Symplekin Specifies Mitotic Fidelity by Supporting Microtubule Dynamics

doi:10.1128/MCB.00758-10

Mol Cell Biol. 2010 November; 30(21): 5135–5144.

Published online 2010 September 7. doi: 10.1128/MCB.00758-10

PMCID: PMC2953045

Symplekin Specifies Mitotic Fidelity by Supporting Microtubule Dynamics ^†

Kathryn M. Cappell,^1,² Brittany Larson,^1,² Noah Sciaky,¹ and Angelique W. Whitehurst^1,²^*

Department of Pharmacology,¹ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina²

^*Corresponding author. Mailing address: 4093 Genetic Medicine Building, CB 7365, Chapel Hill, NC 27599-7365. Phone: (919) 843-3257. Fax: (919) 966-5640. E-mail: awhit1/at/med.unc.edu

Received June 30, 2010; Revised July 22, 2010; Accepted August 23, 2010.

This article has been cited by other articles in PMC.

Abstract

Using a pangenomic loss-of-function screening strategy, we have previously identified 76 potent modulators of paclitaxel responsiveness in non-small-cell lung cancer. The top hit isolated from this screen, symplekin, is a well-established component of the mRNA polyadenylation machinery. Here, we performed a high-resolution phenotypic analysis to reveal the mechanistic underpinnings by which symplekin depletion collaborates with paclitaxel. We find that symplekin supports faithful mitosis by contributing to the formation of a bipolar spindle apparatus. Depletion of symplekin attenuates microtubule polymerization activity as well as expression of the critical microtubule polymerization protein CKAP5 (TOGp). Depletion of additional members of the polyadenylation complex induces similar phenotypes, suggesting that polyadenylation machinery is intimately coupled to microtubule function and thus mitotic spindle formation. Importantly, tumor cells depleted of symplekin display reduced fecundity, but the mitotic defects that we observe are not evident in immortalized cells. These results demonstrate a critical connection between the polyadenylation machinery and mitosis and suggest that tumor cells have an enhanced dependency on these components for spindle assembly.

Pangenomic loss-of-function screening is emerging as an effective tool for revealing the components that support core biological processes, including viral infection, DNA repair, chemotherapeutic responsiveness, melanogenesis, and endocytosis (5-7, 14, 18, 24, 28, 32, 45). A number of screening efforts have focused on identifying those gene products that are required for mitotic progression in both the normal and the tumorigenic settings (26, 27, 29, 30, 36, 45). These screens have successfully returned validated mitotic participants but also have isolated a diverse set of unanticipated genes whose encoded proteins have no previously described role in mitotic progression but instead have well-established roles in processes such as transcription (26, 27, 36, 45), RNA splicing and translation (27, 36, 45), and vesicle transport (27), thereby revealing an unexpected diversity in the compendium of gene products supporting mitosis.

We have recently applied a genome-wide loss-of-function paclitaxel synthetic-lethal strategy to identify cell-autonomous specifiers of chemoresponsiveness in non-small-cell lung cancer (NSCLC) cells (45). This strategy returned a diverse set of gene products, including symplekin (SYMPK), whose depletion was the most potent for sensitizing NSCLC cells to a dose of paclitaxel that has no detectable impact on cell viability. Symplekin is a scaffold protein that supports the assembly of polyadenylation machinery on nascent mRNA transcripts; however, no role for symplekin in drug sensitivity or mitosis has been reported (3). Polyadenylation is essential for the maturation of most pre-mRNAs and regulates mRNA nuclear export, stability, and translation (13, 33, 37). In Xenopus laevis oocytes, the polyadenylation of specific meiotic transcripts is regulated such that their activation occurs only following meiotic maturation signals (9, 20, 44). In mammalian cells, the poly(A) tail length of specific transcripts changes in a cell cycle-dependent manner (37), suggesting that cytoplasmic polyadenylation is a conserved mechanism for exerting translational regulation of gene expression prior to and during cell division.

Here, we have employed a high-resolution phenotypic analysis of symplekin to evaluate the contribution of polyadenylation machinery to mitotic control in the tumor and normal cell settings. We find that symplekin is required to support bipolar spindle formation in multiple NSCLC-derived tumor cells and that symplekin depletion impairs proliferation of NSCLC cells in vivo. The basis of symplekin's contribution to mitotic progression appears to be at the level of microtubule function and expression of a critical component of the microtubule polymerization machinery, CKAP5 (TOGp). Depletion of other polyadenylation components causes similar alterations in CKAP5 expression and mitotic progression. Therefore, our results demonstrate that mitotic fidelity is acutely sensitive to perturbations of polyadenylation machinery and suggest that inhibition of polyadenylation may synergize with current antimitotic agents.

MATERIALS AND METHODS

Cell culture.

H1155, H1299, HCC366, and HCC515 cells were a gift from John Minna. All cell lines had recently been genotyped using short tandem repeat (STR) analysis. Cells were maintained in RPMI medium (Gibco) with 5% fetal bovine serum (FBS) as described previously (45). BJ fibroblasts immortalized with human telomerase reverse transcriptase (hTERT) were a gift from Fred Grinnell (UT-Southwestern). BJ cells were maintained in Dulbecco modified Eagle medium (DMEM) plus 10% FBS.

Cell Titer Glo assays.

Cell Titer Glo assays were performed using independent small interfering RNAs (siRNAs) from the siGENOME Smart pool targeting symplekin as previously described (45).

siRNA transfections.

