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Hexokinase 2 is an RNA-binding protein that regulates mRNA translation independently of glycolysis and induces melanoma cell proliferation

ABSTRACT: Although metabolic benefits of glycolysis have been extensively described in tumor cells, the extra-metabolic functions linked to this energetic pathway in tumor growth and cell proliferation have not been clearly established yet. Recently, some key glycolytic enzymes, such as GAPDH and PKM2, were reported to regulate mRNA translation. Translational control of gene expression is considered as a critical effector in cancer biology, representing a highly promising area of research. Here, we report that Hexokinase 2 (HK2), a glucose kinase that catalyzes the first step of glycolysis at the outer mitochondrial membrane (OMM), is an RNA-binding protein (RBP) that regulates mRNA translation in melanoma cell lines. Polysome profiling experiments followed by RNA sequencing indicate that the translational regulation exerted by HK2 is partly independent of the metabolic status or the glycolytic pathway. We found that HK2 specifically regulates translation of the mRNA encoding SOX10, a transcription factor implicated in the regulation of tumor initiation, maintenance, and progression in melanoma. RNA-protein interaction assays, including crosslinking immunoprecipitation (CLIP), indicate that HK2 is an RBP whose interaction with RNA is independent of its enzymatic activity, its ability to bind glucose or its association with the OMM. HK2 directly interacts with the 5’ untranslated region (5’UTR) of the SOX10 mRNA through a stem-loop RNA secondary structure. Using RNA-protein proximity ligation assays and a fluorescence-based ribosome-bound mRNA mapping method (RIBOmap), we found that high glucose conditions, which promotes the release of HK2 from the OMM, induces an increase in HK2-SOX10 mRNA interaction and SOX10 mRNA translation in the cytoplasm. We further showed that HK2-dependent SOX10 mRNA translation is involved in melanoma cell proliferation and colony formation. Collectively, our data highlight a non-metabolic function of HK2 acting as an RBP and translation regulator.

ORGANISM(S): Homo sapiens

PROVIDER: GSE274146 | GEO | 2025/07/17

REPOSITORIES: GEO

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Project description:Protein-RNA interactions are integral components of nearly every aspect of biology including regulation of gene expression, assembly of cellular architectures, and pathogenesis of human diseases. However, studies in the past few decades have only uncovered a small fraction of the vast landscape of the protein-RNA interactome in any organism, and even less is known about the dynamics of protein-RNA interactions under changing developmental and environmental conditions. Here, we describe the gPAR-CLIP (global photoactivatable-ribonucleoside-enhanced crosslinking and immunopurification) approach for capturing regions of the transcriptome bound by RNA-binding proteins (RBPs) in budding yeast. We report over 13,000 RBP crosslinking sites in untranslated regions (UTR) covering 72% of protein-coding transcripts encoded in the genome, confirming 3M-bM-^@M-^Y UTRs as major sites for RBP interaction. Comparative genomic analyses reveal that RBP crosslinking sites are highly conserved, and RNA folding predictions indicate that secondary structural elements are constrained by protein binding and may serve as generalizable modes of RNA recognition. Finally, 38% of 3M-bM-^@M-^Y UTR crosslinking sites show changes in RBP occupancy upon glucose or nitrogen deprivation, with major impacts on metabolic pathways as well as mitochondrial and ribosomal gene expression. Our study offers an unprecedented view of the pervasiveness and dynamics of protein-RNA interactions in vivo. Duplicate gPAR-CLIP and mRNA-seq libraries were sequenced from yeast strains for each of three conditions: log-phase growth, growth after 2 hour glucose starvation, and growth after 2 hour nitrogen starvation. Additional duplicate mRNA-seq libraries were sequenced from yeast strains grown in the absence of 4-thiouracil. gPAR-CLIP libraries were used to determine regions of mRNA bound by proteins. mRNA-seq libraries served as controls for mRNA abundance. A Puf3p PAR-CLIP library was sequenced to determine how well gPAR-CLIP captured the binding signatures of a single RNA-binding protein.

Project description:Published molecular profiling studies in patients with lymphoma suggested the influence of hypoxia inducible factor-1 alpha (HIF1α) targets in prognosis of DLBCL. Yet, the role of hypoxia in hematological malignancies remains unclear. We observed that activation of HIF1α resulted in global translation repression during hypoxic stress in DLBCL. Protein translation efficiency as measured using 35S-labeled methionine incorporation revealed a ≥50% reduction in translation upon activation of HIF1α. Importantly, translation was not completely inhibited and expression of clinically correlated hypoxia targets such as GLUT1, HK2, and CYT-C was found to be refractory to translational repression under hypoxia in DLBCL cells. Notably, hypoxic induction of these genes was not observed in normal primary B-cells. Translational repression was coupled with a decrease in mitochondrial function. Screening of primary DLBCL patient samples revealed that expression of HK2, which encodes for the enzyme hexokinase 2, was significantly correlated with DLBCL phenotype. Genetic knockdown studies demonstrated that HK2 is required for promoting growth of DLBCL under hypoxic stress. Altogether, our findings provide strong support for the direct contribution of HK2 in B-cell lymphoma development and suggest that HK2 is a key metabolic driver of the DLBCL phenotype.ne incorporation revealed a ≥50% reduction in translation upon activation of HIF1α. Importantly, translation was not completely blunted and expression of clinically correlated hypoxia targets such as GLUT1, HK2, and CYT-C was found to be refractory to translational repression under hypoxia in DLBCL cells. Notably, hypoxic induction of these genes was not observed in normal primary B-cells. Translational repression was coupled with decrease in mitochondrial function. Screening of DLBCL patient samples identified that expression of HK2, which encodes for the enzyme hexokinase 2, was significantly correlated with DLBCL phenotype. Genetic knockdown studies show that HK2 is required for promoting growth of DLBCL under hypoxic stress. Altogether, our findings provide more definitive proof of direct contribution of HK2 in development of B-cell lymphoma and suggest that HK2 is a key metabolic driver of DLBCL phenotype.

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Hexokinase 2 is an RNA-binding protein that regulates mRNA translation independently of glycolysis and induces melanoma cell proliferation

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