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Fragment Ligands of the m⁶A-RNA Reader YTHDF2.

ABSTRACT: We report 17 small-molecule ligands that compete with N6-methyladenosine (m⁶A) for binding to the m⁶A-reader domain of YTHDF2 (YT521-B homology domain family 2). We determined their binding mode at high resolution by X-ray crystallography and quantified their affinity by a fluorescence-based binding assay. 6-Cyclopropyluracil and a pyrazolopyrimidine derivative have favorable ligand efficiencies of 0.47 and 0.38 kcal mol^-1 per non-hydrogen atom, respectively. They represent useful starting points for hit optimization.

SUBMITTER: Nai F

PROVIDER: S-EPMC9466600 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Fragment Ligands of the m6A-RNA Reader YTHDF2.

Nai Francesco F Nachawati Raed R Zálešák František F Wang Xiang X Li Yaozong Y Caflisch Amedeo A

ACS medicinal chemistry letters 20220817 9

We report 17 small-molecule ligands that compete with N6-methyladenosine (m6A) for binding to the m6A-reader domain of YTHDF2 (YT521-B homology domain family 2). We determined their binding mode at high resolution by X-ray crystallography and quantified their affinity by a fluorescence-based binding assay. 6-Cyclopropyluracil and a pyrazolopyrimidine derivative have favorable ligand efficiencies of 0.47 and 0.38 kcal mol-1 per non-hydrogen atom, respective ...[more]

PMID: 36110386

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Project description:Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several m6A readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of m6A in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These m6A-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knocking down one of YTHDF2's key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.

Project description:YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2) is a member of the YTH protein family that binds to N6-methyladenosine (m6A)-modified RNA, regulating RNA stability and restricting viral replication, including Epstein-Barr virus (EBV). PIAS1 is an E3 small ubiquitin-like modifier (SUMO) ligase known as an EBV restriction factor, but its role in YTHDF2 SUMOylation remains unclear. In this study, we investigated the functional regulation of YTHDF2 by PIAS1. We found that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues (K281, K571, and K572). Importantly, PIAS1 synergizes with wild-type YTHDF2, but not a SUMOylation-deficient mutant, to limit EBV lytic replication. Mechanistically, YTHDF2 lacking SUMOylation exhibits reduced binding to EBV transcripts, leading to increased viral mRNA stability. Furthermore, PIAS1 mediates SUMOylation of YTHDF2's paralogs, YTHDF1 and YTHDF3, to restrict EBV replication. These results collectively uncover a unique mechanism whereby YTHDF family proteins control EBV replication through PIAS1-mediated SUMOylation, highlighting the significance of SUMOylation in regulating viral mRNA stability and EBV replication.IMPORTANCEm6A RNA modification pathway plays important roles in diverse cellular processes and viral life cycle. Here, we investigated the relationship between PIAS1 and the m6A reader protein YTHDF2, which is involved in regulating RNA stability by binding to m6A-modified RNA. We found that both the N-terminal and C-terminal regions of YTHDF2 interact with PIAS1. We showed that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues. We also demonstrated that PIAS1 enhances the anti-EBV activity of YTHDF2. We further revealed that PIAS1 mediates the SUMOylation of other YTHDF family members, namely, YTHDF1 and YTHDF3, to limit EBV replication. These findings together illuminate an important regulatory mechanism of YTHDF proteins in controlling viral RNA decay and EBV replication through PIAS1-mediated SUMOylation.

Dataset Information

Fragment Ligands of the m⁶A-RNA Reader YTHDF2.

Publications

Fragment Ligands of the m<sup>6</sup>A-RNA Reader YTHDF2.

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