Efficient generation and reversion of chromosomal translocations using CRISPR/Cas technology.

BACKGROUND: Chromosomal translocations are a hallmark of cancer cells and give rise to fusion oncogenes. To gain insight into the mechanisms governing tumorigenesis, adequate model cell lines are required. RESULTS: We employ the versatile CRISPR/Cas system to engineer cell lines in which chromosomal translocations are either generated de novo (CD74-ROS1) or existing translocations are reverted back to the original configuration (BCR-ABL1). To this end, we co-apply two guide RNAs to artificially generate two breakpoints and screen for spontaneous fusion events by PCR. CONCLUSIONS: The approach we use is efficient and delivers clones bearing translocationsin a predictable fashion. Detailed analysis suggests that the clones display no additional undesired alterations, implying that the approach is robust and precise.

Kinetochore-localized PP1-Sds22 couples chromosome segregation to polar relaxation.

Cell division requires the precise coordination of chromosome segregation and cytokinesis. This coordination is achieved by the recruitment of an actomyosin regulator, Ect2, to overlapping microtubules at the centre of the elongating anaphase spindle. Ect2 then signals to the overlying cortex to promote the assembly and constriction of an actomyosin ring between segregating chromosomes. Here, by studying division in proliferating Drosophila and human cells, we demonstrate the existence of a second, parallel signalling pathway, which triggers the relaxation of the polar cell cortex at mid anaphase. This is independent of furrow formation, centrosomes and microtubules and, instead, depends on PP1 phosphatase and its regulatory subunit Sds22 (refs 2, 3). As separating chromosomes move towards the polar cortex at mid anaphase, kinetochore-localized PP1-Sds22 helps to break cortical symmetry by inducing the dephosphorylation and inactivation of ezrin/radixin/moesin proteins at cell poles. This promotes local softening of the cortex, facilitating anaphase elongation and orderly cell division. In summary, this identifies a conserved kinetochore-based phosphatase signal and substrate, which function together to link anaphase chromosome movements to cortical polarization, thereby coupling chromosome segregation to cell division.

Aurora B kinase promotes cytokinesis by inducing centralspindlin oligomers that associate with the plasma membrane.

In metazoans, cytokinesis is triggered by activation of the GTPase RhoA at the equatorial plasma membrane. ECT-2, the guanine nucleotide exchange factor (GEF) required for RhoA activation, is activated by the centralspindlin complex that concentrates on spindle midzone microtubules. However, these microtubules and the plasma membrane are not generally in apposition, and thus the mechanism by which RhoA is activated at the cell equator remains unknown. Here we report that a regulated pool of membrane-bound, oligomeric centralspindlin stimulates RhoA activation. The membrane-binding C1 domain of CYK-4, a centralspindlin component, promotes furrow initiation in C. elegans embryos and human cells. Membrane localization of centralspindlin oligomers is globally inhibited by PAR-5/14-3-3. This activity is antagonized by the chromosome passenger complex (CPC), resulting in RhoA activation at the nascent cleavage site. Therefore, CPC-directed centralspindlin oligomerization during anaphase induces contractile ring assembly at the membrane.

Functional genomics identifies a requirement of pre-mRNA splicing factors for sister chromatid cohesion.

Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation during cell division. Using functional genomic screening, we identify a set of 26 pre-mRNA splicing factors that are required for sister chromatid cohesion in human cells. Loss of spliceosome subunits increases the dissociation rate of cohesin from chromatin and abrogates cohesion after DNA replication, ultimately causing mitotic catastrophe. Depletion of splicing factors causes defective processing of the pre-mRNA encoding sororin, a factor required for the stable association of cohesin with chromatin, and an associated reduction of sororin protein level. Expression of an intronless version of sororin and depletion of the cohesin release protein WAPL suppress the cohesion defect in cells lacking splicing factors. We propose that spliceosome components contribute to sister chromatid cohesion and mitotic chromosome segregation through splicing of sororin pre-mRNA. Our results highlight the loss of cohesion as an early cellular consequence of compromised splicing. This may have clinical implications because SF3B1, a splicing factor that we identify to be essential for cohesion, is recurrently mutated in chronic lymphocytic leukaemia.

Centralspindlin links the mitotic spindle to the plasma membrane during cytokinesis.

At the end of cell division, cytokinesis splits the cytoplasm of nascent daughter cells and partitions segregated sister genomes. To coordinate cell division with chromosome segregation, the mitotic spindle controls cytokinetic events at the cell envelope. The spindle midzone stimulates the actomyosin-driven contraction of the cleavage furrow, which proceeds until the formation of a microtubule-rich intercellular bridge with the midbody at its centre. The midbody directs the final membrane abscission reaction and has been proposed to attach the cleavage furrow to the intercellular bridge. How the mitotic spindle is connected to the plasma membrane during cytokinesis is not understood. Here we identify a plasma membrane tethering activity in the centralspindlin protein complex, a conserved component of the spindle midzone and midbody. We demonstrate that the C1 domain of the centralspindlin subunit MgcRacGAP associates with the plasma membrane by interacting with polyanionic phosphoinositide lipids. Using X-ray crystallography we determine the structure of this atypical C1 domain. Mutations in the hydrophobic cap and in basic residues of the C1 domain of MgcRacGAP prevent association of the protein with the plasma membrane, and abrogate cytokinesis in human and chicken cells. Artificial membrane tethering of centralspindlin restores cell division in the absence of the C1 domain of MgcRacGAP. Although C1 domain function is dispensable for the formation of the midzone and midbody, it promotes contractility and is required for the attachment of the plasma membrane to the midbody, a long-postulated function of this organelle. Our analysis suggests that centralspindlin links the mitotic spindle to the plasma membrane to secure the final cut during cytokinesis in animal cells.

