Chromosome Segregation Defects Research Articles

ARHGEF17/TEM4 regulates the cell cycle through control of G1 progression.

The Ras homolog (Rho) small GTPases coordinate diverse cellular functions including cell morphology, adhesion and motility, cell cycle progression, survival, and apoptosis via their role in regulating the actin cytoskeleton. The upstream regulators for many of these functions are unknown. ARHGEF17 (also known as TEM4) is a Rho family guanine nucleotide exchange factor (GEF) implicated in cell migration, cell-cell junction formation, and the mitotic checkpoint. In this study, we characterize the regulation of the cell cycle by TEM4. We demonstrate that TEM4-depleted cells exhibit multiple defects in mitotic entry and duration, spindle morphology, and spindle orientation. In addition, TEM4 insufficiency leads to excessive cortical actin polymerization and cell rounding defects. Mechanistically, we demonstrate that TEM4-depleted cells delay in G1 as a consequence of decreased expression of the proproliferative transcriptional co-activator YAP. TEM4-depleted cells that progress through to mitosis do so with decreased levels of cyclin B as a result of attenuated expression of CCNB1. Importantly, cyclin B overexpression in TEM4-depleted cells largely rescues mitotic progression and chromosome segregation defects in anaphase. Our study thus illustrates the consequences of Rho signaling imbalance on cell cycle progression and identifies TEM4 as the first GEF governing Rho GTPase-mediated regulation of G1/S.

RECQ4-MUS81 interaction contributes to telomere maintenance with implications to Rothmund-Thomson syndrome

Replication stress, particularly in hard-to-replicate regions such as telomeres and centromeres, leads to the accumulation of replication intermediates that must be processed to ensure proper chromosome segregation. In this study, we identify a critical role for the interaction between RECQ4 and MUS81 in managing such stress. We show that RECQ4 physically interacts with MUS81, targeting it to specific DNA substrates and enhancing its endonuclease activity. Loss of this interaction, results in significant chromosomal segregation defects, including the accumulation of micronuclei, anaphase bridges, and ultrafine bridges (UFBs). Our data further demonstrate that the RECQ4-MUS81 interaction plays an important role in ALT-positive cells, where MUS81 foci primarily colocalise with telomeres, highlighting its role in telomere maintenance. We also observe that a mutation associated with Rothmund-Thomson syndrome, which produces a truncated RECQ4 unable to interact with MUS81, recapitulates these chromosome instability phenotypes. This underscores the importance of RECQ4-MUS81 in safeguarding genome integrity and suggests potential implications for human disease. Our findings demonstrate the RECQ4-MUS81 interaction as a key mechanism in alleviating replication stress at hard-to-replicate regions and highlight its relevance in pathological conditions such as RTS.

Early onset of septal FtsK localization allows for efficient DNA segregation in SMC-deleted Corynebacterium glutamicum strains.

Structural maintenance of chromosomes (SMC) are ubiquitously distributed proteins involved in chromosome organization. Deletion of smc causes severe growth phenotypes in many organisms. Surprisingly, smc can be deleted in Corynebacterium glutamicum, a member of the Actinomycetota phylum, without any apparent growth phenotype. SMC in C. glutamicum is loaded in a ParB-dependent fashion to the chromosome and functions in replichore cohesion. The unexpected absence of a growth phenotype in the smc mutant prompted us to screen for synthetic interactions within C. glutamicum. We generated a high-density Tn5 library from wild-type and smc-deleted C. glutamicum strains. Transposon sequencing data revealed that the DNA translocase FtsK is essential in an smc-deletion strain. In wild-type cells, FtsK localized to the septa and cell poles, showing polar enrichment during the earlier stages of the life cycle and relocating to the septum in the later stages. However, deletion of smc resulted in an earlier onset of pole-to-septum FtsK relocation, suggesting that prolonged FtsK complex activity is both required and sufficient to compensate for the absence of SMC, thus achieving efficient chromosome segregation in C. glutamicum. Deletion of ParB increases SMC and FtsK mobility. While the change in SMC dynamics aligns with previous data showing ParB's role in SMC loading on DNA, the change in FtsK mobility suggests defects in chromosome segregation. Based on our data, we propose an efficient mechanism for reliable DNA segregation in the absence of replichore arm cohesion in smc mutant cells.IMPORTANCEFaithful DNA segregation is of fundamental importance for life. Bacteria have developed efficient systems to coordinate chromosome compaction, DNA segregation, and cell division. A key factor in DNA compaction is the SMC complex that is found to be essential in many bacteria. In members of the Actinomycetota, smc is dispensable, but the reason for the lack of an smc phenotype in these bacteria remained unclear. We show here that the divisome-associated DNA pump FtsK can compensate for SMC loss and the subsequent loss in correct chromosome organization. In cells with distorted chromosomes, FtsK is recruited and stabilized earlier to the septum, allowing for DNA segregation for a larger part of the cell cycle, until chromosomes are segregated.

