FITC (green): newly synthesized protein CY3 (red): 5′-CY3-labeled siRNAs DAPI (blue): DNA. (H) Immunofluorescence analysis of nascent protein synthesis in control- and YTHDF3-deficient HeLa cells. 5′ UTRs, CDSs and 3 UTRs of mRNAs are individually binned into regions spanning 1% of their total length, and the percentage of YTHDF1 and YTHDF3 binding clusters that fall within each bin is determined. (G) Distribution of YTHDF1 and YTHDF3 binding clusters across the length of mRNA transcripts. (F) YTHDF1 and YTHDF3 binding motifs identfied by HOMER from PAR-CLIP clusters. The enriched terms were ranked by −log 10 ( P-value). (E) Gene ontology analysis of 2 945 mRNAs bound by both YTHDF1 and YTHDF3.
(D) Venn diagram of the overlapping mRNAs with binding clusters of YTHDF1 and YTHDF3 detected by PAR-CLIP-seq. The pull-down RNA products were labeled with biotin at 3′ end (Biotinylation Kit, Thermo) and then visualized by the Chemiluminescent nucleic acid detection module. (C) PAR-CLIP of YTHDF3 protein in control- and YTHDF1-depleted HeLa cells transfected with Myc-YTHDF3 plasmid. An enrichment map was constructed by using Cytoscape with the Enrichment Map plugin. 152 proteins were subjected to DAVID GO analysis. (B) Gene ontology and enrichment analysis of proteins that interact with both YTHDF1 and YTHDF3. Enriched 40S and 60S subunit proteins are highlighted in blue and red, respectively. (A) Scatter plot of proteins bound to YTHDF1 versus YTHDF3. The software itself is very basic but easy to figure out.YTHDF3 promotes translation by interacting with ribosomal proteins. You can even choose which country your IP appears to come from useful if a website blocks certain countries.
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