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

Area of Science:

• Molecular Biology
• RNA Biology
• Cell Biology

Background:

• YTHDF1 (YTH domain-containing family protein 1) is an m6A reader protein primarily studied for its role in mRNA translation efficiency.
• While YTHDF1's involvement in RNA degradation is recognized, the precise mechanisms remain unclear.
• Liquid-liquid phase separation (LLPS) is crucial for forming membraneless cellular compartments, yet its direct link to RNA degradation is under-explored.

Purpose of the Study:

• To elucidate the mechanism by which YTHDF1 mediates mRNA degradation through LLPS.
• To investigate the interplay between YTHDF1, AGO2, and P-body dynamics in post-transcriptional gene regulation.

Main Methods:

• CRISPR/Cas9 gene editing to create YTHDF1 and METTL14 knockout cell lines (HEK293, HeLa).
• 4SU-TT-seq and Actinomycin D/qPCR to assess mRNA half-life.
• Immunofluorescence and Co-immunoprecipitation (Co-IP) to study protein localization and interactions.
• In vitro and in vivo phase separation assays, and Fluorescence Recovery After Photobleaching (FRAP) to analyze droplet dynamics.

Main Results:

• YTHDF1 knockout led to cytoplasmic RNA patch accumulation and prolonged mRNA half-life.
• YTHDF1 co-localizes with AGO2 in P-bodies and interacts via its YTH domain.
• YTHDF1 undergoes LLPS in vitro and in vivo, playing a critical role in P-body formation.
• YTHDF1 deletion shifted AGO2 droplets from liquid to gel/solid states, impacting miRNA-mediated mRNA degradation.

Conclusions:

• YTHDF1 recruits AGO2 through its YTH domain, driving P-body formation via LLPS to facilitate mRNA degradation.
• The absence of YTHDF1 transitions P-bodies to a less dynamic state, causing RNA patches and delayed mRNA decay.
• This study uncovers a novel interaction between YTHDF1 and AGO2, offering new perspectives on YTHDF1's role in mRNA post-transcriptional regulation.