InteractiveFly: GeneBrief

Cenp-C: Biological Overview | References

Faithful transmission of genetic information during mitotic divisions depends on bipolar attachment of sister kinetochores to the mitotic spindle and on complete resolution of sister-chromatid cohesion immediately before the metaphase-to-anaphase transition (see Organization of the animal kinetochore). Separase is thought to be responsible for sister-chromatid separation, but its regulation is not completely understood. Therefore, a screen was carried out for genetic loci that modify the aberrant phenotypes caused by overexpression of the regulatory separase complex subunits Pimples/securin and Three rows in Drosophila. An interacting gene was found to encode a constitutive centromere protein. Characterization of its centromere localization domain revealed the presence of a diverged CENPC motif. While direct evidence for an involvement of this Drosophila Cenp-C homolog in separase activation at centromeres could not be obtained, in vivo imaging clearly demonstrated that it is required for normal attachment of kinetochores to the spindle (Heeger, 2005).

Separase functions as a protease at the metaphase-to-anaphase transition of mitosis. At this crucial cell cycle transition, separase cleaves the α-kleisin subunit (Scc1/Mcd1/Rad21) of the cohesin complex and thereby promotes the final release of sister-chromatid cohesion. The careful control of separase activity during the cell division cycle involves regulatory subunits. Securin is a subunit that accumulates and associates with separase during interphase. It acts as an inhibitor of separase activity. Thus, the rapid degradation of securin at the metaphase-to-anaphase transition via the anaphase-promoting complex/cyclosome (APC/C) pathway of ubiquitin-dependent proteolysis results in separase activation. In Drosophila, securin is encoded by the pimples (pim) gene and the catalytic protease subunit by the Separase (Sse) gene. Drosophila Sse lacks the extensive N-terminal regulatory domain that is present in separases outside the dipterans because the corresponding gene region appears to have evolved into an independent gene, three rows (thr). Drosophila Thr binds to Sse and is required for sister-chromatid separation during mitosis (Heeger, 2005).

The precise role of Thr and the corresponding N-terminal domains in nondipteran separases is not understood. Moreover, Pim and other securins are not just separase inhibitors but also contribute in an unknown positive manner to sister-chromatid separation. In fission yeast, securin recruits separase to the mitotic spindle, and similar observations have been described in other organisms. Separase activation and transport on spindle microtubules might confine its action to the congressed chromosomes in metaphase plates and in particular to the pericentromeric region. This hypothetical scenario might explain why only a minute and preferentially pericentromeric pool of Scc1 appears to be cleaved by separase during mitosis of higher eukaryotic cells, while the large majority of Scc1 remains intact (Heeger, 2005).

To identify additional genes that might contribute to separase regulation and function, a screen was performed for chromosomal regions that act as genetic modifiers of the aberrant phenotypes resulting from overexpression of Pim or a dominant-negative Thr fragment during Drosophila eye development. Molecular characterization of an interacting locus revealed that it encodes a constitutive centromere protein. Mapping of its centromere localization domain in combination with sequence comparisons among Drosophilid orthologs allowed its identification as the most diverged Cenp-C homolog. Cenp-C was originally identified as a human autoantigen localized to centromeres and found to display limited sequence similarity to budding yeast Mif2, which was identified by mutations affecting the fidelity of chromosome transmission during mitosis. Homologs have also been described in nematodes (HCP-4) and plants. For simplicity, Cenp-C has been used as a designation for all these homologs. Interestingly, recent analyses have demonstrated that Cenp-C, as well as Cenp-A, a histone H3 variant present in centromeric nucleosomes, evolve rapidly and adaptively in many lineages, perhaps driven by the rapid evolution of centromeric satellite sequences, and in Drosophila, Cenp-C was supposed to be absent. Apart from providing further support for the striking sequence divergence of ubiquitous eukaryotic centromere components, these findings also raise the possibility that separase activity might be enhanced by such components (Heeger, 2005).

Identification of Drosophila Cenp-C closes a prominent gap in the arguments for homologous centromere organization. Centromeric DNA sequences have evolved extremely rapidly and appear to have driven the coevolution of centromeric proteins during eukaryote evolution. The resulting low sequence similarity between centromeric proteins has effectively concealed the existence of a common set of constitutive eukaryotic centromere proteins until very recently. The first features demonstrated to be shared among fungal, plant, and animal centromeres were centromere-specific histone H3 variants. In addition to Cenp-A homologs, only one further constitutive centromere component, Cenp-C, has so far been shown to be present in each of the three main eukaryotic branches. In combination with the recent identification of related Cenp-H-, Cenp-I-, and Mis12-like proteins in both vertebrates and yeast, these results provide strong support for the notion of a common set of constitutive centromere proteins. These proteins, which are centromeric throughout the cell cycle, appear to provide a foundation for kinetochore assembly and spindle attachment during mitosis by recruiting several distinct multisubunit complexes that also contain highly diverged proteins (Heeger, 2005).