Transfections were performed as described previously (45) with siGENOME Smart pools (ThermoFisher). Cells were transfected for either 72 or 96 h as indicated in the figure legends. As a control, either a mismatch siRNA or an siRNA targeting DLNB14, which has no detectable impact in our assay system, was used. Sequences for all siRNAs utilized can be viewed in Table S1 in the supplemental material.

High-content imaging.

H1155 green fluorescent protein (GFP)-histone 2B-expressing cells were obtained by retroviral transduction. Retrovirus was produced by Fugene (Roche) transfection of 293gp cells with pCLNCX-GFP-H2B (a gift from Gray Pearson, UT-Southwestern) and vesicular stomatitis virus G protein (VSV-G), and virus was harvested at 48 h posttransfection. H1155 cells at 50% confluence were transduced with virus in 4 μg/ml Polybrene, and stably expressing cells were selected using 600 μg/ml Geneticin (Gibco). For imaging, cells were reverse transfected with the indicated siRNAs, plated in a 96-well format, and exposed to paclitaxel at 48 h posttransfection. Twenty-four hours post-paclitaxel treatment, the cells were imaged on a BD Pathway 855 bioimager using a 40× or 20× high-numerical-aperture (high-NA) objective. Images were taken every 15 min for the next 48 h, and an image sequence was generated using ImageJ (39). Manual quantification was used for the indicated parameters.

Flow cytometry.

H1155 cells were fixed in 50% ethanol-phosphate-buffered saline (PBS), washed, and resuspended in propidium iodine (BD) for 30 min. A minimum of 10,000 cells were collected for each condition by Summit 4.3 (Dako), and cell cycle distribution was determined using the ModFit software package (Verity Software House).

Lentivirus production.

Short hairpin RNA (shRNA) clones in the PLKO1 vector were obtained from the RNAi Consortium (Open Biosystems). Lentivirus targeting SYMPK was produced by Fugene-mediated transfection of 293T cells with plasmids for VSV-G, Δ8.9, and shRNAs targeting SYMPK or GFP (SYMPK clones TRCN0000141511 and TRCN0000144902 were effective). Virus was harvested at 48 h posttransfection and used to infect cells at 50% confluence in conjunction with 5 μg/μl Polybrene. Infection rates based on GFP assays performed in parallel were over 90%.

Quantitative real-time RT-PCR.

Total RNA was collected from H1299 cells using the GenElute Mammalian Total RNA Miniprep kit (Sigma). cDNA was synthesized from 2 μg total RNA using the High-Capacity cDNA reverse transcription kit (Applied Biosystems). Real-time reverse transcription-PCR (RT-PCR) used inventoried TaqMan gene expression assays designed to detect mRNA exclusively and the 7500 Fast real-time PCR system (Applied Biosystems). Actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous control, and cells transfected with control siRNA were used for calculating differences in expression by the threshold cycle (2^−ΔΔCT) method. For CKAP5 levels following SYMPK reduction, results are from pooling of results from 3 individual experiments performed in duplicate in which the average endogenous control C_T values between conditions never varied more than 0.3. For measurement of transcript knockdown, experiments were performed in triplicate with C_T values between conditions never varying more than 0.6.

Immunoblotting.

Cells were lysed directly in boiling sample buffer (100 mM Tris-Cl, 4% SDS, 20% glycerol, 0.2% bromophenol blue), subjected to SDS-polyacrylamide gel electrophoresis, and transferred to an Immobilon polyvinylidene difluoride membrane (Sigma). For MG-132 experiments, 20 μM MG-132 (Sigma) was added for 12 h prior to harvesting. Primary antibodies used include anti-SYMPK (BD Biosciences), anti-GAPDH (Santa Cruz), anti-cyclin B1 (Cell Signaling), anti-TACC3 (Santa Cruz), anti-CKAP5 (Abcam), anti-CSTF2 (Abcam), anti-CPSF1 (Santa Cruz), and anti-CPSF3 (Santa Cruz). Secondary antibodies used include peroxidase-conjugated anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch). Densitometry analysis was performed in ImageJ.

Immunofluorescence.

Cells were grown on glass coverslips and fixed at 72 or 96 h posttransfection in 3.7% formaldehyde. Cell were processed for immunofluorescence as described previously (45, 46). To visualize centrosomal proteins (CKAP5, TACC3, c-NAP1, rootletin, and NUMA1), cells were extracted with 0.5% Triton for 30 s, prior to fixation in cold methanol. Primary antibodies used include anti-β-tubulin (Sigma), pericentrin (Abcam), rootletin (Santa Cruz), Aurora-A (Sigma), Ncd80 (Abcam), TACC3 (BioLegend), NUMA1 (Novus), and CKAP5 (Abcam). After washes in PBS containing 0.1% Tween 20 and 10 mg/ml bovine serum albumin (PBTA), slips were placed in Alexa Fluor-conjugated secondary antibody (Invitrogen). Slides were imaged on an Axioimager upright microscope (Zeiss) equipped with a charge-coupled device (CCD) camera.

Microtubule regrowth assay.

