Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation.

Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. Here, we show that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 min, arguing strongly against a splicing-related mitotic function of these factors. We demonstrate that both SFs bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2- and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells.

Drosophila Dgt6 interacts with Ndc80, Msps/XMAP215, and gamma-tubulin to promote kinetochore-driven MT formation.

In centrosome-containing cells, spindle assembly relies on microtubules (MTs) nucleated from both centrosomes and chromosomes. Recent work has suggested that additional spindle MTs can be nucleated by gamma-tubulin ring complexes (gamma-TuRCs) that associate laterally with preexisting spindle MTs, leading to spindle amplification. It has been proposed that in Drosophila S2 cells, gamma-TuRCs are anchored to the spindle MTs by augmin, a multiprotein complex that contains at least eight subunits. Here we show that the Dgt6 component of augmin is primarily required for kinetochore fiber (k-fiber) formation. An analysis of MT regrowth after cold exposure showed that formation of kinetochore-driven k-fibers is severely impaired in Dgt6-depleted cells. In control cells, these fibers are enriched in Dgt6, gamma-tubulin, and Msps/XMAP215. Consistent with these results, Dgt6 coprecipitates with Msps, D-TACC, gamma-tubulin, Ndc80, and Nuf2. However, RNA interference (RNAi)-mediated inhibition of gamma-tubulin, Msps/XMAP215, or Ndc80/Hec1 reduced but did not abolish k-fiber regrowth. These results indicate that Dgt6 plays a pivotal role in kinetochore-driven k-fiber formation, mediating nucleation and/or initial stabilization of chromosome-induced MTs. We propose that Dgt6 binds and stabilizes nascent chromatin-induced MTs, facilitating their interaction with the Ndc80-Nuf2 complex. Dgt6 may also promote elongation of kinetochore-driven k-fibers through its interaction with gamma-tubulin and Msps.

Identification of Drosophila mitotic genes by combining co-expression analysis and RNA interference.

RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression-based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression-based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins.

Mammalian RanBP1 regulates centrosome cohesion during mitosis.

The Ran GTPase plays a central function in control of nucleo-cytoplasmic transport in interphase. Mitotic roles of Ran have also been firmly established in Xenopus oocyte extracts. In this system, Ran-GTP, or the RCC1 exchange factor for Ran, drive spindle assembly by regulating the availability of 'aster-promoting activities'. In previous studies to assess whether the Ran network also influences mitosis in mammalian cells, we found that overexpression of Ran-binding protein 1 (RanBP1), a major effector of Ran, induces multipolar spindles. We now show that these abnormal spindles are generated through loss of cohesion in mitotic centrosomes. Specifically, RanBP1 excess induces splitting of mother and daughter centrioles at spindle poles; the resulting split centrioles can individually organize functional microtubule arrays, giving rise to functional spindle poles. RanBP1-dependent centrosome splitting is specifically induced in mitosis and requires microtubule integrity and Eg5 activity. In addition, we have identified a fraction of RanBP1 at the centrosome. These data indicate that overexpressed RanBP1 interferes with crucial factor(s) that control structural and dynamic features of centrosomes during mitosis and contribute to uncover novel mitotic functions downstream of the Ran network.

E1A deregulates the centrosome cycle in a Ran GTPase-dependent manner.

By means of the yeast two-hybrid system, we have discovered a novel physical interaction between the adenovirus E1A oncoprotein and Ran, a small GTPase which regulates nucleocytoplasmic transport, cell cycle progression, and mitotic spindle organization. Expression of E1A elicits induction of S phase and centrosome amplification in a variety of rodent cell lines. The induction of supernumerary centrosomes requires functional RCC1, the nucleotide exchange factor for Ran and, hence, a functional Ran network. The E1A portion responsible for the interaction with Ran is the extreme NH(2)-terminal region (amino acids 1-36), which is also required for the induction of centrosome amplification. In an in vitro assay with recombinant proteins, wild-type E1A interferes with nucleotide exchange on Ran, whereas an E1A mutant, deleted from the extreme NH(2)-terminal region, does not. In addition, we detected an in vitro interaction between Ran and HPV-16 E7 and SV40 large T antigen, two oncoproteins functionally related to E1A. These findings suggest a common pathway of these oncoproteins in eliciting virus-induced genomic instability.