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.
Synopsis
Correct splicing of cohesin‐loading factor sororin is required for sister chromatid cohesion during interphase. Mutations in a splicing factor essential for cohesion is implicated in chronic lymphocytic leukemia.
Functional genomics identifies a set of 26 spliceosome subunits that are required for sister chromatid cohesion in human cells.
Loss of splicing factors increases the turnover of cohesin complexes on chromatin and abrogates sister chromatid cohesion during interphase.
Compromising splicing reduces the steady‐state levels of sororin, a factor required for the stable association of cohesin with chromatin.
Increasing sororin protein level or removing the cohesin release factor WAPL restores cohesion in cells lacking splicing factors.
The key connections between sister chromatids in human cells are exquisitely sensitive to sororin dosage and to defects in pre‐mRNA splicing.
Recurrent splicing factor mutations in hyperproliferative haematological disorders may affect cohesin turnover on chromatin.
Correct splicing of cohesin‐loading factor sororin is required for sister chromatid cohesion during interphase. Mutations in a splicing factor essential for cohesion is implicated in chronic lymphocytic leukemia.
Two mechanisms safeguard the bipolar attachment of chromosomes in mitosis. A correction mechanism destabilizes erroneous attachments that do not generate tension across sister kinetochores 1. In ...response to unattached kinetochores, the mitotic checkpoint delays anaphase onset by inhibiting the anaphase-promoting complex/cyclosome (APC/CCdc20) 2. Upon satisfaction of both pathways, the APC/CCdc20 elicits the degradation of securin and cyclin B 3. This liberates separase triggering sister chromatid disjunction and inactivates cyclin-dependent kinase 1 (Cdk1) causing mitotic exit. How eukaryotic cells avoid the engagement of attachment monitoring mechanisms when sister chromatids split and tension is lost at anaphase is poorly understood 4. Here we show that Cdk1 inactivation disables mitotic checkpoint surveillance at anaphase onset in human cells. Preventing cyclin B1 proteolysis at the time of sister chromatid disjunction destabilizes kinetochore-microtubule attachments and triggers the engagement of the mitotic checkpoint. As a consequence, mitotic checkpoint proteins accumulate at anaphase kinetochores, the APC/CCdc20 is inhibited, and securin reaccumulates. Conversely, acute pharmacological inhibition of Cdk1 abrogates the engagement and maintenance of the mitotic checkpoint upon microtubule depolymerization. We propose that the simultaneous destruction of securin and cyclin B elicited by the APC/CCdc20 couples chromosome segregation to the dissolution of attachment monitoring mechanisms during mitotic exit.
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•Cyclin B degradation stabilizes kinetochore attachments in anaphase•Cdk1 inactivation prevents mitotic checkpoint engagement after anaphase onset•Chromatid disjunction by separase causes APC/C inhibition if Cdk1 remains active•APC/C links separase activation to dissolution of attachment monitoring mechanisms
Vázquez-Novelle et al. propose that Cdk1-cyclin B activity sets the temporal window for microtubule-kinetochore attachment surveillance. Failure to degrade cyclin B at anaphase onset destabilizes attachments and engages the mitotic checkpoint. APC/C-induced proteolysis could couple sister chromatid disjunction to checkpoint dissolution when sister chromatids split.
The mitotic checkpoint monitors the attachment of kinetochores to microtubules and delays anaphase onset until all sister kinetochores have become attached to opposite poles 1, 2. Correct bipolar ...attachment leads to kinetochore deformation and tension and satisfies the checkpoint 3–6. What prevents mitotic checkpoint reactivation when sister centromeres are split and tension is lost at anaphase onset? Aurora B kinase, the catalytic subunit of the chromosomal passenger protein complex (CPC) 7, acts as a sensor at inner centromeres for the status of attachment 5, 8. Phosphorylation of Aurora B targets at erroneously attached kinetochores elicits the correction of these attachments and the activation of the mitotic checkpoint. At anaphase, the CPC leaves the centromeres and relocates to the spindle midzone 7. This iconic translocation might prevent the checkpoint from reengaging after anaphase onset. To test this hypothesis, we experimentally retained Aurora B and the CPC at the centromere throughout anaphase in human cells. Preventing CPC translocation caused the untimely recruitment of mitotic checkpoint proteins to kinetochores at anaphase in an Aurora B kinase activity-dependent manner. Our results suggest that the relocalization of the CPC, an evolutionarily conserved event in eukaryotes, is a key mechanism that incapacitates the mitotic checkpoint at anaphase.
