Credit:Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation.

Glucose is a vital source of cellular energy, yet its ability to influence protein interactions and its role in regulating these interactions during the differentiation process, when intracellular glucose levels rise, remain understudied. Miao et al. investigated the role of glucose in epidermal differentiation and revealed a previously unrecognized connection between the sugar and the RNA helicase DDX21. The authors employed a combination of mass spectrometry, cell biology, and genetics to uncover the mechanisms by which glucose modulates DDX21 function and ultimately influences the splicing of key differentiation genes.

The researchers utilized azido-glucose click chemistry and affinity chromatography to identify proteins that bind glucose, leading to the discovery of DDX21, a DEAD-box RNA helicase. Further studies demonstrated that glucose directly bound to the ATP-binding domain of DDX21, competing with ATP and altering its protein conformation. This interaction inhibited DDX21's helicase activity and led to the dissociation of DDX21 dimers.

The authors demonstrated that DDX21 was crucial for epidermal differentiation, both in vitro and in 3D human skin tissue organoids. Loss of DDX21 impaired differentiation and slowed keratinocyte proliferation, highlighting its importance in this process.

The study revealed that glucose promotes the re-localization of DDX21 from the nucleolus to the nucleoplasm, where it assembled into larger protein complexes containing RNA splicing factors. This re-localization was associated with an increase in cellular glucose levels during differentiation. CLIP-seq analysis identified a specific RNA motif, SCUGSDGC, as a target of DDX21 binding during differentiation. Glucose-dependent binding of DDX21 to this motif was associated with increased splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings suggested that glucose enabled DDX21 to bind to specific mRNA introns, promoting their splicing and ultimately driving differentiation.

Furthermore, the study showed that glucose elevation during differentiation was associated with a decrease in nuclear ATP levels. However, the ATPase activity of DDX21 was not required for epidermal differentiation, suggesting that glucose binding to DDX21 might play a non-energetic role in this process.

Interestingly, the authors demonstrated that the helicase-dead DDX21K236R mutant could still rescue impaired differentiation and splicing defects caused by DDX21 depletion. This suggested that DDX21's role in regulating mRNA splicing was independent of its helicase activity.

The findings of this study have important implications for understanding the role of glucose in tissue differentiation and homeostasis. The study suggested that glucose acted as a biomolecular cue that modulated the function of proteins like DDX21, influencing cellular processes such as mRNA splicing and ultimately shaping tissue development and function.

Further research is needed to explore the specific mechanisms by which DDX21 regulates mRNA splicing and the broader consequences of glucose-driven DDX21 regulation. Additionally, studies examining the role of DDX21 in differentiation of non-epidermal tissues and the impact of dysregulated tissue glucose levels in diseases like cancer are warranted.

Building on the understanding of how glucose dynamics influence protein function in differentiation, Luo et al. explored the role of circular RNAs (circRNAs) in the metabolic reprogramming of cancer cells under energy stress conditions, with a particular focus on the regulation of glycolysis. Their study highlights the complex interplay between metabolism and gene expression regulation in the context of cancer cell proliferation and survival.

The researchers employed a combination of bioinformatics analysis, cell biology experiments, and mouse models to investigate the role of circDDX21 in cancer metabolism. RNA sequencing analysis identified circDDX21 as one of the top upregulated circRNAs in hepatocellular carcinoma (HCC) cells under glucose deprivation. They confirmed the induction of circDDX21 by glucose deprivation using real-time RT-PCR and Sanger sequencing. The circRNA was localized to the cytoplasm and was resistant to degradation by RNase R, indicating its circular structure.

The authors found that c-Myc, a master transcription factor, mediated the induction of circDDX21 in response to glucose deprivation. ChIP and luciferase reporter assays demonstrated that c-Myc bound to a specific site in the DDX21 gene promoter and transcriptionally upregulated circDDX21 expression.

Functionally, circDDX21 knockdown significantly decreased the glycolytic rate in HCC cells, as measured by extracellular acidification rate (ECAR) assays. Conversely, overexpression of circDDX21 increased the glycolytic rate. Mechanistically, circDDX21 promoted glycolysis by specifically increasing the expression of PGAM1, a rate-limiting enzyme in the glycolytic pathway. Rescue experiments confirmed that the effects of circDDX21 on glycolysis were mediated through the regulation of PGAM1 expression.

Further investigation revealed that circDDX21 increased PGAM1 expression by enhancing PGAM1 mRNA stability. CircDDX21 directly interacted with the RNA-binding protein PABPC1, which also bound to PGAM1 mRNA. CircDDX21 promoted the binding of PABPC1 to PGAM1 mRNA by inhibiting MKRN3-mediated PABPC1 ubiquitination. MKRN3, an E3 ubiquitin ligase, promotes PABPC1 ubiquitination and degradation, thereby reducing its binding to target mRNAs. CircDDX21 competed with MKRN3 for binding to PABPC1, disrupting the MKRN3-PABPC1 interaction and preventing PABPC1 ubiquitination.

The authors further demonstrated that circDDX21 functions as an oncogenic circRNA in hepatocellular carcinoma. CircDDX21 knockdown inhibited cell proliferation and colony formation in vitro, while overexpression of circDDX21 promoted cell growth. In a xenograft mouse model, circDDX21 knockdown significantly inhibited tumor growth, and this effect was reversed by PGAM1 overexpression. Consistent with its role in promoting hepatocellular carcinogenesis, circDDX21 was highly expressed in clinical HCC tissues compared to adjacent normal tissues, and its expression was positively correlated with PGAM1 expression.

This study identified circDDX21 as a novel regulator of glycolysis in cancer cells. CircDDX21 was induced by glucose deprivation through c-Myc-mediated transcriptional activation. It promoted glycolysis by increasing PGAM1 expression through the stabilization of PGAM1 mRNA. CircDDX21 interacted with PABPC1 and disrupted its association with MKRN3, thereby inhibiting PABPC1 ubiquitination and enhancing its binding to PGAM1 mRNA. CircDDX21 functioned as an oncogenic circRNA in hepatocellular carcinoma, promoting cell proliferation and tumor growth. These findings suggested that circDDX21 might serve as a potential therapeutic target for hepatocellular carcinoma.

In conclusion, these studies have uncovered novel insights into the regulation of glycolysis and differentiation by circDDX21 and DDX21. The intricate interplay between RNA-binding proteins, circRNAs, and mRNA splicing highlights the complexity of cellular metabolism and opens up new avenues for therapeutic intervention in cancer and other diseases.

Reference:

1. Miao W, Porter DF, Lopez-Pajares V, Siprashvili Z, Meyers RM, Bai Y, Nguyen DT, Ko LA, Zarnegar BJ, Ferguson ID, Mills MM, Jilly-Rehak CE, Wu CG, Yang YY, Meyers JM, Hong AW, Reynolds DL, Ramanathan M, Tao S, Jiang S, Flynn RA, Wang Y, Nolan GP, Khavari PA. Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation. Cell. 2023 Jan 5;186(1):80-97.e26.

2. Luo J, Yang Y, Zhang G, Fang D, Liu K, Mei Y, Wang F. Energy stress-induced circDDX21 promotes glycolysis and facilitates hepatocellular carcinogenesis. Cell Death Dis. 2024 May 21;15(5):354.