Microtubule nucleation and organization by the centrosome require ␥-tubulin, a protein that exists in a macromolecular complex called the ␥-tubulin ring complex (␥TuRC). We report characterization of CDK5RAP2, a novel centrosomal protein whose mutations have been linked to autosomal recessive primary microcephaly. In somatic cells, CDK5RAP2 localizes throughout the pericentriolar material in all stages of the cell cycle. When overexpressed, CDK5RAP2 assembled a subset of centrosomal proteins including ␥-tubulin onto the centrosomes or under the microtubule-disrupting conditions into microtubule-nucleating clusters in the cytoplasm. CDK5RAP2 associates with the ␥TuRC via a short conserved sequence present in several related proteins found in a range of organisms from fungi to mammals. The binding of CDK5RAP2 is required for ␥TuRC attachment to the centrosome but not for ␥TuRC assembly. Perturbing CDK5RAP2 function delocalized ␥-tubulin from the centrosomes and inhibited centrosomal microtubule nucleation, thus leading to disorganization of interphase microtubule arrays and formation of anastral mitotic spindles. Together, CDK5RAP2 is a pericentriolar structural component that functions in ␥TuRC attachment and therefore in the microtubule organizing function of the centrosome. Our findings suggest that centrosome malfunction due to the CDK5RAP2 mutations may underlie autosomal recessive primary microcephaly. INTRODUCTIONIn animal cells, the centrosome is the primary microtubule (MT) organizing center (MTOC), which plays a key role in the control of the temporal and spatial distribution of MT networks (Ou and Rattner, 2004;Doxsey et al., 2005;Luders and Stearns, 2007). Typically, centrosomes are positioned at the focus of a radial array of MTs during interphase and are incorporated into spindle poles during mitosis. Interphase centrosomes are composed of a pair of centrioles embedded in a cloud of electron-dense pericentriolar material (PCM). The centriole has a well-defined structure with MT triplets arranged into a cylinder, whereas the organization of the PCM is less apparent. Early studies have shown the existence of a salt (2 M KI)-insoluble scaffold/matrix underlying the PCM (Moritz et al., 1998;Schnackenberg et al., 1998). This matrix, formed with a high content of large coiled-coil proteins, provides binding sites for the tubulin family member ␥-tubulin and other proteins associated with centrosomal functions, such as MT nucleation. Through the cell cycle, the PCM varies in volume and the MT-nucleating activity, which are smallest in G1-phase and biggest during mitosis. In addition, proteins have a precise and cell cycle-specific placement within the PCM that has been observed as a tubular structure located along the surface of the centriole and around its proximal end but not around its distal end (Ou and Rattner, 2000;Ou et al., 2003). The outer surface of the PCM is dynamic and PCM proteins found in the cytoplasm transit to the PCM either by diffusion or via MTs.Distributed throughout the PCM, ␥-tubulin ...

Background: 53BP1 counteracts BRCA1 in DNA repair. Results: RIF1 acts downstream of 53BP1 and counteracts BRCA1 in DNA end resection. It also has a 53BP1-independent role in regulating BLM chromatin association. Conclusion: RIF1 is the major downstream effector of 53BP1. Significance: These results reveal that RIF1 antagonizes BRCA1, functions in DNA end protection, and prevents homologous recombination repair.

Tamoxifen, an estrogen receptor (ER) antagonist, is the mainstay treatment of breast cancer and the development of resistance represents a major obstacle for a cure. Although lncRNAs such as HOTAIR have been implicated in breast tumorigenesis, their roles in chemotherapy resistance remain largely unknown. In this study, we report that HOTAIR is up-regulated in tamoxifen-resistant breast cancer tissues compared to their primary counterparts. Mechanistically, HOTAIR is a direct target of ER-mediated transcriptional repression and is thus restored upon the blockade of ER signaling, either by hormone deprivation or tamoxifen treatment. Interestingly, this elevated HOTAIR increases ER protein level and thus enhances ER occupancy on the chromatin and potentiates its downstream gene regulation. HOTAIR overexpression is sufficient to activate the ER transcriptional program even under hormone-deprived conditions. Functionally, we found that HOTAIR overexpression increases breast cancer cell proliferation, whereas its depletion significantly impairs cell survival and abolishes tamoxifen-resistant cell growth. In conclusion, the lncRNA HOTAIR is directly repressed by ER and its up-regulation promotes ligand-independent ER activities and contributes to tamoxifen resistance.

SUMMARY Understanding the mechanisms of androgen receptor (AR) activation in the milieu of low androgen is critical to effective treatment of castration-resistant prostate cancer (CRPC). Here, we report HOTAIR as an androgen-repressed lncRNA, and, as such, it is markedly upregulated following androgen deprivation therapies and in CRPC. We further demonstrate a distinct mode of lncRNA-mediated gene regulation, wherein HOTAIR binds to the AR protein to block its interaction with the E3 ubiquitin ligase MDM2, thereby preventing AR ubiquitination and protein degradation. Consequently, HOTAIR expression is sufficient to induce androgen-independent AR activation and drive the AR-mediated transcriptional program in the absence of androgen. Functionally, HOTAIR overexpression increases, whereas HOTAIR knockdown decreases, prostate cancer cell growth and invasion. Taken together, our results provide compelling evidence of lncRNAs as drivers of androgen-independent AR activity and CRPC progression, and they support the potential of lncRNAs as therapeutic targets.