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► CPC relocation prevents mitotic checkpoint engagement at anaphase onset ► Centromeric CPC retention causes recruitment of Bub1, BubR1, and Mps1 to kinetochores ► This untimely mitotic checkpoint response at anaphase requires Aurora B activity ► CPC retention at centromeres does not destabilize K fibers or delay mitotic exit
Two closely connected mechanisms safeguard the fidelity of chromosome segregation in eukaryotic cells. The mitotic checkpoint monitors the attachment of kinetochores to microtubules and delays ...anaphase onset until all sister kinetochores have become attached to opposite poles. In addition, an error correction mechanism destabilizes erroneous attachments that do not lead to tension at sister kinetochores. Aurora B kinase, the catalytic subunit of the CPC (chromosomal passenger complex), acts as a sensor and effector in both pathways. In this review we focus on a poorly understood but important aspect of mitotic control: what prevents the mitotic checkpoint from springing into action when sister centromeres are split and tension is suddenly lost at anaphase onset? Recent work has shown that disjunction of sister chromatids, in principle, engages the mitotic checkpoint, and probably also the error correction mechanism, with potentially catastrophic consequences for cell division. Eukaryotic cells have solved this 'anaphase problem' by disabling the mitotic checkpoint at the metaphase-to-anaphase transition. Checkpoint inactivation is in part due to the reversal of Cdk1 (cyclin-dependent kinase 1) phosphorylation of the CPC component INCENP (inner centromere protein; Sli15 in budding yeast), which causes the relocation of the CPC from centromeres to the spindle midzone. These findings highlight principles of mitotic checkpoint control: when bipolar chromosome attachment is reached in mitosis, the checkpoint is satisfied, but still active and responsive to loss of tension. Mitotic checkpoint inactivation at anaphase onset is required to prevent checkpoint re-engagement when sister chromatids split.
Abstract
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.
Synopsis
image
Correct splicing of cohesin‐loading factor sororin is required for sister chromatid cohesion during interphase. Mutations in a splicing factor essential for cohesion is implicated in chronic lymphocytic leukemia.
Functional genomics identifies a set of 26 spliceosome subunits that are required for sister chromatid cohesion in human cells.
Loss of splicing factors increases the turnover of cohesin complexes on chromatin and abrogates sister chromatid cohesion during interphase.
Compromising splicing reduces the steady‐state levels of sororin, a factor required for the stable association of cohesin with chromatin.
Increasing sororin protein level or removing the cohesin release factor WAPL restores cohesion in cells lacking splicing factors.
The key connections between sister chromatids in human cells are exquisitely sensitive to sororin dosage and to defects in pre‐
mRNA
splicing.
Recurrent splicing factor mutations in hyperproliferative haematological disorders may affect cohesin turnover on chromatin.