H1299 cells were treated with 11 μM nocodazole (Calbiochem) at 96 h posttransfection. After 2 h of treatment, cells were placed in fresh medium and allowed to recover for the indicated time period. Cells were washed in PHEM buffer {60 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)], 25 mM HEPES, 10 mM EGTA, 2.0 mM MgCl₂, 1.0 μM paclitaxel}, and depolymerized tubulin was extracted with 0.2% Triton in PHEM buffer for 1 min. Extracted cells were washed in 1× PBS and fixed in 3.7% formaldehyde for 15 min. An immunofluorescence assay for β-tubulin and pericentrin was performed after permeabilization in 0.5% Triton and blocking in PBTA. Quantification of mean β-tubulin fluorescence intensity in the region of the centrosome was measured in ImageJ. For the measurement, pericentrin staining was used to identify the centrosomes of each cell and a circle of constant diameter across all samples was drawn around the centrosome to measure the intensity of the β-tubulin fibers emanating from the centrosome. ImageJ was utilized to measure the β-tubulin fluorescence in the circle with a minimum of 40 cells per treatment group.

Tumor xenografts.

H1299 cells stably expressing luciferin (H1299_luc) were a gift from John Minna (UT-Southwestern). H1299_luc cells were transduced with lentivirus targeting shGFP or SYMPK, and viable cells were harvested at 5 days postinfection for injection into nude mice. Two million cells/mouse were injected subcutaneously in the right flank, and mice were treated with 10 μl/gram luciferin and imaged biweekly with bioluminescent imaging (BLI) (34) until tumor development. Tumor growth was thereafter monitored biweekly by BLI. Tumor volume was calculated as length × width² × 0.5 from BLI measurements. All animals were treated in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines instituted at The University of North Carolina at Chapel Hill.

RESULTS

Symplekin is required for mitotic spindle integrity.

Symplekin was originally identified as a statistically significant modulator of paclitaxel sensitivity by using a high-throughput chemosensitizer screening platform. Thus, we first validated symplekin for off-target consequences by verifying that multiple independent siRNA sequences were capable of inducing synthetic lethality (Fig. (Fig.11 A). In addition, we validated that symplekin protein levels were depleted (Fig. (Fig.1A).1A). Because we have previously observed that RNA interference (RNAi) paclitaxel sensitizer screens can return components whose depletion induces a mitotic arrest (45), we evaluated the integrity of the mitotic spindle in symplekin-depleted H1155 cells treated with 10 nM paclitaxel. As observed for other targets identified by our whole-genome screening effort, symplekin depletion led to an increase in the accumulation of mitotic figures (Fig. (Fig.1B).1B). This accumulation was also detected at the population level by flow cytometry, which revealed a marked increase in 4N DNA content of symplekin-depleted cells exposed to 10 nM paclitaxel compared to that of control-transfected cells (Fig. (Fig.1C).1C). Immunoblot analysis revealed elevated levels of cyclin B1, a substrate of the anaphase-promoting complex (APC), the E3 ligase whose activity is restricted in the presence of improper chromosome alignment (Fig. (Fig.1D).1D). Thus, the observed accumulation of mitotic cells in the symplekin-depleted samples could indicate an aberrant mitosis. Indeed, symplekin-depleted H1155 cells exposed to 10 nM paclitaxel displayed a high frequency of multipolar spindles characterized by disorganized tubulin and multiple centrosomes compared to the structure of control-transfected cells (Fig. (Fig.1E).1E). These data suggest that symplekin function is directly coupled to the ability of cells to form a normal bipolar spindle.

FIG. 1.

SYMPK is required for mitotic spindle integrity after exposure to paclitaxel (Pac). (A) Whole-cell extracts of H1155 cells transfected with indicated siRNAs were immunoblotted to detect endogenous SYMPK (left panel). Viability assay in H1155 cells transfected (more ...)

Symplekin is required for high-fidelity mitosis.

Given our observations that symplekin contributes to bipolar spindle formation, we directly assessed the consequence of symplekin depletion on mitotic progression in real time by live imaging of H1155 cells stably expressing the chromatin marker GFP-H2B. By performing single-cell lineage tracing, we measured both the length and outcome of mitosis in symplekin- and control siRNA-transfected cells (Fig. (Fig.2).2). As expected, control or symplekin depletion alone had little effect on either mitotic fate or mitotic timing (Fig. 2A and B). However, symplekin-depleted cells exposed to 10 nM paclitaxel exhibited a significantly prolonged mitosis compared to those of control-transfected and paclitaxel-treated cells (Fig. (Fig.2B).2B). The outcome of this prolonged mitosis was aberrant in 75% of the individual cells studied. In particular, instead of the formation of 2 daughter cells, symplekin-depleted samples underwent either apoptosis, micronucleation, or a multipolar mitosis following mitotic arrest (Fig. 2A and C). Taken together, these observations suggest that symplekin supports mitotic spindle formation and mitotic progression in NSCLC cells.

FIG. 2.

SYMPK is required for high-fidelity mitotic progression in tumor cells. (A) H1155 cells expressing GFP-histone H2B were transfected with indicated siRNAs. At 48 h posttransfection, cells were exposed to 10 nM paclitaxel or carrier and imaged by live time-lapse (more ...)

Symplekin is necessary for mitosis in diverse NSCLC genetic settings.

To determine if the impacts of symplekin are unique to H1155 cells or represent a common mechanism observed across diverse genetic backgrounds, we assayed mitotic defects following symplekin depletion in H1299 cells, which were derived from an NSCLC lymph node metastasis. H1299 cells undergo a mitotic arrest followed by bypass of an activated APC and form micronucleated daughter cells when exposed to doses of paclitaxel of 10 nM and greater. Similar to the synthetic lethality seen in H1155 cells, H1299 cells depleted of symplekin and exposed to 10 nM paclitaxel displayed an increase in the frequency of micro- and multinucleated cells compared to that of the control (Fig. (Fig.33 A). We do not observe the same effects in the normal-tissue-derived human bronchial epithelial cell line HBEC3 (Fig. (Fig.3B3B).

