YTHDF1 promotes mRNA degradation via YTHDF1‐AGO2 interaction and phase separation

<jats:title>Abstract</jats:title><jats:sec><jats:title>Objectives</jats:title><jats:p>YTHDF1 is known as a m<jats:sup>6</jats:sup>A reader protein, and many researches of YTHDF1 focused on the regulation of mRNA translation efficiency. However, YTHDF1 is also related to RNA degradation, but how YTHDF1 regulates mRNA degradation is indefinite. Liquid‐liquid phase separation (LLPS) underlies the formation of membraneless compartments in mammal cells, and there are few reports focused on the correlation of RNA degradation with LLPS. In this research, we focused on the mechanism of YTHDF1 degraded mRNA through LLPS.</jats:p></jats:sec><jats:sec><jats:title>Materials and Methods</jats:title><jats:p>The CRISPR/Cas9 knock out system was used to establish the <jats:italic>YTHDF1</jats:italic> knock out (<jats:italic>YTHDF1</jats:italic>‐<jats:italic>KO</jats:italic>) cell lines (HEK293 and HeLa) and <jats:italic>METTL14</jats:italic> knock out (<jats:italic>METTL14</jats:italic>‐<jats:italic>KO</jats:italic>) cell line (HEK293). 4SU‐TT‐seq was used to check the half‐life changes of mRNAs. Actinomycin D and qPCR were used to test the half‐life changes of individual mRNA. RNA was stained with SYTO RNA‐select dye in wild type (WT) and <jats:italic>YTHDF1</jats:italic>‐<jats:italic>KO HeLa</jats:italic> cell lines. Co‐localization of YTHDF1 and AGO2 was identified by immunofluorescence. The interaction domain of YTHDF1 and AGO2 was identified by western blot. Phase separation of YTHDF1 was performed <jats:italic>in vitro</jats:italic> and <jats:italic>in vivo</jats:italic>. Fluorescence recovery after photobleaching (FRAP) was performed on droplets as an assessment of their liquidity.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>In this research, we found that deletion of <jats:italic>YTHDF1</jats:italic> led to massive RNA patches deposited in cytoplasm. The results of 4SU‐TT‐seq showed that deletion of <jats:italic>YTHDF1</jats:italic> would prolong the half‐life of mRNAs. Immunofluorescence data showed that YTHDF1 and AGO2 could co‐localize in P‐body, and Co‐IP results showed that YTHDF1 could interact with AGO2 through YT521‐B homology (YTH) domain. We confirmed that YTHDF1 could undergo phase separation <jats:italic>in vitro</jats:italic> and <jats:italic>in vivo</jats:italic>, and compared with AGO2, YTHDF1 was more important in P‐body formation. The FRAP results showed that liquid AGO2 droplets would convert to gel/solid when <jats:italic>YTHDF1</jats:italic> was deleted. As AGO2 plays important roles in miRISCs, we also found that miRNA‐mediate mRNA degradation is related to YTHDF1.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>YTHDF1 recruits AGO2 through the YTH domain. YTHDF1 degrades targeting mRNAs by promoting P‐body formation through LLPS. The deletion of <jats:italic>YTHDF1</jats:italic> causes the P‐body to change from liquid droplets to gel/solid droplets, and form AGO2/RNA patches, resulting in a degradation delay of mRNAs. These findings reveal a previously unrecognized crosstalk between YTHDF1 and AGO2, raising a new sight of mRNA post‐transcriptional regulation by YTHDF1.</jats:p></jats:sec>