BRCA2 fine-tunes the spindle assembly checkpoint through reinforcement of BubR1 acetylation.

Germline mutations that inactivate BRCA2 promote early-onset cancer with chromosome instability. Here, we report that BRCA2 regulates the spindle assembly checkpoint (SAC). Previously, we reported that BubR1 acetylation is essential for SAC activity. In this study we show that BRCA2 recruits the PCAF acetyltransferase and aids in BubR1 acetylation during mitosis. In the absence of BRCA2, BubR1 acetylation is abolished, and the level of BubR1 decreases during mitosis. Similarly, Brca2-deficient mouse embryonic fibroblasts exhibited weak SAC activity. Transgenic mice that were engineered to have interruptions in the BRCA2-BubR1 association exhibited marked decrease of BubR1 acetylation, weakened SAC activity, and aneuploidy. These transgenic mice developed spontaneous tumors at 40% penetrance. Moreover, immunohistochemical analyses of human breast cancer specimens suggested that BRCA2 mutation and BubR1 status is closely linked. Our results provide an explanation for how mutation of BRCA2 can lead to chromosome instability without apparent mutations in SAC components.

The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA.

Individuals with BRCA2 mutations are predisposed to breast cancers owing to genome instability. To determine the functions of BRCA2, the human protein was purified. It was found to bind selectively to single-stranded DNA (ssDNA), and to ssDNA in tailed duplexes and replication fork structures. Monomeric and dimeric forms of BRCA2 were observed by EM. BRCA2 directed the binding of RAD51 recombinase to ssDNA, reduced the binding of RAD51 to duplex DNA and stimulated RAD51-mediated DNA strand exchange. These observations provide a molecular basis for the role of BRCA2 in the maintenance of genome stability.

Evidence that the tumor-suppressor protein BRCA2 does not regulate cytokinesis in human cells.

Germline mutations in the tumor-suppressor gene BRCA2 predispose to breast and ovarian cancer. BRCA2 plays a well-established role in maintaining genome stability by regulating homologous recombination. BRCA2 has more recently been implicated in cytokinesis, the final step of cell division, but the molecular basis for this remains unknown. We have used time-lapse microscopy, recently developed cytokinesis assays and BAC recombineering (bacterial artificial chromosome recombinogenic engineering) to investigate the function and localization of BRCA2 during cell division. Our analysis suggests that BRCA2 does not regulate cytokinesis in human cells. Thus, cytokinesis defects are unlikely to contribute to chromosomal instability and tumorigenesis in BRCA2-related cancers.

A novel mutant of the Sup35 protein of Saccharomyces cerevisiae defective in translation termination and in GTPase activity still supports cell viability.

BACKGROUND: When a stop codon is located in the ribosomal A-site, the termination complex promotes release of the polypeptide and dissociation of the 80S ribosome. In eukaryotes two proteins eRF1 and eRF3 play a crucial function in the termination process. The essential GTPase Sup35p, the eRF3 release factor of Saccharomyces cerevisiae is highly conserved. In particular, we observed that all eRF3 homologs share a potential phosphorylation site at threonine 341, suggesting a functional role for this residue. The goal of this study was to determine whether this residue is actually phosphorylated in yeast and if it is involved in the termination activity of the protein. RESULTS: We detected no phosphorylation of the Sup35 protein in vivo. However, we show that it is phosphorylated by the cAMP-dependent protein kinase A on T341 in vitro. T341 was mutated to either alanine or to aspartic acid to assess the role of this residue in the activity of the protein. Both mutant proteins showed a large decrease of GTPase activity and a reduced interaction with eRF1/Sup45p. This was correlated with an increase of translational readthrough in cells carrying the mutant alleles. We also show that this residue is involved in functional interaction between the N- and C-domains of the protein. CONCLUSION: Our results point to a new critical residue involved in the translation termination activity of Sup35 and in functional interaction between the N- and C-domains of the protein. They also raise interesting questions about the relation between GTPase activity of Sup35 and its essential function in yeast.

Different modes of stop codon restriction by the Stylonychia and Paramecium eRF1 translation termination factors.

In universal-code eukaryotes, a single-translation termination factor, eukaryote class-1 polypeptide release factor (eRF1), decodes the three stop codons: UAA, UAG, and UGA. In some ciliates, like Stylonychia and Paramecium, eRF1s exhibit UGA-only decoding specificity, whereas UAG and UAA are reassigned as sense codons. Because variant-code ciliates may have evolved from universal-code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of the protein. Using both in vitro and in vivo assays, we show here that chimeric molecules composed of the N-terminal domain of Stylonychia eRF1 fused to the core domain (MC domain) of human eRF1 retained specificity toward UGA; this unambiguously associates eRF1 stop codon specificity to the nature of its N-terminal domain. Functional analysis of eRF1 chimeras constructed by swapping ciliate N-terminal domain sequences with the matching ones from the human protein highlighted the crucial role of the tripeptide QFM in restricting Stylonychia eRF1 specificity toward UGA. Using the site-directed mutagenesis, we show that Paramecium eRF1 specificity toward UGA resides within the NIKS (amino acids 61-64) and YxCxxxF (amino acids 124-131) motifs. Thus, we establish that eRF1 from two different ciliates relies on different molecular mechanisms to achieve specificity toward the UGA stop codon. This finding suggests that eRF1 restriction of specificity to only UGA might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UAG and UAA to sense codons.