FIG. 3.

SYMPK is necessary for mitosis in tumor cells from multiple genetic backgrounds. (A) H1299 cells were transfected with the indicated siRNAs for 48 h followed by exposure to paclitaxel for 24 h. Cells were fixed and stained with β-tubulin and DAPI (more ...)

We next sought to determine the impacts of prolonged suppression of symplekin expression in NSCLC cells. To this end, we stably repressed symplekin expression in H1299 cells by using an shRNA-mediated system where we pooled two effective shRNAs targeting symplekin (Fig. (Fig.3C).3C). Stably repressing symplekin expression in the H1299 NSCLC cell line led to an increase in micronucleation in the absence of paclitaxel (Fig. (Fig.3C).3C). In our original screening cell line, H1155, stable repression of symplekin led to an increase in mitotic figures in the absence of paclitaxel (Fig. (Fig.3D).3D). Extending this analysis to two additional NSCLC cell lines and immortalized BJ fibroblasts revealed that the generation of micronucleated cells following prolonged symplekin depletion is a common phenomenon in NSCLC cells but not in normal diploid fibroblasts immortalized with hTERT (Fig. (Fig.3D).3D). Thus, while the transient impacts of symplekin depletion are observable only in the presence of a microtubule-disrupting agent, prolonged symplekin depletion alone increases the frequency of aberrant mitosis and mitotic arrest in tumor cells but not normal cells.

Loss of symplekin impairs tumor formation in a mouse xenograft model.

To directly test if the mitotic dysfunction that we observe in multiple NSCLC cell backgrounds could result in reduced neoplastic potential, we evaluated the ability of H1299 cells stably depleted of symplekin to form xenograft tumors in nude mice. Luciferase-expressing H1299 cells were injected into nude mice 5 days following infection with shRNAs targeting symplekin or GFP. Tumor development was monitored for 4 weeks by twice-weekly imaging of tumors. While 100% of control-infected cells formed subcutaneous tumors, only 50% of mice injected with symplekin-depleted cells established tumors (Fig. (Fig.44 A). Furthermore, at 5 weeks postinjection, the symplekin hairpin tumors were significantly smaller than those in control cells (Fig. (Fig.4B),4B), suggesting that the asymmetric divisions that we observe in vitro may accumulate over time to reduce tumor cell fecundity.

FIG. 4.

Depletion of SYMPK impairs tumor growth in vivo. (A) Two million luciferase-expressing H1299 cells infected with lentivirus harboring control or SYMPK shRNAs were injected in the right flank of Harlan nude mice. The picture shows bioluminescence imaging (more ...)

Symplekin modulates microtubule polymerization.

The formation of a normal, bipolar spindle apparatus is exquisitely dependent on proper microtubule function, which is significantly altered in the presence of chemotherapeutic drugs such as paclitaxel (23). Given the symplekin-paclitaxel synthetic-lethal phenotype that we observed in NSCLC cells, we probed microtubule polymerization efficiency in H1299 cells following transient depletion of symplekin by using a microtubule regrowth assay. Here, H1299 cells transfected with control or symplekin siRNAs were exposed to a high dose of nocodazole to induce microtubule depolymerization. Microtubules were depolymerized to similar degrees in both control- and symplekin-transfected samples (Fig. (Fig.55 A and B). However, after 10 min of recovery, symplekin-depleted cells displayed little microtubule regrowth from their centrosomes. A similar trend was observed in mitotic cells, where growth of microtubules from both the spindle poles and the kinetochores was significantly attenuated in symplekin-depleted samples (Fig. 5A and B). Thus, depletion of symplekin significantly alters microtubule polymerization, a process that is essential for normal spindle formation.

FIG. 5.

Depletion of SYMPK reduces microtubule stability. (A) H1299 cells transfected with indicated siRNAs for 96 h were treated with 11 μM nocodazole for 2 h. Cells were fixed at 0 and 10 min post-nocodazole washout and stained with β-tubulin (more ...)

CKAP5 expression is sensitive to depletion of symplekin.

Symplekin is a multifunctional protein implicated in transcription and translation as well as signaling at tight junctions (3, 21, 25, 43). The localization pattern of symplekin has previously been described at the tight junctions and in the nucleus (25), the latter of which is a pattern that we also observe in H1299 cells (see Fig. S1A in the supplemental material). During mitosis, symplekin is distributed diffusely throughout the nucleoplasm and does not appear to localize to a specific mitotic structure at the microscopic resolution employed in this study (Fig. S1A), suggesting that symplekin does not impact mitosis through a direct association with the mitotic machinery. Given the role of symplekin in gene expression (8, 21, 38) and the errors observed in spindle integrity, mitotic progression, and microtubule nucleation, we hypothesized that symplekin could be impacting expression of proteins required for mitotic spindle formation. To assess this possibility, we performed a functional analysis of a panel of proteins required for proper centrosomal maturation and microtubule nucleation (1, 4, 10, 15, 17, 19, 47, 48). Symplekin depletion alone had no detectable effect on the localization and expression of almost all proteins studied (Fig. S1B). However, symplekin depletion had a profound effect on the expression of CKAP5 as detected by both immunofluorescence (Fig. (Fig.66 A) and immunoblot analysis (Fig. 6B and C). CKAP5 displays an elevated expression pattern in tumor cells (11) and localizes to the centrosome, where it is thought to enhance microtubule polymerization and nucleation (12, 22). CKAP5 depletion significantly decreases the viability of H1155 and H1299 cells (Fig. S2A). This effect is likely due to the potent impact of CKAP5 on microtubule polymerization and stability in H1299 and other cell types (Fig. S2B) (16, 19, 31).