The extensive sequence divergence characteristically observed among homologous eukaryotic centromere and kinetochore proteins is striking, especially in the light of their common fundamental cellular function. The average amino acid identity observed in a genome-wide comparison of D. melanogaster and D. pseudoobscura ortholog pairs is 77% and only 38% in case of the Cenp-C pair. Moreover, based on the ratio between radical charge mutations and conservative substitutions in D. melanogaster and D. pseudoobscura ortholog pairs, Cenp-C is one of 44 genes likely to have evolved under positive selection. Except for a few very restricted regions, comparison of D. melanogaster Cenp-C with the orthologs from D. erecta and D. yakuba, which are closer relatives than D. pseudoobscura and thus amenable to d_N/d_S analyses, did not reveal strong evidence for positive selection, in contrast to the recent findings in plant and mammalian lineages. However, these d_N/d_S analyses ignore insertions and deletions (indels), which have occurred considerably more often during Cenp-C evolution in Drosophilids than in the mammalian lineage. Most of the indels are observed within the minimally conserved central regions of Drosophilid Cenp-C. Similar variabilities resulting from recurrent exon duplications have been observed in the central region of the plant Cenp-C genes (Heeger, 2005).

The adaptively evolving regions of mammalian Cenp-C have been shown to bind to DNA in vitro, consistent with the proposed coevolution of centromeric DNA and protein sequences (Talbert, 2004). However, this DNA binding in vitro is not sequence-specific, suggesting that interactions with centromere-specific proteins contribute to centromere localization of Cenp-C. As in other organisms, Cid/Cenp-A is also required for centromere localization of Cenp-C in Drosophila. High-resolution light microscopy of mitotic chromosomes has indicated that human Cenp-C covers the poleward-oriented peripheral region of the Cenp-A-containing centromeric chromatin (Blower, 2002). Direct interactions between Cenp-A and Cenp-C have not yet been demonstrated in any organism. Attempts with yeast two-hybrid experiments were also unsuccessful (Heeger, 2005).

The CENPC motif has recently been identified as the only region conserved among the Cenp-C orthologs from fungi, plant, and animals (Talbert, 2004). In Drosophilids, even this short motif of ~24 amino acids is not fully conserved in its C-terminal part. These results suggest that this CENPC motif is crucial for centromere localizationA single-point mutation affecting one of the invariant positions in the CENPC motif interferes with centromere localization of the C-terminal domain of Cenp-C in a transfection assay. This mutation was identified as the only missense mutation interfering with centromere localization after extensive random mutagenesis. Further experiments will reveal whether and how the CENPC motif contacts Cid/Cenp-A nucleosomes. It is emphasized, however, that also in Drosophila Cenp-C, other regions than the CENPC motif clearly contribute to efficient centromere localization. Centromere localization of the CN subregion (1009-1205), for instance, is only detected in a transfection assay in live but not in fixed cells, while centromere localization of the larger C region (1009-1411) is resistant to fixation (Heeger, 2005).

The highest conservation among Drosophilid Cenp-C proteins is observed within the N-terminal third, which is neither required nor sufficient for normal centromere localization. Nevertheless, prolonged overexpression of this domain in proliferating eye and wing imaginal disc cells results in severe defects. The conserved N-terminal Cenp-C domains (R and DH) might bind to kinetochore proteins and titrate these away from the centromere when overexpressed. Biochemical and genetic characterizations in Saccharomyces cerevisiae and Caenorhabditis elegans have suggested that Cenp-C is not only associated with Cenp-A, but that it also recruits the next layer of kinetochore proteins, in particular the Mis12/Mtw1 and Ndc80 complexes, which remain to be identified in Drosophila (Heeger, 2005).

As in yeast (Meluh, 1995), chicken (Fukagawa, 1997; Fukagawa, 1999), and mice (Kalitsis, 1998), Cenp-C is also an essential gene in Drosophila. Antibody microinjection experiments in mammalian cells (Tomkiel, 1994); RNA interference in C. elegans (Moore, 2001; Oegema, 2001); and phenotypic analysis in yeast (Brown, 1993), chicken cells (Fukagawa, 2001), and mutant Drosophila embryos demonstrate that Cenp-C is required for normal chromosome segregation during mitosis. in vivo imaging in Cenp-C mutant embryos discloses these defects in detail. Previously, in vivo imaging has also been applied in C. elegans CENP-C(RNAi) embryos (Moore, 2001; Oegema, 2001). The formation of holocentric chromosomes and transient Cenp-C recruitment only during mitosis differentiates C. elegans from other metazoans like Drosophila and mammalian cells. Moreover, in contrast to the findings in C. elegans, chromosome congression into a central plane is still observed in the Drosophila Cenp-C mutants. Presumably, this chromosome congression reflects the function of residual maternally provided Cenp-C, which is still detectable at this stage of analysis. Moreover, time-lapse analyses demonstrate that chromosome congression is not entirely normal in the Cenp-C mutant embryos. Metaphase plate formation is delayed and often does not lead to the highly ordered arrangement of all chromosomes characteristically observed before anaphase onset in wild type. Occasional chromosomes fail to achieve bipolar attachment in the Cenp-C mutants. These chromosomes do not segregate normally during anaphase. Cenp-C is, therefore, clearly required for normal attachment of kinetochores to the mitotic spindle (Heeger, 2005).

