Discovery Reveals that O-GlcNAc Glycan Modification Inhibits P-Body Formation and Maintains Pluripotency in ES Cells (Shoko Nishihara, Director, Glycan and Life Systems Integration Center / Graduate School of Science and Engineering)

Key Findings

- In mouse ES cells, proteasome activator subunit 3 (Psme3) is O‑GlcNAcylated at serine 111 (Ser111).
- O‑GlcNAc modification at Ser111 of Psme3 inhibits the formation of processing bodies (P‑bodies), a type of RNA granule, through promoting degradation of the DEAD-box polypeptide 6 (Ddx6).
- Loss of O‑GlcNAc modification at Ser111 of Psme3 leads to increased Ddx6 levels and P‑body formation, driving ES cells toward differentiation.

The research group led by Professor Shoko Nishihara, Director of the Glycan and Life Systems Integration Center, Department of Life Sciences, Graduate School of Science and Engineering, and Department of Symbiotic Creative Engineering, Faculty of Science and Engineering, Soka University, in collaboration with the research group of Professor Kazuo Yamamoto at the University of Tokyo, has discovered that O-GlcNAc glycan modification inhibits the formation of P-bodies, thereby maintaining the pluripotency of embryonic stem cells.
Recent studies increasingly highlight the crucial role of glycan modifications in determining stem cell characteristics. This study revealed that O‑GlcNAc modification at Ser111 of proteasome activator subunit 3 (Psme3) is essential for maintaining pluripotency in mouse ES cells. O‑GlcNAcylation at Ser111 promotes degradation of DEAD-box polypeptide 6 (Ddx6), which is required for P‑body assembly, thereby reducing P‑body formation. As a result, mRNAs encoding core transcription factors involved in maintaining pluripotency, such as Klf4 and Klf2, are translated, sustaining the pluripotent state.
Conversely, mutation of Ser111 that inhibits O‑GlcNAcylation stabilizes Ddx6, increases P‑body formation, lowers protein levels of core pluripotency transcription factors, and leads mouse ES cells to exit pluripotency and proceed toward differentiation.
This study clarifies that O‑GlcNAc modification at Ser111 of Psme3 regulates P‑body homeostasis, serving as a critical proteasome-regulating mechanism controlling ES cell pluripotency. The formation of RNA granules, which exclude mRNAs from translation, is an important process involved in dramatic cellular switching such as differentiation, and this novel finding reveals its regulation by O‑GlcNAc modification.
The results of this research were published in the American scientific journal Cell Reports on July 13, 11:00 AM (EST).

Part of this research was supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI), Grant Number 18K06139, and by the joint research fund of the Glycan and Life Systems Integration Center, Soka University.

Significance of the Research Findings

P-bodies, a type of RNA granule, are membrane-less cytoplasmic organelles that regulate stem cell identity. They arise through liquid–liquid phase separation, and their formation requires DEAD-box polypeptide 6 (Ddx6), an RNA helicase. Proteasome activator subunit 3 (Psme3) has been known to regulate the homeostasis of membrane-less organelles formed via liquid–liquid phase separation, such as Cajal bodies and nuclear speckles. In addition, the function of O-GlcNAc, the only glycan modification found in the cytoplasm, in stem cells has been gradually elucidated. However, a link among these three factors had never been anticipated.
In this study, the researchers demonstrated that, in mouse embryonic stem (ES) cells, Psme3 interacts with Ddx6, and that O-GlcNAc modification of Psme3 at Ser111 mediates Ddx6 degradation. This finding establishes a connection between O-GlcNAc, the proteasome, and the homeostasis of P-bodies in ES cells. The results provide new insights into the biology of P-bodies and the roles of the proteasome and O-GlcNAc glycosylation in pluripotency networks, contributing to the foundation of developmental biology and stem cell biology.

Background of the Research

The pluripotency of mouse embryonic stem (ES) cells is tightly regulated by a complex network of exogenous and endogenous factors. O-linked β-N-acetylglucosamine (O-GlcNAc) is the only glycan modification found on cytoplasmic and nuclear proteins and plays an essential role in regulating fundamental cellular processes. O-GlcNAc is added to the serine or threonine residues of proteins by O-GlcNAc transferase and removed by O-GlcNAcase. Although several research groups, including ours, have reported its functions in ES cells, the wide diversity of O-GlcNAc–modified proteins has hindered comprehensive analysis of its roles in maintaining pluripotency.
In this study, we first employed a highly sensitive O-GlcNAc detection method developed by our collaborator Dr. Yamamoto to comprehensively identify O-GlcNAc–modified proteins in mouse ES cells and epiblast-like cells (EpiLCs) derived from them. Interestingly, the pattern of O-GlcNAc–modified proteins differed between ES cells and EpiLCs, the latter being slightly more differentiated yet still pluripotent. Given that the relationship between proteasome regulation by O-GlcNAc and the pluripotency network of ES cells remained unknown, we selected proteasome activator subunit 3 (Psme3) from the detected proteins for further analysis. Psme3 was found to be O-GlcNAc–modified in both cell types maintaining pluripotency.
To explore Psme3’s functional partners, we expressed Psme3 as a FLAG-tag fusion protein and identified its interacting proteins by immunoprecipitation followed by mass spectrometry. This revealed DEAD-box polypeptide 6 (Ddx6), an essential factor for P-body assembly, as well as Grbp2, Fxr1, and Rpl7, all known to interact with Ddx6. These findings suggested a potential role for Psme3 in P-body regulation. Indeed, overexpression of Psme3 led to degradation of Ddx6 and a reduction in P-body numbers.
Mass spectrometry and immunoprecipitation confirmed that Psme3 was O-GlcNAc–modified at serine 111 (Ser111). We then generated a mutant Psme3 lacking O-GlcNAc modification at Ser111 and compared it to wild-type Psme3 in ES cells. The mutant exhibited markedly reduced O-GlcNAc modification and diminished interaction with Ddx6, indicating that O-GlcNAcylation at Ser111 controls both Psme3–Ddx6 interaction and subsequent Ddx6 degradation. Furthermore, ES cells overexpressing the mutant showed suppressed Ddx6 degradation, increased P-body numbers, decreased protein levels of core pluripotency transcription factors such as Klf4 and Klf2, and transitioned out of the pluripotent state toward differentiation.
Taken together, these results reveal that O-GlcNAcylation of Psme3 at Ser111 acts as a molecular switch that regulates ES cell pluripotency by maintaining P-body homeostasis.

Published in: Cell Reports, July 13 issue
Article Title: Site-specific O-GlcNAcylation of Psme3 maintains mouse stem cell pluripotency by impairing P-body homeostasis
Authors: Federico Pecori, Nanako Kondo, Chika Ogura, Taichi Miura, Masahiko Kume, Youhei Minamijima, Kazuo Yamamoto, Shoko Nishihara