The microtubule-severing enzyme spastin regulates spindle dynamics to promote chromosome segregation in Trypanosoma brucei.

Microtubule-severing enzymes play essential roles in regulating diverse cellular processes, including mitosis and cytokinesis, by modulating microtubule dynamics. In the early branching protozoan parasite Trypanosoma brucei , microtubule-severing enzymes are involved in cytokinesis and flagellum length control during different life cycle stages, but none of them have been found to regulate mitosis in any life cycle form. Here, we report the biochemical and functional characterization of the microtubule-severing enzyme spastin in the procyclic form of T. brucei . We demonstrate that spastin catalyzes microtubule severing in vitro and ectopic overexpression of spastin disrupts spindle microtubules in vivo in trypanosome cells, leading to defective chromosome segregation. Knockdown of spastin impairs spindle integrity and disrupts chromosome alignment in metaphase and chromosome segregation in anaphase. We further show that the function of spastin requires the catalytic AAA-ATPase domain, the microtubule-binding domain, and the microtubule interacting and trafficking domain, and that the association of spastin with spindle depends on the microtubule-binding domain. Together, these results uncover an essential role for spastin in chromosome segregation by regulating spindle dynamics in this unicellular eukaryote.

Comparison of clinical artificial oocyte activation protocols on mouse egg activation and embryo development.

Artificial oocyte activation (AOA) with Ca2+ ionophores is an experimental procedure that benefits patients who fail to obtain fertilized eggs. However, the impact of non-physiological Ca2+ increases on cellular events involved in egg-embryo transition and early development remains poorly understood. Using the mouse model, this study compares common Ca2+ ionophore protocols applied in clinical practice - one or two exposures to A23187 or a single exposure to ionomycin - focusing on embryonic development and cellular events associated with egg activation. All groups of ionophore-activated eggs exhibit lower levels of first mitotic division compared to those activated by spermatozoa or SrCl2, attributable to the variations in Ca2+ dynamics during activation. At the cellular level, these eggs presented defects in spindle morphology and chromosome segregation during meiosis progression, associated with lower levels of cytoplasmic ATP, without changes in reactive oxygen species (ROS). These findings highlight the importance of optimizing Ca2+ management in AOA protocols.

Sequestration of ribosomal subunits as inactive 80S by targeting eIF6 limits mitotic exit and cancer progression.

Moderating the pool of active ribosomal subunits is critical for maintaining global translation rates. A factor crucial for modulating the 60S ribosomal subunit is eukaryotic translation initiation factor-6 (eIF6). Release of eIF6 from the 60S subunit is essential to permit 60S interactions with the 40S subunit. Here, using the eIF6-N106S mutant, we show that disrupting eIF6 interaction with the 60S subunit leads to an increase in vacant 80S ribosomes. It further highlights a dichotomy in the anti-association activity of eIF6 that is distinct from its role in 60S subunit biogenesis and shows that nucleolar localization of eIF6 is not dependent on BCCIP chaperone and uL14. Limiting active ribosomal pools markedly deregulates translation especially in mitosis and leads to chromosome segregation defects, mitotic exit delays and mitotic catastrophe. Ribo-seq analysis of eIF6-N106S mutant shows a significant downregulation in the translation efficiencies of mitotic factors and specifically transcripts with long 3' untranslated regions. eIF6-N106S mutation also limits cancer invasion, and this role is correlated with overexpression of eIF6 only in high-grade invasive cancers suggesting that deregulation of eIF6 is probably not an early event in cancers. Thus, this study highlights the segregation of eIF6 functions and its role in moderating 80S ribosome availability for translation, mitosis and cancer progression.

Mutations in tumor suppressor genes Vhl and Rassf1a cause DNA damage, chromosomal instability and induce gene expression changes characteristic of clear cell renal cell carcinoma.