FIG. 6.

SYMPK depletion leads to loss of CKAP5. (A) H1299 cells transfected with indicated siRNAs were fixed at 80 h posttransfection and immunostained for CKAP5 (red), Aurora-A (green), and DAPI. (B) Whole-cell lysates from H1299 cells transfected with indicated (more ...)

Given symplekin's role in transcription and translation, we evaluated CKAP5 transcript levels in symplekin-depleted cells. Symplekin depletion did not affect the level of CKAP5 mRNA, suggesting a posttranscriptional mode of regulation (Fig. (Fig.6D).6D). To determine whether the effects on CKAP5 were mediated by increased degradation, we evaluated CKAP5 protein levels in symplekin-depleted cells exposed to the proteosome inhibitor MG-132. CKAP5 levels were globally increased in MG-132-treated cells; however, proteasome inhibition was not sufficient to rescue the reduced CKAP5 levels observed in symplekin-depleted cells (Fig. (Fig.6E).6E). Since CKAP5 stabilizes microtubules primarily by opposing the depolymerizing activity of mitotic centromere-associated kinesin (MCAK), we evaluated the impact of codepletion of SYMPK and MCAK on the microtubule network. In H1299 cells, MCAK depletion impairs microtubule depolymerization by nocodazole, as has previously been reported (2). Importantly, codepletion of MCAK and SYMPK results in the depolymerization of microtubules in the presence of nocodazole (Fig. (Fig.6F6F).

Multiple polyadenylation components collaborate with paclitaxel.

Symplekin is a core component of the polyadenylation machinery, which has recently been implicated in regulating mitotic progression through phase-specific changes in poly(A) tail length (37). To determine if attenuation of the polyadenylation complex in general can collaborate with paclitaxel, we retrospectively examined the impact of the 14 core polyadenylation proteins in our original genome-wide paclitaxel sensitivity screen (45). In addition to symplekin, depletion of the polyadenylation proteins CPSF1, CSTF2, and CPSF3 all enhanced paclitaxel sensitivity to some degree in our primary screen. In H1299 cells, depletion of both CPSF1 and CPSF3 reduced CKAP5 protein levels as detected by immunoblot analysis (Fig. (Fig.77 A), suggesting that CKAP5 expression is exquisitely sensitive to perturbations of the polyadenylation complex. However, the sensitization observed with CSTF2 is uncoupled from the CKAP5 phenotype. Additionally, H1299 cells depleted of CSTF2, CPSF1, or CPSF3 and exposed to paclitaxel demonstrated a significant increase in the occurrence of multi- and micronucleated cells (Fig. (Fig.7B).7B). To determine if these subunits impacted mitotic progression in a manner similar to that of symplekin, we employed our time-lapse imaging system in H1155 GFP-H2B cells to evaluate mitotic outcomes. As with symplekin, CPSF1, CPSF3, and CSTF2 depletion increased the frequency of abnormal mitotic exits (Fig. (Fig.7C).7C). Surprisingly, CSTF2 depletion exhibited a higher prevalence of multipolar divisions than did depletion of the other components. Thus, altered expression of multiple polyadenylation components has acute effects on mitotic fidelity.

FIG. 7.

Polyadenylation is required for CKAP5 expression and faithful mitosis. (A) Whole-cell lysates from H1299 cells transfected with indicated siRNAs for 96 h were immunoblotted for CKAP5 expression. (B) H1299 cells were transfected with the indicated siRNAs (more ...)

DISCUSSION

We have uncovered an intimate functional connection between polyadenylation machinery and the formation of a bipolar spindle needed for accurate segregation of chromosomes. These mitotic defects are due, at least in part, to attenuation of the microtubule polymerization machinery and loss of microtubule dynamicity, which is essential for chromosome capture and alignment. The observation that depletion of multiple components of the polyadenylation complex leads to mitotic defects demonstrates an unanticipated and critical contribution of this complex to mitotic progression by supporting microtubule dynamics. This selectivity is a bit surprising since polyadenylation is likely an essential housekeeping process required for many biological processes. However, hypomorphic analyses, such as RNAi, allow us to uncover processes that are most vulnerable to alterations of a given housekeeping function. Our results, taken together with the identification of symplekin as the most statistically significant sensitizer to paclitaxel in the genome, suggest that polyadenylation machinery is tightly coupled to mitotic progression.

In particular, we find that the protein expression level of a critical mitotic component, CKAP5, is sensitive to depletion of polyadenylation machinery. While we have not yet determined if CKAP5 protein expression can be directly regulated by polyadenylation, we find that symplekin depletion does not appear to affect CKAP5 transcript abundance or protein turnover. Thus, the changes that we observe in CKAP5 protein expression could be due to an alteration in translation initiation or mRNA stability, which could be a direct result of the depletion of key polyadenylation components. Alternatively, perturbations in polyadenylation machinery, which may be impacting a large set of transcripts (37), could alter endogenous mechanisms that regulate CKAP5 protein levels. In either case, we have revealed that mitotic integrity, microtubule dynamics, and CKAP5 levels are sensitive to alterations in the polyadenylation machinery.

