It is currently believed that certain messenger RNAs (mRNAs) are localized to distinct subcellular regions to efficiently target their encoded proteins. However, this simplistic model does not explain why in certain scenarios mRNA localization is dispensable for proper protein distribution. In other cases, mRNA localization is accompanied by translational silencing and degradation by the localization machinery. Here we propose that in certain scenarios mRNAs are localized so that they can either be stabilized and translated, or silenced and degraded, in response to the needs of the subcellular locale. In these cases, the localized mRNA, and its cadre of associated factors, act as a rheostat that regulates protein production and/or mRNA stability in response to the needs of its immediate subcellular environment. WIREs RNA 2017, 8:e1416. doi: 10.1002/wrna.1416 For further resources related to this article, please visit the WIREs website.

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...

With the discovery of the double helical structure of DNA, a shift occurred in how biologists investigated questions surrounding cellular processes, such as protein synthesis. Instead of viewing biological activity through the lens of chemical reactions, this new field used biological information to gain a new profound view of how biological systems work. Molecular biologists asked new types of questions that would have been inconceivable to the older generation of researchers, such as how cellular machineries convert inherited biological information into functional molecules like proteins. This new focus on biological information also gave molecular biologists a way to link their findings to concepts developed by genetics and the modern synthesis. However, by the late 1960s this all changed. Elevated rates of mutation, unsustainable genetic loads, and high levels of variation in populations, challenged Darwinian evolution, a central tenant of the modern synthesis, where adaptation was the main driver of evolutionary change. Building on these findings, Motoo Kimura advanced the neutral theory of molecular evolution, which advocates that selection in multicellular eukaryotes is weak and that most genomic changes are neutral and due to random drift. This was further elaborated by Jack King and Thomas Jukes, in their paper “Non-Darwinian Evolution”, where they pointed out that the observed changes seen in proteins and the types of polymorphisms observed in populations only become understandable when we take into account biochemistry and Kimura’s new theory. Fifty years later, most molecular biologists remain unaware of these fundamental advances. Their adaptionist viewpoint fails to explain data collected from new powerful technologies which can detect exceedingly rare biochemical events. For example, high throughput sequencing routinely detects RNA transcripts being produced from almost the entire genome yet are present less than one copy per thousand cells and appear to lack any function. Molecular biologists must now reincorporate ideas from classical biochemistry and absorb modern concepts from molecular evolution, to craft a new lens through which they can evaluate the functionality of transcriptional units, and make sense of our messy, intricate, and complicated genome.

In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels–providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)—and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation.