# The role of YTHDF phase separation in the regulation of m6A mRNA stability

> **NIH NIH F31** · WEILL MEDICAL COLL OF CORNELL UNIV · 2020 · $45,520

## Abstract

PROJECT SUMMARY
N6-methyladenosine (m6a) is the most abundant nucleotide modification in the transcriptome and has been
implicated in development and cancer. A major function of m6A is to reduce the stability of mRNAs. In the
cytoplasm, m6A is bound by the YTHDF proteins, which consist of two domains: a YTH domain, which binds
m6A, and a low complexity domain, rich in disorder-promoting amino acids. The low complexity domain allows
the YTHDF proteins to undergo `phase separation,' a phenomenon in which protein- and RNA-rich droplets
form in the cytoplasm. By forming multivalent interactions with mRNAs rich in m6A, YTHDF-mediated phase
separation allows the sequestration of m6A-mRNAs in phase-separated droplets in cells. However, it is not
known how the phase separation potential of the YTHDF proteins is controlled or if this process contributes to
m6A mRNA instability. Analysis of the low complexity domain of the YTHDF proteins reveals several prion-like
domains and putative phosphorylation sites. In this proposal, I seek to address the following key question: how
do the prion-like domains and phosphorylation of the YTHDFs control phase separation, and is phase
separation necessary for m6A mRNA degradation? To explore this, I will first determine how mutations in the
low complexity domain and at putative phosphorylation sites of YTHDF proteins affect in vitro phase separation
potential. These experiments will determine the importance of certain domains and phosphorylation sites in
enhancing and/or reducing the phase separation potential of the YTHDFs. Second, I will test how these
mutations affect YTHDF phase separation and localization in living cells. These experiments will show how
changes in in vitro phase separation potential affect cellular localization and phase separation potential of the
YTHDFs in cells. Third, I will determine the effect that these mutations in the YTHDFs have on the abundance
and stability of m6A mRNAs in cells lacking endogenous YTHDF protein expression. This will test if the
YTHDFs with altered phase separation potential and/or phosphorylation sites influence m6A mRNA decay.
Collectively, the proposed experiments will test if mutations that affect YTHDF phase separation potential
and/or localization in cells alters the stability of m6A mRNAs, and if this process can be regulated by YTHDF
phosphorylation. The outcomes of this study will substantially improve our understanding of the role that the
YTHDF proteins and phase separation play in influencing m6A mRNA stability.

## Key facts

- **NIH application ID:** 10068097
- **Project number:** 1F31CA254763-01
- **Recipient organization:** WEILL MEDICAL COLL OF CORNELL UNIV
- **Principal Investigator:** Ryan J Ries
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $45,520
- **Award type:** 1
- **Project period:** 2020-07-10 → 2023-07-09

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10068097

## Citation

> US National Institutes of Health, RePORTER application 10068097, The role of YTHDF phase separation in the regulation of m6A mRNA stability (1F31CA254763-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10068097. Licensed CC0.

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