An emerging paradigm is that polyadenylation may play an important role in tumorigenesis. Most transcripts have alternative polyadenylation (APA) sites, which allow for regulation of the 3′ untranslated region (UTR) length. APA has been correlated with proliferation, as activation of T cells induces a global shortening of 3′ UTRs (40), and with tumorigenesis, where truncation of the 3′ UTR is widespread, potentially conferring resistance to microRNA (miRNA)-mediated silencing to support oncogenic phenotypes (35, 42). In addition, symplekin expression in lung and colon cancer cells is elevated compared to that in normal cells, indicating that an upregulation of polyadenylation machinery could be a frequent event during tumorigenesis (8, 41). Our findings that symplekin depletion induces mitotic defects in tumor cells, but not in normal cells, suggest that the demand for cell division in tumor cells may heighten the dependency on polyadenylation machinery to maintain the molecular framework that supports mitosis. Collectively, these results suggest that polyadenylation may be an acquired vulnerability in tumor cells. Our results indicate that this pressure point may be best observed and exploited through combinatorial targeting of mitosis. Antimitotics, such as paclitaxel, are first-line chemotherapeutics whose effectiveness is limited by significant toxicity and acquired resistance. Thus, therapeutic regimens that combine antimitotics with polyadenylation inhibitors may have an enhanced effectiveness for cytotoxicity in tumor cells while decreasing adverse events in normal tissues.

Supplementary Material

[Supplemental material]

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Acknowledgments

We thank Charlene Ross for support with animal studies and Mark Cronan for assistance with live-cell imaging.

This work was supported by the PHS grant CA128926, from the NCI to A.W.W., as well as the North Carolina University Cancer Research Fund. K.M.C. was supported by the NCI training grant CA071341-14 and general medicine training grant GM008719.

Footnotes

Published ahead of print on 7 September 2010.

^†Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

1. Bahe, S., Y. D. Stierhof, C. J. Wilkinson, F. Leiss, and E. A. Nigg. 2005. Rootletin forms centriole-associated filaments and functions in centrosome cohesion. J. Cell Biol. 171:27-33. [PMC free article] [PubMed]

2. Bakhoum, S. F., G. Genovese, and D. A. Compton. 2009. Deviant kinetochore microtubule dynamics underlie chromosomal instability. Curr. Biol. 19:1937-1942. [PMC free article] [PubMed]

3. Barnard, D. C., K. Ryan, J. L. Manley, and J. D. Richter. 2004. Symplekin and xGLD-2 are required for CPEB-mediated cytoplasmic polyadenylation. Cell 119:641-651. [PubMed]

4. Barr, A. R., and F. Gergely. 2007. Aurora-A: the maker and breaker of spindle poles. J. Cell Sci. 120:2987-2996. [PubMed]

5. Bartz, S. R., Z. Zhang, J. Burchard, M. Imakura, M. Martin, A. Palmieri, R. Needham, J. Guo, M. Gordon, N. Chung, P. Warrener, A. L. Jackson, M. Carleton, M. Oatley, L. Locco, F. Santini, T. Smith, P. Kunapuli, M. Ferrer, B. Strulovici, S. H. Friend, and P. S. Linsley. 2006. Small interfering RNA screens reveal enhanced cisplatin cytotoxicity in tumor cells having both BRCA network and TP53 disruptions. Mol. Cell. Biol. 26:9377-9386. [PMC free article] [PubMed]

6. Brass, A. L., D. M. Dykxhoorn, Y. Benita, N. Yan, A. Engelman, R. J. Xavier, J. Lieberman, and S. J. Elledge. 2008. Identification of host proteins required for HIV infection through a functional genomic screen. Science 319:921-926. [PubMed]

7. Brass, A. L., I. C. Huang, Y. Benita, S. P. John, M. N. Krishnan, E. M. Feeley, B. J. Ryan, J. L. Weyer, L. van der Weyden, E. Fikrig, D. J. Adams, R. J. Xavier, M. Farzan, and S. J. Elledge. 2009. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 139:1243-1254. [PMC free article] [PubMed]

8. Buchert, M., M. Papin, C. Bonnans, C. Darido, W. S. Raye, V. Garambois, A. Pelegrin, J. F. Bourgaux, J. Pannequin, D. Joubert, and F. Hollande. 2010. Symplekin promotes tumorigenicity by up-regulating claudin-2 expression. Proc. Natl. Acad. Sci. U. S. A. 107:2628-2633. [PubMed]

9. Cao, Q., and J. D. Richter. 2002. Dissolution of the maskin-eIF4E complex by cytoplasmic polyadenylation and poly(A)-binding protein controls cyclin B1 mRNA translation and oocyte maturation. EMBO J. 21:3852-3862. [PubMed]

10. Chang, P., M. Coughlin, and T. J. Mitchison. 2009. Interaction between poly(ADP-ribose) and NuMA contributes to mitotic spindle pole assembly. Mol. Biol. Cell 20:4575-4585. [PMC free article] [PubMed]

11. Charrasse, S., M. Mazel, S. Taviaux, P. Berta, T. Chow, and C. Larroque. 1995. Characterization of the cDNA and pattern of expression of a new gene over-expressed in human hepatomas and colonic tumors. Eur. J. Biochem. 234:406-413. [PubMed]

12. Charrasse, S., M. Schroeder, C. Gauthier-Rouviere, F. Ango, L. Cassimeris, D. L. Gard, and C. Larroque. 1998. The TOGp protein is a new human microtubule-associated protein homologous to the Xenopus XMAP215. J. Cell Sci. 111:1371-1383. [PubMed]