RASSF1A is frequently biallelically inactivated in clear cell renal cell carcinoma (ccRCC) due to loss of chromosome 3p and promoter hypermethylation. Here we investigated the cellular and molecular consequences of single and combined deletion of the Rassf1a and Vhl tumor suppressor genes to model the common ccRCC genotype of combined loss of function of RASSF1A and VHL. In mouse embryonic fibroblasts and in primary kidney epithelial cells, double deletion of Rassf1a and Vhl caused chromosomal segregation defects and increased formation of micronuclei, demonstrating that pVHL and RASSF1A function to maintain genomic integrity. Combined Rassf1a and Vhl deletion in kidney epithelial cells in vivo increased proliferation and caused mild tubular disorganization, but did not lead to the development of kidney tumors. Single cell RNA-sequencing unexpectedly revealed that Rassf1a or Vhl deletion both induce the expression of an overlapping set of genes in a sub-population of proximal tubule cells. Many of these genes are also upregulated in the Vhl/Trp53/Rb1 deficient mouse model of ccRCC. In other subsets of proximal tubule cells, combined Vhl/Rassf1a deletion induced the expression of additional genes that were not upregulated in each of the single knockouts. The expression of the human homologues of Rassf1a-regulated genes correlate negatively with RASSF1 expression levels in human ccRCC. Our results suggest that the loss of RASSF1A function establishes a ccRCC-characteristic gene expression pattern.

The histone H3.3 K27M mutation suppresses Ser31phosphorylation and mitotic fidelity, which can directly drive gliomagenesis.

Serine 31 is a phospho-site unique to the histone H3.3 variant; mitotic phospho-Ser31 is restricted to pericentromeric heterochromatin, and disruption of phospho-Ser31 results in chromosome segregation defects and loss of p53-dependant G1 cell-cycle arrest.1,2,3,4 Ser31 is proximal to the H3.3 lysine 27-to-methionine (K27M) mutation that drives ∼80% of pediatric diffuse midline gliomas.5,6,7,8,9,10,11,12 Here, we show that expression of the H3.3 K27M mutant in normal, diploid cells results in increased chromosome missegregation and failure to arrest in the following G1. Expression of a non-phosphorylatable S31A mutant also drives chromosome missegregation, while the expression of a double K27M+ phosphomimetic S31E mutant restores mitotic fidelity and the p53 response to chromosome missegregation. We show that patient-derived H3.3 K27M tumor cells have decreased mitotic Ser31 phosphorylation and increased frequency of chromosome missegregation. CRISPR reversion of the K27M mutation to wild type (WT) restores phospho-Ser31 levels and results in a decrease in chromosome missegregation. However, inserting an S31A mutation by CRISPR into these revertant cells disrupts mitotic fidelity. Invitro and invivo analyses reveal that Chk1-the mitotic Ser31 kinase-is preferentially retained at pericentromeres in K27M-expressing tumor cells, compared with MLysine27-to-methionine mutation (M27K) isogenic revertants, correlating with both diminished phospho-Ser31 and mitotic defects. Interestingly, whereas M27K revertant cells do not form xenograft tumors in mice, H3.3 S31A cells do, similar to those formed by H3.3 K27M cells. Replication-competent avian leukosis virus splice-acceptor (RCAS)/cellular receptor for subgroup A avian sarcoma and leukosis virus (TVA) mice expressing S31A also form diffuse midline gliomas morphologically indistinguishable from K27M tumors. Together, our results reveal that the H3.3 K27M mutant alters H3.3 Ser31 phosphorylation, which, in turn, has profound impacts on chromosome segregation/cell-cycle regulation.

Exploring the role of oxidative stress and mitochondrial dysfunction in β-damascone-induced aneuploidy

