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Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae
Published on: February 16, 2015
YTHDF1 promotes mRNA degradation via YTHDF1-AGO2 interaction and phase separation
Objectives:
YTHDF1 is known as a m6 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.
Materials And Methods:
The CRISPR/Cas9 knock out system was used to establish the YTHDF1 knock out (YTHDF1-KO) cell lines (HEK293 and HeLa) and METTL14 knock out (METTL14-KO) 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 YTHDF1-KO HeLa 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 in vitro and in vivo. Fluorescence recovery after photobleaching (FRAP) was performed on droplets as an assessment of their liquidity.
Results:
In this research, we found that deletion of YTHDF1 led to massive RNA patches deposited in cytoplasm. The results of 4SU-TT-seq showed that deletion of YTHDF1 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 in vitro and in vivo, 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 YTHDF1 was deleted. As AGO2 plays important roles in miRISCs, we also found that miRNA-mediate mRNA degradation is related to YTHDF1.
Conclusions:
YTHDF1 recruits AGO2 through the YTH domain. YTHDF1 degrades targeting mRNAs by promoting P-body formation through LLPS. The deletion of YTHDF1 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.
Insights
YTHDF1 protein recruits AGO2, promoting P-body formation via liquid-liquid phase separation (LLPS) to degrade mRNA. Loss of YTHDF1 alters P-bodies, delaying mRNA degradation and revealing new mRNA regulation insights.
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.
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