFs in HeLa cells. Numbers of DCP1A foci per cell were quantified with CellProfiler 3.0. Number of cells analyzed: siCtrl (32), siMETTL3 (49), siDF1 (57), siDF2 (56), siDF3 (46), siDF1/2/3 (41). c Boxplots showing changes in RNA abundance after triple knockdown of YTHDFs, grouped by both m⁶A modification status and P-body enrichment (depleted: “Depl”, enriched: “Enr”).Red boxes denote m⁶A methylated transcripts and gray boxes denote unmethylated transcripts. d Scatter plots showing enrichment of m⁶A-modified transcripts in the P-body enriched pool. Top: EDC3 dataset, bottom: EDC4 dataset. Red dots denote m⁶A methylated transcripts and gray dots denote unmethylated transcripts. e Boxplots showing changes in RNA abundance after DDX6 knockdown in control (left) or YTHDF1-3 (right) knockdown HeLa cells grouped by both m⁶A modification status and P-body enrichment in WT cells (depleted: “Depl”, enriched: “Enr”). Red boxes denote m⁶A methylated transcripts and gray boxes denote unmethylated transcripts. f Boxplots showing changes in RNA abundance after DDX6 overexpression in control (left) or YTHDF1-3 (right) knockdown HeLa cells grouped by both m⁶A modification status and P-body enrichment in WT cells (depleted: “Depl”, enriched: “Enr”). Red boxes denote m⁶A methylated transcripts and gray boxes denote unmethylated transcripts. For boxplots, the center line represents the median, the box limits show the upper and lower quartiles, and whiskers represent 1.5 × interquartile range. P values were determined by a Mann-Whitney-Wilcoxon test