Abstract
Interacting with proteins is a crucial way for long noncoding RNAs (lncRNAs) to exert their biological responses. Here we report a high throughput strategy to characterize lncRNA interacting proteins in vivo by combining tobramycin affinity purification and mass spectrometric analysis (TOBAP-MS). Using this method, we identify 140 candidate binding proteins for lncRNA highly upregulated in liver cancer (HULC). Intriguingly, HULC directly binds to two glycolytic enzymes, lactate dehydrogenase A (LDHA) and pyruvate kinase M2 (PKM2). Mechanistic study suggests that HULC functions as an adaptor molecule that enhances the binding of LDHA and PKM2 to fibroblast growth factor receptor type 1 (FGFR1), leading to elevated phosphorylation of these two enzymes and consequently promoting glycolysis. This study provides a convenient method to study lncRNA interactome in vivo and reveals a unique mechanism by which HULC promotes Warburg effect by orchestrating the enzymatic activities of glycolytic enzymes.
Highlights
Cellular metabolism reprogramming is a major hallmark of cancer[1]
To evaluate the identification results, we examined three common proteins identified by both methods (TGM2, H2A and protein disulfide-isomerase A3 (PDIA3)) and another two randomly selected proteins observed by TOBAP-MS (DDX58 and LGALS3BP)
The results showed that stimulation by FGF induced the trans-localization of lactate dehydrogenase A (LDHA), Pyruvate kinase M2 (PKM2) and highly upregulated lncRNAs in liver cancer (HULC) to the cell membrane, where they co-localized with fibroblast growth factor receptor type 1 (FGFR1) (Fig. 5f)
Summary
Cellular metabolism reprogramming is a major hallmark of cancer[1]. To sustain tumor growth, cancer cells have an enhanced demand for nutrients to support dramatically elevated cell proliferation. Instead of using the tricarboxylic acid (TCA) cycle in mitochondria to generate ATP, cancer cells preferentially convert glucose to lactate through glycolysis, even in the presence of oxygen, which provides more metabolites for cell proliferation[2,3] This aerobic glycolysis process plays important roles in tumorigenesis and tumor progression[4,5]. ChIPR, CHART, and RAP all use antisense oligonucleotides to isolate the endogenous lncRNA of interest These methods usually require large input cell numbers and may not be competent for lncRNA molecules with low abundances, and they may suffer from false positives induced by interference from other RNAs containing homolog sequences[22]. We establish a high throughput strategy to characterize the interacting proteome of lncRNA-HULC by combining tobramycin affinity purification and quantitative mass spectrometry analysis (TOBAP-MS) Using this method, we identify 140 potential HULC interacting proteins and build a highly connected interactome network. Mechanistic study validates these interactions and reveals that HULC-promoted aerobic glycolysis by directly binding to LDHA and PKM2 and modulating their enzymatic activities
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