BackgroundThe rose ketone β-damascone (β-Dam) elicits positive results in the in vitro micronucleus (MN) assay using human lymphocytes, but shows negative outcomes in the Ames test and combined in vivo MN and comet assays. This has led to the interpretation that the in vitro MN result is a misleading positive result. Oxidative stress has been suggested as an indirect mode of action (MoA) for in vitro MN formation, with the α, β-unsaturated carbonyl moiety of the β-Dam chemical structure expected to cause misleading positive results through this MoA. In this study, we investigated the role of oxidative stress in β-Dam-induced in vitro MN formation by co-treatment with the antioxidant N-acetyl-l-cysteine (NAC), thereby highlighting a possible link between mitochondrial dysfunction and aneugenicity.Resultsβ-Dam induced MN formation in both CHL/IU and BEAS-2B cells, with the response completely inhibited by co-treatment with NAC. Moreover, β-Dam induced oxidative stress-related reporter activity in the ToxTracker assay and increased reactive oxygen species levels, while decreasing glutathione levels, in BEAS-2B cells in the high-content analysis. All of these effects were suppressed by NAC co-treatment. These findings indicate that β-Dam elicits oxidative stress, which causes DNA damage and ultimately leads to MN induction. However, no significant DNA damage-related reporter activities were observed in the ToxTracker assay, nor was there an increased number of γH2AX foci in the high-content analysis. These data suggest that MN formation is not a DNA-reactive MoA. Considering recent reports of aneuploidy resulting from chromosome segregation defects caused by mitochondrial dysfunction, we investigated if β-Dam could cause such dysfunction. We observed that the mitochondrial membrane potential was dose-dependently impaired in BEAS-2B cells exposed to β-Dam.ConclusionsThese findings suggest that the oxidative stress induced by β-Dam exposure may be explained through an aneugenic MoA via mitochondrial dysfunction, thereby contributing to MN formation in mammalian cells.

Nuclear localization of MTHFD2 is required for correct mitosis progression

Subcellular compartmentalization of metabolic enzymes establishes a unique metabolic environment that elicits specific cellular functions. Indeed, the nuclear translocation of certain metabolic enzymes is required for epigenetic regulation and gene expression control. Here, we show that the nuclear localization of the mitochondrial enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) ensures mitosis progression. Nuclear MTHFD2 interacts with proteins involved in mitosis regulation and centromere stability, including the methyltransferases KMT5A and DNMT3B. Loss of MTHFD2 induces severe methylation defects and impedes correct mitosis completion. MTHFD2 deficient cells display chromosome congression and segregation defects and accumulate chromosomal aberrations. Blocking the catalytic nuclear function of MTHFD2 recapitulates the phenotype observed in MTHFD2 deficient cells, whereas restricting MTHFD2 to the nucleus is sufficient to ensure correct mitotic progression. Our discovery uncovers a nuclear role for MTHFD2, supporting the notion that translocation of metabolic enzymes to the nucleus is required to meet precise chromatin needs.

Pachytene piRNAs control discrete meiotic events during spermatogenesis and restrict gene expression in space and time.

Pachytene piRNAs, a Piwi-interacting RNA subclass in mammals, are hypothesized to regulate non-transposon sequences during spermatogenesis. Caenorhabditis elegans piRNAs, the 21URNAs, are implicated in regulating coding sequences; the messenger RNA targets and biological processes they control during spermatogenesis are largely unknown. We demonstrate that loss of 21URNAs compromises homolog pairing and makes it permissive for nonhomologous synapsis resulting in defects in crossover formation and chromosome segregation during spermatogenesis. We identify Polo-like kinase 3 (PLK-3), among others, as a 21URNA target. 21URNA activity restricts PLK-3 protein to proliferative cells, and expansion of PLK-3 in pachytene overlaps with the meiotic defects. Removal of plk-3 results in quantitative genetic suppression of the meiotic defects. One discrete 21URNA inhibits PLK-3 expression in late pachytene cells. Together, these results suggest that the 21URNAs function as pachytene piRNAs during C. elegans spermatogenesis. We identify their targets and meiotic events and highlight the remarkable intricacy of this multi-effector mechanism during spermatogenesis.

Nr5a2 is dispensable for zygotic genome activation but essential for morula development.

Early embryogenesis is driven by transcription factors (TFs) that first activate the zygotic genome and then specify the lineages constituting the blastocyst. Although the TFs specifying the blastocyst's lineages are well characterized, those playing earlier roles remain poorly defined. Using mouse models of the TF Nr5a2, we show that Nr5a2-/- embryos arrest at the early morula stage and exhibit altered lineage specification, frequent mitotic failure, and substantial chromosome segregation defects. Although NR5A2 plays a minor but measurable role during zygotic genome activation, it predominantly acts as a master regulator at the eight-cell stage, controlling expression of lineage-specifying TFs and genes involved in mitosis, telomere maintenance, and DNA repair. We conclude that NR5A2 coordinates proliferation, genome stability, and lineage specification to ensure correct morula development.

Kinesin-7 CENP-E mediates centrosome organization and spindle assembly to regulate chromosome alignment and genome stability.

