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
). 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
). 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.