Evidently, the insufficiently attached chromosomes in Cenp-C mutant embryos are unable to inhibit the onset of anaphase, even though the mitotic spindle checkpoint appears to be at least partially functional in Cenp-C mutants at the analyzed stage. However, assembly of mitotic spindle checkpoint proteins might fail, particularly on the kinetochores of those chromosomes that do not attach correctly to the mitotic spindle (Heeger, 2005).

In principle, the chromosome segregation defects observed in the Cenp-C mutants might not only reflect impaired interactions between kinetochores and spindle. Segregation of sister chromatids to the spindle poles also depends on complete resolution of sister-chromatid cohesion at the metaphase-to-anaphase transition. This final separation of sister chromatids is thought to be achieved by separase-mediated cleavage of the Scc1/Rad21 subunit of those cohesin complexes that perdure in the pericentromeric region until the metaphase-to-anaphase transition. Several observations are consistent with the idea that a localized full activation of separase might be assisted by centromeric proteins. Accordingly, mutations in Cenp-C might reduce separase activity and thereby explain the genetic interactions with the regulatory separase subunits Pim/securin and Thr that led to the identification of Drosophila Cenp-C. No coimmunoprecipitation of Cenp-C with separase complex proteins was observed and no effects of Cenp-C mutations on Pim and Thr levels was observed. This analysis has, therefore, not exposed clear evidence for separase activation at centromeres. In contrast, Cenp-C is clearly required for normal chromosome attachment to the mitotic spindle, and thus the observed genetic interactions most likely reflect a summation of negative effects on the efficiency of sister-chromatid separation (by separase) and segregation (by the spindle). However, it is emphasized that the results certainly do not rule out a local activation of separase within the centromeric region (Heeger, 2005).

Spatial organization of a ubiquitous eukaryotic kinetochore protein network in Drosophila chromosomes

Chromosome segregation during meiosis and mitosis depends on the assembly of functional kinetochores within centromeric regions. Centromeric DNA and kinetochore proteins show surprisingly little sequence conservation despite their fundamental biological role. However, identification in Drosophila of the most diverged orthologs identified so far, which encode components of a kinetochore protein network including the Ndc80 and Mis complexes, further emphasizes the notion of a shared eukaryotic kinetochore design. To determine its spatial organization, quantitative light microscopy was used to analyzed hundreds of native chromosomes from transgenic Drosophila strains coexpressing combinations of red and green fluorescent fusion proteins, fully capable of providing the essential wild-type functions. Thereby, Cenp-A/Cid, Cenp-C, Mis12 and the Ndc80 (CG9938) complex were mapped along the inter sister kinetochore axis with a resolution below 10 nm. The C terminus of Cenp-C was found to be near but well separated from the innermost component Cenp-A/Cid. The N terminus of Cenp-C is further out, clustered with Mis12 and the Spc25 end of the rod-like Ndc80 complex, which is known to bind to microtubules at its other more distal Ndc80/Nuf2 end (Schittenhelm, 2007).

Identification of Drosophila kinetochore proteins further exposes hidden similarities of kinetochore design in eukaryotes. In addition to the previously known, highly diverged Cenp-A/Cid and Cenp-C homologs, Drosophila expresses similarly diverged homologs of the Mis12 and Ndc80 complex network, which is also present in yeast, C. elegans, vertebrates, and presumably in plants as well. These ubiquitous CKC components have been localized along the intersister kinetochore axis with unprecedented spatial resolution. Early Drosophila embryos allow an efficient isolation of native mitotic chromosomes and thereby imaging with reduced background. Moreover, transgenic strains allow the expression of fluorescent fusion proteins, which were demonstrated to be fully functional by genetic complementation tests (Schittenhelm, 2007).