Las fosfatasas Cdc14 desempeñan funciones relacionadas con la regulación del ciclo celular en diversos organismos eucariotas, actuando como antagonistas de la actividad CDK (quinasa dependiente de ...ciclina). En las levaduras Saccharomyces cerevisiae y Schizosaccharomyces pombe, respectivamente, Cdc14 y Flp1/Clp1 participan en la regulación de la salida de mitosis, promoviendo la inhibición de la actividad CDK mitótica mediante mecanismos de acción diferentes.Con el fin de estudiar las funciones de las proteínas Cdc14 humanas, hCdc14A y hCdc14B, comenzamos analizando la homología funcional entre éstas y Flp1. De este modo comprobamos que ambas proteínas humanas complementan el fenotipo correspondiente a la falta de Flp1 en S. pombe, mediante mecanismos diferentes. La fosfatasa hCdc14A, como Flp1, desfosforila a Cdc25 (activador de los complejos CDK mitóticos) e induce así la reducción de sus niveles proteicos; mientras que el mecanismo de la complementación por hCdc14B no se conoce.A continuación, determinamos que hCdc14A también es capaz de desfosforilar in vitro al menos a dos de las proteínas Cdc25 humanas en residuos fosforilados por Cdk1, a hCdc25A (en Serina 115 y Serina 320) y a hCdc25C (en residuos no determinados). Además, probamos que la desfosforilación de hCdc25A por hCdc14A en estos dos residuos identificados promueve su degradación.Por último, estudiamos los efectos que tienen la sobre-expresión y del silenciamiento de la expresión de hCdc14A sobre la progresión por mitosis en varias líneas celulares humanas. Determinamos así que hCdc14A, de forma dependiente de su actividad fosfatasa, participa en la regulación de los complejos CDK mitóticos a la entrada en mitosis, a través de la inhibición de la actividad catalítica de hCdc25A. Obtuvimos también datos que sugieren que hCdc14A podría inducir la inactivación de los complejos CDK mitóticos a la salida de mitosis en parte, posiblemente, a través de la desfosforilación y consiguiente degradación de hCdc25A.
Cdc14 phosphatases are involved in regulation of the eukaryotic cell cycle, acting as antagonists of CDK (Cyclin-Dependent Kinases) activity. In yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively, Cdc14 and Flp1/Clp1 control mitotic exit, promoting mitotic CDK activity inhibition by different mechanisms. In order to study the functions developed by human Cdc14 proteins, hCdc14A and hCdc14B, we analyzed functional homology among hCdc14s and Flp1. Both human proteins complement the lack of Flp1 in S. pombe cells, by different molecular mechanisms. hCdc14A phosphatase, as Flp1 does, dephosphorylates Cdc25 (mitotic CDK activator) and thereby induces its degradation, while how hCdc14B complements the lack of Flp1 remains unknown.hCdc14A also dephosphorylates in vitro at least two of human Cdc25 proteins, previously phosphorylated by Cdk1, hCdc25A (on Serines 115 and 320) and hCdc25C (on unknown residues). hCdc25A dephosphorylation on this two residues by hCdc14A promotes its degradation.Finally, we also studied how the deregulation of hCdc14A levels (by overexpression or gene silencing) affects mitotic progression in human cell lines. We concluded that hCdc14A, in a phosphatase activity-dependent manner, is involved in the regulation of mitotic CDK complexes at the entry into mitosis, by inhibiting hCdc25A phosphatase activity. Our results also suggested that hCdc14A could induce mitotic CDK complexes inactivation at mitotic exit in part, possibly, by dephosphorylating hCdc25A and promoting its degradation.
Budding and fission yeast Cdc14 homologues, a conserved family of serine-threonine phosphatases, play a role in the inactivation of mitotic cyclin-dependent kinases (CDKs) by molecularly distinct ...mechanisms. Saccharomyces cerevisiae Cdc14 protein phosphatase inactivates CDKs by promoting mitotic cyclin degradation and the accumulation of a CDK inhibitor to allow budding yeast cells to exit from mitosis. Schizosaccharomyces pombe Flp1 phosphatase down-regulates CDK/cyclin activity, controlling the degradation of the Cdc25 tyrosine phosphatase for fission yeast cells to undergo cytokinesis. In the present work, we show that human Cdc14 homologues (hCdc14A and hCdc14B) rescued flp1-deficient fission yeast strains, indicating functional homology. We also show that hCdc14A and B interacted in vivo with S. pombe Cdc25 and that hCdc14A dephosphorylated this mitotic inducer both in vitro and in vivo. Our results support a Cdc14 conserved inhibitory mechanism acting on S. pombe Cdc25 protein and suggest that human cells may regulate Cdc25 in a similar manner to inactivate Cdk1-mitotic cyclin complexes.
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