13. Chatterjee, S., and J. K. Pal. 2009. Role of 5′- and 3′-untranslated regions of mRNAs in human diseases. Biol. Cell 101:251-262. [PubMed]

14. Collinet, C., M. Stoter, C. R. Bradshaw, N. Samusik, J. C. Rink, D. Kenski, B. Habermann, F. Buchholz, R. Henschel, M. S. Mueller, W. E. Nagel, E. Fava, Y. Kalaidzidis, and M. Zerial. 2010. Systems survey of endocytosis by multiparametric image analysis. Nature 464:243-249. [PubMed]

15. Dictenberg, J. B., W. Zimmerman, C. A. Sparks, A. Young, C. Vidair, Y. Zheng, W. Carrington, F. S. Fay, and S. J. Doxsey. 1998. Pericentrin and gamma-tubulin form a protein complex and are organized into a novel lattice at the centrosome. J. Cell Biol. 141:163-174. [PMC free article] [PubMed]

16. Dionne, M. A., A. Sanchez, and D. A. Compton. 2000. ch-TOGp is required for microtubule aster formation in a mammalian mitotic extract. J. Biol. Chem. 275:12346-12352. [PubMed]

17. Du, Q., L. Taylor, D. A. Compton, and I. G. Macara. 2002. LGN blocks the ability of NuMA to bind and stabilize microtubules. A mechanism for mitotic spindle assembly regulation. Curr. Biol. 12:1928-1933. [PubMed]

18. Ganesan, A. K., H. Ho, B. Bodemann, S. Petersen, J. Aruri, S. Koshy, Z. Richardson, L. Q. Le, T. Krasieva, M. G. Roth, P. Farmer, and M. A. White. 2008. Genome-wide siRNA-based functional genomics of pigmentation identifies novel genes and pathways that impact melanogenesis in human cells. PLoS Genet. 4:e1000298. [PMC free article] [PubMed]

19. Gergely, F., V. M. Draviam, and J. W. Raff. 2003. The ch-TOG/XMAP215 protein is essential for spindle pole organization in human somatic cells. Genes Dev. 17:336-341. [PubMed]

20. Groisman, I., Y. S. Huang, R. Mendez, Q. Cao, W. Theurkauf, and J. D. Richter. 2000. CPEB, maskin, and cyclin B1 mRNA at the mitotic apparatus: implications for local translational control of cell division. Cell 103:435-447. [PubMed]

21. Hofmann, I., M. Schnolzer, I. Kaufmann, and W. W. Franke. 2002. Symplekin, a constitutive protein of karyo- and cytoplasmic particles involved in mRNA biogenesis in Xenopus laevis oocytes. Mol. Biol. Cell 13:1665-1676. [PMC free article] [PubMed]

22. Holmfeldt, P., S. Stenmark, and M. Gullberg. 2004. Differential functional interplay of TOGp/XMAP215 and the KinI kinesin MCAK during interphase and mitosis. EMBO J. 23:627-637. [PubMed]

23. Jordan, M. A., and L. Wilson. 2004. Microtubules as a target for anticancer drugs. Nat. Rev. Cancer 4:253-265. [PubMed]

24. Karlas, A., N. Machuy, Y. Shin, K. P. Pleissner, A. Artarini, D. Heuer, D. Becker, H. Khalil, L. A. Ogilvie, S. Hess, A. P. Maurer, E. Muller, T. Wolff, T. Rudel, and T. F. Meyer. 2010. Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 463:818-822. [PubMed]

25. Keon, B. H., S. Schafer, C. Kuhn, C. Grund, and W. W. Franke. 1996. Symplekin, a novel type of tight junction plaque protein. J. Cell Biol. 134:1003-1018. [PMC free article] [PubMed]

26. Kittler, R., L. Pelletier, A. K. Heninger, M. Slabicki, M. Theis, L. Miroslaw, I. Poser, S. Lawo, H. Grabner, K. Kozak, J. Wagner, V. Surendranath, C. Richter, W. Bowen, A. L. Jackson, B. Habermann, A. A. Hyman, and F. Buchholz. 2007. Genome-scale RNAi profiling of cell division in human tissue culture cells. Nat. Cell Biol. 9:1401-1412. [PubMed]

27. Kittler, R., G. Putz, L. Pelletier, I. Poser, A. K. Heninger, D. Drechsel, S. Fischer, I. Konstantinova, B. Habermann, H. Grabner, M. L. Yaspo, H. Himmelbauer, B. Korn, K. Neugebauer, M. T. Pisabarro, and F. Buchholz. 2004. An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature 432:1036-1040. [PubMed]

28. Krishnan, M. N., A. Ng, B. Sukumaran, F. D. Gilfoy, P. D. Uchil, H. Sultana, A. L. Brass, R. Adametz, M. Tsui, F. Qian, R. R. Montgomery, S. Lev, P. W. Mason, R. A. Koski, S. J. Elledge, R. J. Xavier, H. Agaisse, and E. Fikrig. 2008. RNA interference screen for human genes associated with West Nile virus infection. Nature 455:242-245. [PMC free article] [PubMed]

29. Kwon, M., S. A. Godinho, N. S. Chandhok, N. J. Ganem, A. Azioune, M. Thery, and D. Pellman. 2008. Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes. Genes Dev. 22:2189-2203. [PubMed]

30. Leber, B., B. Maier, F. Fuchs, J. Chi, P. Riffel, S. Anderhub, L. Wagner, A. D. Ho, J. L. Salisbury, M. Boutros, and A. Kramer. 2010. Proteins required for centrosome clustering in cancer cells. Sci. Transl. Med. 2:33ra38. [PubMed]