The position of fluorescent signal maxima has been determined within the kinetochore of native chromosomes released from embryos expressing fluorescent CKC fusion proteins. The CKC map is based on averaged data from hundreds of analyzed chromosomes. Therefore, its interpretation depends critically on the variability of kinetochore organization in individual chromosomes. For instance, in principle, a given component might be localized on the inner kinetochore side in 50% of the chromatids and on the outer side in the other half of the chromatids, resulting in a misleading central positioning in the CKC map. Theoretically, such variability should widen the distribution of the distances measured in individual chromosomes. However, kinetochore width is smaller than the spreading of the image of a point light source in the microscope, and several additional factors (like background, noise, pixelation) further limit the precision of the measurements. The effect of positional variability on distribution width of the measured values would therefore be very subtle. Moreover, none of the known CKC proteins has been firmly demonstrated to be a spatially invariable kinetochore component, precluding comparisons to an established standard distribution. However, the reproducible trilaminar structure of the kinetochore during prometaphase, that has been documented by EM, argues strongly against extensive organizational variability. It is emphasized that the difficulties in detecting subtle alterations in the distribution width of the measurements obtained for a given CKC component has important consequences even under the assumption that the spatial distribution of CKC components is essentially invariable in individual kinetochores. These difficulties prevent conclusions concerning the width occupied by a given CKC component within a kinetochore. For instance, Mis12 could either be confined to a single layer in the middle of the kinetochore or spread throughout the kinetochore, and both localization patterns would result in a central signal maximum. However, biochemical analyses of kinetochore proteins have so far revealed highly specific interactions, arguing strongly for a precise and restricted localization of CKC components. The following discussion is therefore based on the unproven but likely assumption that the kinetochore represents a precisely defined layered structure (Schittenhelm, 2007).

Based on previous analyses, Cenp-A, Cenp-C, and Mis12 are thought to be components of the inner plate of the characteristic trilaminar kinetochore structure apparent in the EM (Kline, 2006; Vos, 2006). The analyses indicate a significant separation between the inner most CKC component Cenp-A and all other CKC components analyzed here. Recently, Cenp-A nucleosomes purified from human cells were found to be intimately associated with the five proteins Cenp-M, Cenp-N, Cenp-T, Cenp-U, and Cenp-H in addition to Cenp-C (Foltz, 2006; Izuta, 2006; Okada, 2006). The apparent space between Cenp-A and Cenp-C might therefore be occupied by some of those proteins (Schittenhelm, 2007).

Many immunolocalization studies, including a recent study with Drosophila cells (Maiato, 2006), have failed to detect a comparable extensive spatial separation between Cenp-A/Cid and Cenp-C. However, immunolocalization with human chromosomes also revealed little overlap between Cenp-A and Cenp-C, with the latter extending over the top and bottom of a Cenp-A cylinder (Blower, 2002). Antigen accessibility problems, which were not excluded by Blower (2002), cannot affect the concurrent findings (Schittenhelm, 2007).

In this paper, Cenp-C is shown to be spread in a polar orientation across a central CKC region. The C-terminal domain of Cenp-C, which contains the most conserved region including the CENP-C motif (Talbert, 2004; Heeger, 2005), points toward the centromeric DNA. These C-terminal sequences are connected via minimally conserved spacer sequences to the N-terminal domain which is oriented toward the kinetochore spindle fibers. The N-terminal region of D. melanogaster Cenp-C contains some blocks which are highly conserved among Drosophilids (Heeger, 2005). These blocks might be involved in recruiting the next layer of kinetochore proteins which are suggested to include the Ndc80 and Mis12 complexes. Mis12 is close to the N-terminal Cenp-C region. Moreover, the Ndc80 complex component Spc25 (CG7242) appears to be even a bit closer but well separated by about 20 nm from the other Ndc80 component Nuf2 (CG8902). Apart from a polar Cenp-C orientation, these analyses therefore also indicate a polar orientation for the Ndc80 complex (Schittenhelm, 2007).

The tetrameric Ndc80 complex has a highly elongated, rod-like structure in vitro (Ciferri, 2005; Wei, 2005). The globular N-terminal domains of Ndc80 and Nuf2 are present on one end of the rod. The remainder of these two subunits forms an extended coiled coil which is further prolonged at its C-terminal end by binding to the N-terminal coiled coil region of the Spc24/Spc25 dimer. Closely associated C-terminal globular domains of Spc24 and Spc25 (Wei, 2006) form the other end of the rod. Scanning force microscopy and EM analyses have indicated that the coiled coil region separating the globular domains at the end of the Ndc80 complex has an extension of about 40 nm (Ciferri, 2005; Wei, 2005). This is twofold longer than the distance observed between fluorescent proteins at the N and C termini of Nuf2 and Spc25 in kinetochores of native Drosophila chromosomes. Many of the elongated Ndc80 complexes might not be perfectly oriented along the spindle axis, especially as the kinetochores in the preparations used in this study are not under tension. Such a nonuniform orientation could result in spatial distributions of the N and C termini of Nuf2 and Spc25, respectively, with signal maxima that are more closely spaced than their separation within an isolated complex. An analysis of the positions of CKC components in chromosomes that are bi-oriented within the spindle and under tension would clearly be of interest. However, the increased background levels present in living embryos have so far precluded such analyses (Schittenhelm, 2007).

The observed polar orientation of the Ndc80 complex within the kinetochore confirms the findings of a recent independent study (Deluca. 2006). Moreover, the observation that Ndc80 and Nuf2 kinetochore localization is no longer observed in the absence of Spc24 or Spc25 is consistent but does not prove an orientation of the complex with inner Spc24/Spc25 and outer Ndc80/Nuf2 globular domains, because absence of Spc24 or Spc25 for instance might simply result in an instability of other complex components, as often observed in the case of stable complexes (Schittenhelm, 2007 and references therein).