Chromosome congression and alignment are essential for cell cycle progression and genomic stability. Kinesin-7 CENP-E, a plus-end-directed kinesin motor, is required for chromosome biorientation, congression and alignment in cell division. However, it remains unclear how chromosomes are aligned and segregated in the absence of CENP-E in mitosis. In this study, we utilize the CRISPR-Cas9 gene editing method and high-throughput screening to establish CENP-E knockout cell lines and reveal that CENP-E deletion results in defects in chromosome congression, alignment and segregation, which further promotes aneuploidy and genomic instability in mitosis. Both CENP-E inhibition and deletion lead to the dispersion of spindle poles, the formation of the multipolar spindle and spindle disorganization, which indicates that CENP-E is necessary for the organization and maintenance of spindle poles. In addition, CENP-E heterozygous deletion in spleen tissues also leads to the accumulation of dividing lymphocytes and cell cycle arrest in vivo. Furthermore, CENP-E deletion also disrupts the localization of key kinetochore proteins and triggers the activation of the spindle assembly checkpoint. In summary, our findings demonstrate that CENP-E promotes kinetochore-microtubule attachment and spindle pole organization to regulate chromosome alignment and spindle assembly checkpoint during cell division.

MiR-31-mediated local translation at the mitotic spindle is important for early development.

miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including β-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis.

Embryonic genome instability upon DNA replication timing program emergence

Faithful DNA replication is essential for genome integrity1–4. Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis5–8. However, the regulation of DNA replication remains poorly understood in early mammalian embryos. Here we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and identified an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing program, and the entire genome replicated gradually and uniformly using extremely slow-moving replication forks. In 4-cell embryos, a somatic-cell-like replication timing program commenced abruptly. However, the fork speed was still slow, S phase was extended, and markers of replication stress, DNA damage and repair increased. This was followed by an increase in break-type chromosome segregation errors specifically during the 4-to-8-cell division with breakpoints enriched in late-replicating regions. These errors were rescued by nucleoside supplementation, which accelerated fork speed and reduced the replication stress. By the 8-cell stage, forks gained speed, S phase was no longer extended and chromosome aberrations decreased. Thus, a transient period of genomic instability exists during normal mouse development, preceded by an S phase lacking coordination between replisome-level regulation and megabase-scale replication timing regulation, implicating a link between their coordination and genome stability.

β-TrCP-Mediated Proteolysis of Mis18β Prevents Mislocalization of CENP-A and Chromosomal Instability.

Restricting the localization of evolutionarily conserved histone H3 variant CENP-A to the centromere is essential to prevent chromosomal instability (CIN), an important hallmark of cancers. Overexpressed CENP-A mislocalizes to non-centromeric regions and contributes to CIN in yeast, flies, and human cells. Centromeric localization of CENP-A is facilitated by the interaction of Mis18β with CENP-A specific chaperone HJURP. Cellular levels of Mis18β are regulated by β-transducin repeat containing protein (β-TrCP), an F-box protein of SCF (Skp1, Cullin, F-box) E3-ubiquitin ligase complex. Here, we show that defects in β-TrCP-mediated proteolysis of Mis18β contributes to the mislocalization of endogenous CENP-A and CIN in a triple-negative breast cancer (TNBC) cell line, MDA-MB-231. CENP-A mislocalization in β-TrCP depleted cells is dependent on high levels of Mis18β as depletion of Mis18β suppresses mislocalization of CENP-A in these cells. Consistent with these results, endogenous CENP-A is mislocalized in cells overexpressing Mis18β alone. In summary, our results show that β-TrCP-mediated degradation of Mis18β prevents mislocalization of CENP-A and CIN. We propose that deregulated expression of Mis18β may be one of the key mechanisms that contributes to chromosome segregation defects in cancers.

RNA polymerase I mutant affects ribosomal RNA processing and ribosomal DNA stability

ABSTRACT Transcription is a major contributor to genomic instability. The ribosomal RNA (rDNA) gene locus consists of a head-to-tail repeat of the most actively transcribed genes in the genome. RNA polymerase I (RNAPI) is responsible for massive rRNA production, and nascent rRNA is co-transcriptionally assembled with early assembly factors in the yeast nucleolus. In Saccharomyces cerevisiae, a mutant form of RNAPI bearing a fusion of the transcription factor Rrn3 with RNAPI subunit Rpa43 (CARA-RNAPI) has been described previously. Here, we show that the CARA-RNAPI allele results in a novel type of rRNA processing defect, associated with rDNA genomic instability. A fraction of the 35S rRNA produced in CARA-RNAPI mutant escapes processing steps and accumulates. This accumulation is increased in mutants affecting exonucleolytic activities of the exosome complex. CARA-RNAPI is synthetic lethal with monopolin mutants that are known to affect the rDNA condensation. CARA-RNAPI strongly impacts rDNA organization and increases rDNA copy number variation. Reduced rDNA copy number suppresses lethality, suggesting that the chromosome segregation defect is caused by genomic rDNA instability. We conclude that a constitutive association of Rrn3 with transcribing RNAPI results in the accumulation of rRNAs that escape normal processing, impacting rDNA organization and affecting rDNA stability.

HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos.

Human fertility is suboptimal, partly due to error-prone divisions in early cleavage-stages that result in aneuploidy. Most human pre-implantation are mosaics of euploid and aneuploid cells, however, mosaic embryos with a low proportion of aneuploid cells have a similar likelihood of developing to term as fully euploid embryos. How embryos manage aneuploidy during development is poorly understood. This knowledge is crucial for improving fertility treatments and reducing developmental defects. To explore these mechanisms, we established a new mouse model of chromosome mosaicism to study the fate of aneuploid cells during pre-implantation development. We previously used the Mps1 inhibitor reversine to generate aneuploidy in embryos. Here, we found that treatment with the more specific Mps1 inhibitor AZ3146 induced chromosome segregation defects in pre-implantation embryos, similar to reversine. However, AZ3146-treated embryos showed a higher developmental potential than reversine-treated embryos. Unlike reversine-treated embryos, AZ3146-treated embryos exhibited transient upregulation of Hypoxia Inducible-Factor-1A (HIF1A) and lacked p53 upregulation. Pre-implantation embryos develop in a hypoxic environment in vivo, and hypoxia exposure in vitro reduced DNA damage in response to Mps1 inhibition and increased the proportion of euploid cells in the mosaic epiblast. Inhibiting HIF1A in mosaic embryos also decreased the proportion of aneuploid cells in mosaic embryos. Our work illuminates potential strategies to improve the developmental potential of mosaic embryos.

Modulation of ribosomal subunit associations by eIF6 is critical for mitotic exit and cancer progression.

Moderating the pool of active ribosomal subunits is critical for maintaining global translation rates. A factor crucial for modulating the 60S ribosomal subunits is eukaryotic translation initiation factor 6. Release of eIF6 from 60S is essential to permit 60S interactions with 40S. Here, using the N106S mutant of eIF6, we show that disrupting eIF6 interaction with 60S leads to an increase in vacant 80S. It further highlights a dichotomy in the anti-association activity of eIF6 that is distinct from its role in 60S biogenesis and shows that the nucleolar localization of eIF6 is not dependent on uL14-BCCIP interactions. Limiting active ribosomal pools markedly deregulates translation especially in mitosis and leads to chromosome segregation defects, mitotic exit delays and mitotic catastrophe. Ribo-Seq analysis of the eIF6-N106S mutant shows a significant downregulation in the translation efficiencies of mitotic factors and specifically transcripts with long 3'UTRs. eIF6-N106S mutation also limits cancer invasion, and this role is correlated with the overexpression of eIF6 only in high-grade invasive cancers suggesting that deregulation of eIF6 is probably not an early event in cancers. Thus, this study highlights the segregation of eIF6 functions and its role in moderating 80S availability for mitotic translation and cancer progression.

A role for condensin-mediator interaction in mitotic chromosomal organization.

Eukaryotic genomes are organized by condensin into 3D chromosomal architectures suitable for chromosomal segregation during mitosis. However, molecular mechanisms underlying the condensin-mediated chromosomal organization remain largely unclear. Here, we investigate the role of newly identified interaction between the Cnd1 condensin and Pmc4 mediator subunits in fission yeast, Schizosaccharomyces pombe. We develop a condensin mutation, cnd1-K658E, that impairs the condensin-mediator interaction and find that this mutation diminishes condensinmediated chromatin domains during mitosis and causes chromosomal segregation defects. The condensin-mediator interaction is involved in recruiting condensin to highly transcribed genes and mitotically activated genes, the latter of which demarcate condensin-mediated domains. Furthermore, this study predicts that mediator-driven transcription of mitotically activated genes contributes to forming domain boundaries via phase separation. This study provides a novel insight into how genome-wide gene expression during mitosis is transformed into the functional chromosomal architecture suitable for chromosomal segregation.