Pyruvate Kinase M Links Glucose Availability to Protein Synthesis

Abstract

How human cells coordinate various metabolic processes, such as glycolysis and protein translation, remains unclear. One key insight is that various metabolic enzymes have been found to associate with mRNAs, however whether these enzymes regulate mRNA biology in response to changes in cellular metabolic state remains unknown. Here we report that the glycolytic enzyme, pyruvate kinase M (PKM), inhibits the translation of 7% of the transcriptome in response to elevated levels of glucose and pyruvate.Our data suggest that in the presence of glucose and pyruvate, PKM associates with ribosomes that are synthesizing stretches of polyacidic nascent polypeptides and stalls the elongation step of translation.PKM-regulated mRNAs encode proteins required for the cell cycle and may explain previous results linking PKM to cell cycle regulation. Our study uncovers an unappreciated link between glycolysis and the ribosome that likely coordinates the intake of glycolytic metabolites with the regulation of protein synthesis and the cell cycle. Results and Discussion Mass spectrometry analysis of ER and cytosolic polysomes and mRNPsOver the past decade, numerous proteins that lack RNA binding domains, such as metabolic enzymes, have been shown to exhibit mRNA and ribosome binding 1-12 . It is unclear whether these unconventional RNA binding proteins (RBP) associate with transcripts and ribosomes in a spatially defined manner, or if they link metabolic states to mRNA stability or translation. To determine the spatial distribution of RNA-, and ribosome-binding proteins, we isolated ER and cytosolic fractions from human osteosarcoma (U2OS) cells ( Figure 1A) and sedimented crude polysomes. The cytosol and ER represent the major division in cellular protein synthesis, each containing distinct pools of mRNAs and unique translational regulatory systems [13][14][15][16] . These isolated polysomes were then treated with RNase to liberate RNA-binding proteins ("RNA-bound fraction"), and resedimented to pellet ribosomes and associated proteins ("Ribosome-bound fraction"; Figure 1B). We then analyzed the composition of the RNA-bound and Ribosome-bound fractions ( Figure 1B-C) by mass spectrometry, as previously described 17 . In parallel we also isolated messenger ribonuclear protein (mRNP) complexes from the ER and cytosol using oligo-dT affinity chromatography ("mRNP-bound"; Figure 1B, D, "dT") and again analyzed these fractions by mass spectrometry. To control for non-specific binding to the oligo-dT resin we also performed the affinity chromatography with beads lacking any nucleic acid ( Figure 1B, "Mock Beads", 1D "B"). Our purification conditions, done in the absence of crosslinking, enabled recovery of proteins that are directly and indirectly bound to mRNAs and/or ribosomes.After statistical processing (see Methods), 370 proteins were present in the RNA-bound fraction, 414 were present in the ribosome-bound fraction, and 2690 proteins were enriched in the mRNPassociated fraction. Upon further manual curation (see methods), 496 protei...

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“…Recent characterization of the ribosome-associated interactome identified PKM2, LDHA, and ALDOA as being part of a macromolecular complex that controls mRNA location, stability, and translation (Simsek et al, 2017). Enrichment of glycolytic enzymes with ribosomes has been later confirmed, including both spliced isoforms of pyruvate kinase (PKM1 and 2) (Kejiou et al, 2019). Global characterization of the PKM2:RNA interactome has identified hundreds of protein-encoding mRNAs, although the role of PKM2 as modulator of mRNA translation remains far from being clear.…”

“…Simsek et al found that PKM2 binds preferentially with ribosomal RNAs and mRNAs associated to the endoplasmic reticulum (ER) promoting the translation of ER and secreted proteins (Simsek et al, 2017). Similarly, Kejiou et al found that PKM2 associates to polysomes in an RNA-dependent manner (Kejiou et al, 2019). However, they did not observe an enrichment of rRNAs associated with PKM2.…”

“…Further analysis of mRNA translation suggested that PKM2 could act as a repressor by controlling elongation of charged nascent peptides. This was linked to the availability of glucose and cell cycle progression (Kejiou et al, 2019). Although further investigations are needed to clarify whether PKM2 preferentially controls translation of cytosolic or ER‐associated and secreted proteins, PKM2 could be part of a more extended mechanism that blocks protein synthesis during cell starvation.…”

The mutual interaction of glycolytic enzymes and RNA in post-transcriptional regulation

“…But also human PKM was found to associate to ribosomes. In 2019 Kejiou and co-workers discovered that PKM regulates polysome association of mRNAs related to cell cycle (Kejiou et al, 2019). It binds the coding sequence of transcripts with polyacidic stretches and stalls ribosome elongation in human osteosarcoma (U2OS) cells in response to pyruvate and glucose.…”

“…PKM binding activity and translational regulation is cell cycle-dependent. Binding to ribosomal RNA is also critically discussed by the authors but lacks solid support by their data ( Kejiou et al 2019 ).…”