In budding yeast, the Ndc80 complex has been proposed to function as a connection between the inner components (CBF3 complex, Cenp-A/Cse4 nucleosome, Cenp-C/Mif2, Mis12/MIND complex) and the Dam/DASH complex which is required for bi-orientation and appears to form a ring around the single microtubule attaching to a yeast kinetochore. More recently, bacterial expression of the C. elegans KMN network composed of the Spc105/KNL-1, Mis12 and Ndc80 complexes has led to a convincing identification of two independent sites in this protein network that can bind directly to microtubules in vitro (Cheeseman, 2006). One of these microtubule binding sites is present within Spc105/KNL-1. The other is found within the globular N-terminal Ndc80 domain (Cheeseman, 2006) which is known to be within the outer kinetochore plates where kinetochore microtubules terminate (DeLuca, 2005). In vitro, the Ndc80 complex binds to microtubules at an angle (Cheeseman, 2006). A corresponding orientation of the Ndc80 complex within the kinetochore is fully consistent with the finding that the separation of the terminal globular domains of Spc25 and Nuf2 along the intersister kinetochore axis appears to be less than their separation along the axis of isolated complexes (Ciferri, 2005; Wei, 2005). Accordingly, the 'barbed end' of microtubules decorated with the Ndc80 complex would be predicted to correspond to the plus end (Schittenhelm, 2007 and references therein).

In conclusion, in addition to the identification of Drosophila Ndc80 and Mis12 complex components, this work provides a highly resolved structural framework integrating the most widely studied ubiquitous CKC components and a precise method for a future incorporation of additional proteins (Schittenhelm, 2007).

Centromeric α-satellite RNA is essential for the nucleolar localization of CENPC1 and INCENP

The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. This study demonstrates that centromeric α-satellite RNA and proteins CENPC1 and INCENP accumulate in the human interphase nucleolus in an RNA polymerase I-dependent manner. The nucleolar targeting of CENPC1 and INCENP requires α-satellite RNA, as evident from the delocalization of both proteins from the nucleolus in RNase-treated cells, and the nucleolar relocalization of these proteins following α-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, the nucleolar localization sequences on CENPC1 and INCENP have been identified. Evidence that CENPC1 is an RNA-associating protein that binds α-satellite RNA in an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and 'RNA replenishment' experiments, α-satellite RNA is show to be a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere. These data suggest that centromere satellite RNA directly facilitates the accumulation and assembly of centromere-specific nucleoprotein components at the nucleolus and mitotic centromere, and that the sequestration of these components in the interphase nucleolus provides a regulatory mechanism for their timely release into the nucleoplasm for kinetochore assembly at the onset of mitosis (Wong, 2007).

The centromere is a specialized structure on chromosomes for microtubule attachment to ensure the equal partitioning of chromosomes during cell division. This structure comprises two defined domains: the central core for the assembly of the kinetochore and the flanking pericentric heterochromatin for centromere cohesion. In Schizosaccharomyces pombe, the outer centromeric repeat sequences give rise to small interfering RNAs (siRNA) that participate in chromatin repression. The depletion of Dicer (a nuclease required for the processing of siRNAs) in a chicken cell line leads to the disruption of heterochromatin assembly and cohesion. However, Dicer depletion has no observable effect on the binding of the core kinetochore proteins CENPA and CENPC1, indicating that while the evolutionarily conserved RNA interference (RNAi) machinery is crucial for the establishment of the pericentric heterochromatin, it may not be essential for the core kinetochore region. A recent study in maize has further described the association of single-stranded centromeric transposable element and repeat RNA with the core kinetochore complex that is distinct from those at pericentric heterochromatin; however, the functional significance of the observed centromere RNA transcripts is unclear. Furthermore, little is known about the subnuclear distribution of centromere RNA, and the pathway and significance, if any, of such RNA in kinetochore formation and function (Wong, 2007).

The nucleolus is a specialized organelle of the interphase nucleus where ribosomal DNA is transcribed, pre-rRNA is processed, and ribosome subunits are assembled. In addition, this organelle is known to serve a variety of other essential cellular and cell cycle control functions by allowing the timely sequestration of specific trans-acting factors and the biogenesis of many cellular ribonucleoprotein particles (RNPs) including transfer tRNAs, small nuclear snRNAs, different mRNAs, and telomerase RNA. Specifically, numerous studies have directly demonstrated the accumulation of different centromere-associated proteins at the nucleolus, including borealin (See Drosophila Borealin-related), PARP1, and PARP2 (Wong, 2007 and references therein).

This study shows the enrichment of centromeric α-satellite RNA and centromere proteins CENPC1 and INCENP in the interphase nucleolus. Evidence is presented that the centromere satellite RNA is required for the assembly of centromere-associated nucleoprotein components at the nucleolus and kinetochore (Wong, 2007).

