Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.

Whether protein translation occurs in the nucleus is contentious. To address this question, we developed the ribopuromycylation method (RPM), which visualizes translation in cells via standard immunofluorescence microscopy. The RPM is based on ribosome-catalyzed puromycylation of nascent chains immobilized on ribosomes by antibiotic chain elongation inhibitors followed by detection of puromycylated ribosome-bound nascent chains with a puromycin (PMY)-specific monoclonal antibody in fixed and permeabilized cells. The RPM correlates localized translation with myriad processes in cells and can be applied to any cell whose translation is sensitive to PMY. In this paper, we use the RPM to provide evidence for translation in the nucleoplasm and nucleolus, which is regulated by infectious and chemical stress.

For cells to function properly the process of translating RNA messengers into proteins needs to be accurate, on the whole. Yet work in HeLa cells now shows that about 1% of methionine residues used in protein synthesis are aminoacylated to 'textbook-incorrect' tRNAs. Surprisingly, the proportion of Met-misacylated tRNAs increases significantly when cells are under stress through viral infection or treatment with viral or bacterial Toll-like receptor ligands. Tests with other amino acids indicate that the phenomenon is limited to Met, and as Met residues are known to protect proteins against damage from reactive oxygen species, one possibility is that Met-misacylation is a natural protective response to cellular stress. Accurate transfer RNA (tRNA) aminoacylation is necessary for translational fidelity; however, the accuracy of tRNA aminoacylation in vivo is uncertain. In mammalian cells, approximately 1% of methionine residues used in protein synthesis are now shown to be aminoacylated to non-methionyl-tRNAs. Furthermore, misacylation of methionine increases up to tenfold upon exposing cells to viruses, toll-like receptor ligands or oxidative stress. Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings1,2. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower3,4,5,6. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage7, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.

Significance: In neurons, many mRNAs are transported to synapses in a translationally repressed state, allowing for the spatial and temporal regulation of protein synthesis required for synaptic plasticity. It has been assumed that these mRNAs are repressed at the initiation step of translation. Here we provide evidence for a second mechanism whereby these mRNAs are instead repressed at elongation/termination awaiting translational reactivation upon appropriate synaptic signals. Our results establish that a form of translation-dependent synaptic plasticity, which is dysregulated in neurodevelopmental and psychiatric pathologies, occurs independently of translation initiation. Elucidating the upstream pathways that lead to repression and reactivation of elongation/termination on these mRNAs may provide new avenues for the design of therapies targeting neurodevelopmental disorders.

Here, we address the question of why the influenza A virus hemagglutinin (HA) does not escape immunity by hyperglycosylation. Uniquely among dozens of monoclonal antibodies specific for A/Puerto Rico/8/34, escape from H28-A2 neutralization requires substitutions introducing N-linked glycosylation at residue 131 or 144 in the globular domain. This escape decreases viral binding to cellular receptors, which must be compensated for by additional substitutions in HA or neuraminidase that enable viral replication. Sequence analysis of circulating H1 influenza viruses confirms the in vivo relevance of our findings: natural occurrence of glycosylation at residue 131 is always accompanied by a compensatory mutation known to increase HA receptor avidity. In vaccinated mice challenged with WT vs. H28-A2 escape mutants, the selective advantage conferred by glycan-mediated global reduction in antigenicity is trumped by the costs of diminished receptor avidity. These findings show that, although N-linked glycosylation can broadly diminish HA antigenicity, fitness costs restrict its deployment in immune evasion.

Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N⁶,2’-O-dimethyladenosine (m⁶A_m) demethylase activity. While m⁶A_m is strategically located next to the m⁷G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m⁶A_m level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m⁶A_m methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m⁶A_m modification in stem-like properties acquisition. FTO-mediated regulation of m⁶A_m marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m⁶A_m modification and its potential adverse consequences for colorectal cancer management.