31. Lee, M. J., F. Gergely, K. Jeffers, S. Y. Peak-Chew, and J. W. Raff. 2001. Msps/XMAP215 interacts with the centrosomal protein D-TACC to regulate microtubule behaviour. Nat. Cell Biol. 3:643-649. [PubMed]

32. Li, Q., A. L. Brass, A. Ng, Z. Hu, R. J. Xavier, T. J. Liang, and S. J. Elledge. 2009. A genome-wide genetic screen for host factors required for hepatitis C virus propagation. Proc. Natl. Acad. Sci. U. S. A. 106:16410-16415. [PubMed]

33. Mandel, C. R., Y. Bai, and L. Tong. 2008. Protein factors in pre-mRNA 3′-end processing. Cell. Mol. Life Sci. 65:1099-1122. [PMC free article] [PubMed]

34. Marash, L., N. Liberman, S. Henis-Korenblit, G. Sivan, E. Reem, O. Elroy-Stein, and A. Kimchi. 2008. DAP5 promotes cap-independent translation of Bcl-2 and CDK1 to facilitate cell survival during mitosis. Mol. Cell 30:447-459. [PubMed]

35. Mayr, C., and D. P. Bartel. 2009. Widespread shortening of 3′UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell 138:673-684. [PMC free article] [PubMed]

36. Neumann, B., T. Walter, J. K. Heriche, J. Bulkescher, H. Erfle, C. Conrad, P. Rogers, I. Poser, M. Held, U. Liebel, C. Cetin, F. Sieckmann, G. Pau, R. Kabbe, A. Wunsche, V. Satagopam, M. H. Schmitz, C. Chapuis, D. W. Gerlich, R. Schneider, R. Eils, W. Huber, J. M. Peters, A. A. Hyman, R. Durbin, R. Pepperkok, and J. Ellenberg. 2010. Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes. Nature 464:721-727. [PMC free article] [PubMed]

37. Novoa, I., J. Gallego, P. G. Ferreira, and R. Mendez. 2010. Mitotic cell-cycle progression is regulated by CPEB1 and CPEB4-dependent translational control. Nat. Cell Biol. 12:447-456. [PubMed]

38. Radford, H. E., H. A. Meijer, and C. H. de Moor. 2008. Translational control by cytoplasmic polyadenylation in Xenopus oocytes. Biochim. Biophys. Acta 1779:217-229. [PMC free article] [PubMed]

39. Rasband, W. S. 1997-2009. ImageJ. National Institutes of Health, Bethesda, MD. http://rsb.info.nih.gov/ij/.

40. Sandberg, R., J. R. Neilson, A. Sarma, P. A. Sharp, and C. B. Burge. 2008. Proliferating cells express mRNAs with shortened 3′ untranslated regions and fewer microRNA target sites. Science 320:1643-1647. [PMC free article] [PubMed]

41. Shen, J., C. Behrens, I. I. Wistuba, L. Feng, J. J. Lee, W. K. Hong, and R. Lotan. 2006. Identification and validation of differences in protein levels in normal, premalignant, and malignant lung cells and tissues using high-throughput Western array and immunohistochemistry. Cancer Res. 66:11194-11206. [PubMed]

42. Singh, P., T. L. Alley, S. M. Wright, S. Kamdar, W. Schott, R. Y. Wilpan, K. D. Mills, and J. H. Graber. 2009. Global changes in processing of mRNA 3′ untranslated regions characterize clinically distinct cancer subtypes. Cancer Res. 69:9422-9430. [PMC free article] [PubMed]

43. Takagaki, Y., and J. L. Manley. 2000. Complex protein interactions within the human polyadenylation machinery identify a novel component. Mol. Cell. Biol. 20:1515-1525. [PMC free article] [PubMed]

44. Tay, J., R. Hodgman, M. Sarkissian, and J. D. Richter. 2003. Regulated CPEB phosphorylation during meiotic progression suggests a mechanism for temporal control of maternal mRNA translation. Genes Dev. 17:1457-1462. [PubMed]

45. Whitehurst, A. W., B. O. Bodemann, J. Cardenas, D. Ferguson, L. Girard, M. Peyton, J. D. Minna, C. Michnoff, W. Hao, M. G. Roth, X. J. Xie, and M. A. White. 2007. Synthetic lethal screen identification of chemosensitizer loci in cancer cells. Nature 446:815-819. [PubMed]

46. Whitehurst, A. W., R. Ram, L. Shivakumar, B. Gao, J. D. Minna, and M. A. White. 2008. The RASSF1A tumor suppressor restrains anaphase-promoting complex/cyclosome activity during the G1/S phase transition to promote cell cycle progression in human epithelial cells. Mol. Cell. Biol. 28:3190-3197. [PMC free article] [PubMed]

47. Williams, B., G. Leung, H. Maiato, A. Wong, Z. Li, E. V. Williams, C. Kirkpatrick, C. F. Aquadro, C. L. Rieder, and M. L. Goldberg. 2007. Mitch—a rapidly evolving component of the Ndc80 kinetochore complex required for correct chromosome segregation in Drosophila. J. Cell Sci. 120:3522-3533. [PubMed]

48. Zimmerman, W., and S. J. Doxsey. 2000. Construction of centrosomes and spindle poles by molecular motor-driven assembly of protein particles. Traffic 1:927-934. [PubMed]

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