This study demonstrates the nucleolar accumulation of the constitutive centromere protein CENPC1, the centromere-associated chromosomal passenger protein INCENP, and centromeric α-satellite RNA. The nucleolus localization sequence (NoLS) motifs for both of these proteins coincide with the nucleus localization sequence (NLS) motifs, and are found at the N-terminal portions of the proteins. These NoLS motifs share significant homology with those of other nucleolus-associated proteins. Some of these proteins, including borealin, PARP1 and PARP2, and INCENP, also bind directly to the centromere (Wong, 2007).

The presence of centromeric α-satellite RNA is closely associated with the nucleolar localization of CENPC1 and INCENP, since RNase treatment induces a complete disruption of this localization. More specifically, in the RNA 'rescue' study, it was shown that the incubation of RNase-treated cells with α-satellite RNA results in the in situ targeting of myc-tagged CENPC1 and INCENP recombinant proteins to the nucleolus. Experiments involving ActD treatment further demonstrate that the nucleolar accumulation of α-satellite RNA, CENPC1, and INCENP is dependent on active RNA polymerase I transcription. Previous studies have similarly shown that the nucleolar localization of centromere proteins borealin and PARP1 and PARP2 is dependent on active nucleolar transcriptional activity. Recent studies in S. pombe have shown that RNA polymerase II and IV (a unique RNA polymerase in plants) are essential for the generation of siRNAs. The transcription machinery that modulates the transcription of centromere repeats or siRNA synthesis in vertebrates is still unknown. However, it is unlikely that RNA polymerase I is directly driving the transcription of the nucleolar α-satellite RNA, as indicated by nuclear run-on transcription data. Furthermore, the depletion of α-satellite RNA-FISH signals is unlikely to be due to the down-regulation of some specific proteins, since the conditions used for ActD treatment did not affect the expression levels of proteins in general, as exemplified by those of CENPC1 and INCENP. In this context, the mechanism of action for the RNA polymerase I remains undefined, although it may be directly involved in providing a structural network or complex that retains the various centromere components in the nucleolus (Wong, 2007).

The nucleolus serves the important function of ribosome synthesis. Increasing numbers of studies now show that it is also a multitasking organelle that engages in other important cellular functions. An example of the pluri-functionality of the nucleolus is the cell cycle-dependent nucleolar localization of the telomere components hTERT (reverse transcriptase catalytic subunit of telomerase), hTR (telomerase RNA), and telomeric DNA-binding protein TERF2, where these components have been shown to be released from the nucleolus at late S and G2/M phase at a time that coincides with telomere elongation. The data suggest that the nucleolus may similarly sequester centromeric components such as centromere RNA and proteins for timely delivery to the chromosomes for kinetochore assembly at mitosis. The centromere proteins, in particular the chromosomal passenger proteins such as borealin, PARP1 and PARP2, and INCENP, display dynamic changes in distribution patterns during various stages of the cell cycle. Although these proteins only bind the centromere following mitotic onset, they are expressed much earlier in mid-S to G2 phase and have been shown to accumulate in the nucleolus. During this period, the nucleolus may serve a repository role for the temporal storage of centromere RNA and proteins, and control the timely release of these components into the nucleoplasm for kinetochore assembly at M phase. It is also possible that the assembly of functional kinetochore nucleoprotein complexes may take place in the nucleolus (Wong, 2007).

Treatment of metaphase cells with single-stranded RNA-specific RNases (but not with a double-stranded RNA-specific RNase) results in the significant but incomplete delocalization of CENPC1, and the complete delocalization of two centromere-associated chromosomal passenger proteins INCENP and survivin, from the metaphase centromere. Similar treatments have no significant effect on the centromeric localization of CENPA, CENPB, and CENPE. These results indicate that the assembly of INCENP and survivin, and to an extent CENPC1, but not CENPA, CENPB, and CENPE, at the metaphase centromere is dependent on the presence of single-stranded RNA (Wong, 2007).

Several lines of evidence suggest that at least a component of this single-stranded RNA is directly transcribed from the centromeric α-satellite DNA, and that this α-satellite RNA transcript associates directly with CENPC1. (1) Recombinant GST-fusion protein truncation constructs and site-specific mutagenesis were used to show that CENPC1 binds single-stranded α-satellite RNA probes derived from chromosomes 2, 4, 9, or 13/21, and ranging in size from 167-342 nucleotides (nt). This RNA-binding activity resides within the central region (amino acids 426-551) as well as the C-terminal portion of the protein. (2) By performing RT-PCR on RNA isolated following RNA-ChIP using an anti-CENPC1 antibody, in vivo binding of single-stranded α-satellite RNA with the CENPC1-associated kinetochore protein complex has been demonstrated. The RT-PCR results indicate that the kinetochore-associated α-satellite RNA has a size range corresponding to multiples of the 171-nt α-satellite repeating unit. These studies show the presence of single-stranded α-satellite RNA of 171 nt and larger and belonging to the different chromosome-specific α-satellite subfamilies. However, the possibility cannot be excluded that shorter RNA or α-satellite RNA of other sequences may be present, as the present detection may bias against these RNAs (Wong, 2007).

(3) It has been shown that the in situ replenishment of single-stranded α-satellite RNA significantly restores the relocalization of myc-tagged CENPC1 and INCENP to the centromeres of some of the chromosomes following RNase treatment. The incomplete rescue of the recruitment of the myc-tagged recombinant proteins to the kinetochore could be due to the inappropriate folding or modification of the recombinant proteins or replenishing RNA used. Further investigations are needed to discern these possibilities and define the characteristics and possible heterogeneity associated with these RNA transcripts (Wong, 2007).

(4) It has been shown that wild-type GFP-CENPC1, but not the mutant construct with mutated residues within the NoLs domain, is able to partially restore the functionality of the CENPC1-depleted kinetochore in the CENPC1 RNAi-knockdown cells and rescue the cells from the inhibition of cell growth. The failure of the over-expression of the mutated GFP-CENPC1 protein in improving the rate of cell growth suggests that the NoLS domain is essential for the proper function of CENPC1 (Wong, 2007).

Together, this analysis indicates that, in addition to the important role played by the short, double-stranded siRNA at the pericentric heterochromatin, a distinct class of longer, single-stranded centromeric α-satellite RNA is an important structural component of the human centromere (Wong, 2007).

Central to this model is the finding that centromeric α-satellite RNA is essential for the enrichment of centromere proteins CENPC1, INCENP, and/or survivin at the nucleolus and mitotic centromere. It is proposed that the nucleolar sequestration of centromere-specific nucleoprotein components provides a regulatory mechanism for the timely release of these components into the nucleoplasm for kinetochore assembly at the onset of mitosis. The finding that CENPC1 binds centromere satellite RNA, whereas previous in vitro studies have shown that CENPC1 is also DNA-binding (Yang, 1996; Politi, 2002), together suggest that this protein has a dual RNA- and DNA-binding function. It is therefore possible that one pool of CENPC1 may play a constitutive centromere DNA-binding role that persists throughout the cell cycle, while a second pool of this protein may act directly as, or be part of, a chaperone mechanism to relocate centromeric α-satellite RNA and centromere proteins, including INCENP, from the nucleolus onto the mitotic centromere. The RNA dependence of the nucleolar localization of both CENPC1 and INCENP suggests that these proteins may associate with the centromeric α-satellite RNA and be assembled into a nucleoprotein complex in the nucleolus. This nucleolar chaperone complex may further include other centromere-associated proteins such as borealin, PARP1, and PARP2, since these components have also been shown to accumulate in the nucleolus. In further support of the proposed dual role of CENPC1, significantly higher quantitative immunofluorescence signals have consistently been observed for CENPC1 (but not other constitutive centromere proteins examined, including CENPA and CENPB) on the metaphase centromere compared with those of the interphase centromere. Of interest, a recent study (Kwon, 2007) has shown that CENPC1 is targeted to interphase or metaphase centromeres through interactions with different sets of centromere proteins, providing further evidence that CENPC1 is localized to the centromere via two independent pathways during interphase or the mitosis stage. The present study has shown that treatment with single-stranded RNA-specific RNases results in a significant, but not complete, delocalization of CENPC1 from the metaphase centromere, consistent with the idea that only the RNA-, but not the DNA-, associated pool of CENPC1 (and components of its complex, such as INCENP) is sensitive to the RNase treatment. The data demonstrate that survivin does not localize to the nucleolus, although its localization to the metaphase centromere is also RNA-dependent. This can be explained by the incorporation of survivin into the metaphase chromosomes following the breakdown of the nucleolar membrane, via INCENP and/or borealin; previous work (Klein, 2006) has shown that survivin forms a functional complex with these two proteins for targeting to the centromere (Wong, 2007).

Search PubMed for articles about Drosophila Cenp-C

Blower, M. D., Sullivan, B. A. and Karpen, G. H (2002) Conserved organization of centromeric chromatin in flies and humans. Dev Cell 2: 319-330. Medline abstract: 11879637

Brown, M. T., Goetsch, L., and Hartwell, L. H. (1993). MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae. J. Cell Biol. 123: 387-403. Medline abstract: 8408221

Cheeseman, I. M., Chappie, J. S., Wilson-Kubalek, E. M. and Desai, A. (2006). The conserved KMN network constitutes the core microtubule-binding site of the kinetochore. Cell 127: 983-997. Medline abstract: 17129783

Ciferri, C., et al. (2005). Architecture of the human ndc80-hec1 complex, a critical constituent of the outer kinetochore. J. Biol. Chem. 280: 29088-29095. Medline abstract: 15961401

Deluca, J. G., et al. (2006). Kinetochore microtubule dynamics and attachment stability are regulated by hec1. Cell 127: 969-982. Medline abstract: 17129782

Foltz, D. R., et al. (2006) The human CENP-A centromeric nucleosome-associated complex. Nat Cell Biol 8: 458-469. Medline abstract: 16622419

Fukagawa, T. and Brown, W.R. (1997). Efficient conditional mutation of the vertebrate CENP-C gene. Hum. Mol. Genet. 6: 2301-2308. Medline abstract: 9361037

Fukagawa, T., Pendon, C., Morris, J., and Brown, W. (1999). CENP-C is necessary but not sufficient to induce formation of a functional centromere. EMBO J. 18: 4196-4209. Medline abstract: 10428958

Fukagawa, T., Regnier, V., and Ikemura, T. (2001). Creation and characterization of temperature-sensitive CENP-C mutants in vertebrate cells. Nucleic Acids Res. 29: 3796-3803. Medline abstract: 11557811

Heeger, S., et al. (2005). Genetic interactions of Separase regulatory subunits reveal the diverged Drosophila Cenp-C homolog. Genes Dev 19: 2041-2053. Medline abstract: 16140985

Izuta, H., et al. (2006). Comprehensive analysis of the ICEN (interphase centromere complex) components enriched in the CENP-A chromatin of human cells. Genes Cells 11: 673-684. Medline abstract: 16716197

Kalitsis, P., Fowler, K. J., Earle, E., Hill, J. and Choo, K. H. (1998). Targeted disruption of mouse centromere protein C gene leads to mitotic disarray and early embryo death. Proc. Natl. Acad. Sci. 95(3): 1136-41. PubMed citation: 9448298

Klein, U. R., Nigg, E. A. and Gruneberg, U. (2006). Centromere targeting of the chromosomal passenger complex requires a ternary subcomplex of Borealin, Survivin, and the N-terminal domain of INCENP. Mol. Biol. Cell 17(6): 2547-58. Medline abstract: 16571674

Kline, S. L., Cheeseman, I. M., Hori, T., Fukagawa, T. and Desai, A. (2006). The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation. J. Cell Biol 173: 9-17. Medline abstract: 16585270

Kwon, M. S., Hori, T., Okada, M., and Fukagawa, T. (2007). CENP-C is involved in chromosome segregation, mitotic checkpoint function, and kinetochore assembly. Mol. Biol. Cell 18: 2155-2168. Medline abstract: 17392512

Maiato, H., et al. (2006). The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells. Chromosoma 115: 469-480. Medline abstract: 16909258

Meluh, P.B. and Koshland, D. (1995). Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C. Mol. Biol. Cell 6: 793-807. Medline abstract: 7579695

Moore, L. L. and Roth, M. B. (2001). HCP-4, a CENP-C-like protein in Caenorhabditis elegans, is required for resolution of sister centromeres. J. Cell Biol. 153: 1199-1208. Medline abstract: 11402064

Oegema, K., Desai, A., Rybina, S., Kirkham, M., and Hyman, A. A. (2001). Functional analysis of kinetochore assembly in Caenorhabditis elegans. J. Cell Biol. 153: 1209-1226. Medline abstract: 11402065

Okada, M,. et al. (2006). The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres. Nat Cell Biol 8: 446-457. Medline abstract: 16622420

Politi, V., Perini, G., Trazzi, S., Pliss, A., Raska, I., Earnshaw, W.C., and Della Valle, G. (2002). CENP-C binds the alpha-satellite DNA in vivo at specific centromere domains. J. Cell Sci. 115: 2317-2327. Medline abstract: 12006616

Schittenhelm, R. B., et al. (2007). Spatial organization of a ubiquitous eukaryotic kinetochore protein network in Drosophila chromosomes. Chromosoma 116(4): 385-402. Medline abstract: 17333235

Talbert, P. B., Bryson, T. D., and Henikoff, S. (2004). Adaptive evolution of centromere proteins in plants and animals. J. Biol. 3: 18. Medline abstract: 15345035

Tomkiel, J., Cooke, C. A., Saitoh, H., Bernat, R. L., and Earnshaw, W. C. (1994). CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase. J. Cell Biol. 125: 531-545. Medline abstract: 8175879

Vos, L. J., Famulski, J. K. and Chan, G. K. (2006). How to build a centromere: from centromeric and pericentromeric chromatin to kinetochore assembly. Biochem Cell Biol 84: 619-639. Medline abstract: 16936833

Wei, R. R., Sorger, P. K. and Harrison, S. C. (2005). Molecular organization of the Ndc80 complex, an essential kinetochore component. Proc. Natl. Acad. Sci. 102: 5363-5367. Medline abstract: 15809444

Wong, L. H., et al. (2007). Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere. Genome Res. 17(8): 1146-60. Medline abstract: 17623812

Yang, C. H., Tomkiel, J., Saitoh, H., Johnson, D. H. and Earnshaw, W. C. (1996). Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C. Mol. Cell. Biol. 16: 3576-3586. Medline abstract: 8668174

date revised: 12 January 2008

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