Involvement of satellite I noncoding RNA in regulation of chromosome segregation

Human centromeres consist of repetitive sequences from which satellite I noncoding RNAs are transcribed. We found that knockdown of satellite I RNA causes abnormal chromosome segregation and generation of nuclei with a grape‐shape phenotype. Co‐immunoprecipitation experiments showed that satellite I RNA associates with Aurora B, a component of the chromosome passenger complex (CPC) regulating proper attachment of microtubules to kinetochores, in mitotic HeLa cells. Satellite I RNA was also shown to associate with INCENP, another component of the CPC. In addition, depletion of satellite I RNA resulted in up‐regulation of kinase activity of Aurora B and delocalization of the CPC from the centromere region. These results suggest that satellite I RNA is involved in chromosome segregation through controlling activity and centromeric localization of Aurora B kinase.     

RBMX is a component of the centromere noncoding RNP complex involved in cohesion regulation

Satellite I RNA, a noncoding (nc)RNA transcribed from repetitive regions in human centromeres, binds to Aurora kinase B and forms a ncRNP complex required for chromosome segregation. To examine its function in this process, we purified satellite I ncRNP complex from nuclear extracts prepared from asynchronized or mitotic (M) phase‐arrested HeLa cells and then carried out LC/MS to identify proteins bound to satellite I RNA. RBMX (RNA‐binding motif protein, X‐linked), which was isolated from M phase‐arrested cells, was selected for further characterization. We found that RBMX associates with satellite I RNA only during M phase. Knockdown of RBMX induced premature separation of sister chromatid cohesion and abnormal nuclear division. Likewise, knockdown of satellite I RNA also caused premature separation of sister chromatids during M phase. The amounts of RBMX and Sororin, a cohesion regulator, were reduced in satellite I RNA‐depleted cells. These results suggest that satellite I RNA plays a role in stabilizing RBMX and Sororin in the ncRNP complex to maintain proper sister chromatid cohesion.     

C. elegans condensin promotes mitotic chromosome architecture, centromere organization, and sister chromatid segregation during mitosis and meiosis

Chromosome segregation and X-chromosome gene regulation in Caenorhabditis elegans share the component MIX-1, a mitotic protein that also represses X-linked genes during dosage compensation. MIX-1 achieves its dual roles through interactions with different protein partners. To repress gene expression, MIX-1 acts in an X-chromosome complex that resembles the mitotic condensin complex yet lacks chromosome segregation function. Here we show that MIX-1 interacts with a mitotic condensin subunit, SMC-4, to achieve chromosome segregation. The SMC-4/MIX-1 complex positively supercoils DNA in vitro and is required for mitotic chromosome structure and segregation in vivo. Thus, C. elegans has two condensin complexes, one conserved for mitosis and another specialized for gene regulation. SMC-4 and MIX-1 colocalize with centromere proteins on condensed mitotic chromosomes and are required for the restricted orientation of centromeres toward spindle poles. This cell cycle-dependent localization requires AIR-2/AuroraB kinase. Depletion of SMC-4/MIX-1 causes aberrant mitotic chromosome structure and segregation, but not dramatic decondensation at metaphase. Moreover, SMC-4/MIX-1 depletion disrupts sister chromatid segregation during meiosis II but not homologous chromosome segregation during meiosis I, although both processes require chromosome condensation. These results imply that condensin is not simply required for compaction, but plays a more complex role in chromosome architecture that is essential for mitotic and meiotic sister chromatid segregation.     

Condensin I and II behaviour in interphase nuclei and cells undergoing premature chromosome condensation

Condensin is an integral component of the mitotic chromosome condensation machinery, which ensures orderly segregation of chromosomes during cell division. In metazoans, condensin exists as two complexes, condensin I and II. It is not yet clear what roles these complexes may play outside mitosis, and so we have examined their behaviour both in normal interphase and in premature chromosome condensation (PCC). We find that a small fraction of condensin I is retained in interphase nuclei, and our data suggests that this interphase nuclear condensin I is active in both gene regulation and chromosome condensation. Furthermore, live cell imaging demonstrates condensin II dramatically increases on G1 nuclei following completion of mitosis. Our PCC studies show condensins I and II and topoisomerase II localise to the chromosome axis in G1-PCC and G2/M-PCC, while KIF4 binding is altered. Individually, condensins I and II are dispensable for PCC. However, when both are knocked out, G1-PCC chromatids are less well structured. Our results define new roles for the condensins during interphase and provide new information about the mechanism of PCC.     

Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere

The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. Here, we demonstrate that centromeric alpha-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 alpha-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 alpha-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, we have identified the nucleolar localization sequences on CENPC1 and INCENP. We present evidence that CENPC1 is an RNA-associating protein that binds alpha-satellite RNA by an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and "RNA replenishment" experiments, we show that alpha-satellite RNA is a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere. Our 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.     

The interaction between the helicase DHX33 and IPS-1 as a novel pathway to sense double-stranded RNA and RNA viruses in myeloid dendritic cells

In eukaryotes, there are at least 60 members of the DExD/H helicase family, many of which are able to sense viral nucleic acids. By screening all known family members, we identified the helicase DHX33 as a novel double-stranded RNA （dsRNA） sensor in myeloid dendritic cells （mDCs）. The knockdown of DHX33 using small heteroduplex RNA （shRNA） blocked the ability of mDCs to produce type I interferon （IFN） in response to poly hC and reovirus. The HELICc domain of DHX33 was shown to bind poly hC. The interaction between DHX33 and IPS-1 is mediated by the HELICc region of DHX33 and the C-terminal domain of IPS-1 （also referred to MAVS and VISA）. The inhibition of DHX33 expression by RNA interference blocked the poly hC-induced activation of MAP kinases, NF-KB and IRF3. The interaction between the helicase DHX33 and IPS-1 was independent of RIG-I/MDA5 and may be a novel pathway for sensing poly I-C and RNA viruses in mDCs.     

Comprehensive analysis of the ICEN (Interphase Centromere Complex) components enriched in the CENP-A chromatin of human cells

The centromere is a chromatin structure essential for correct segregation of sister chromatids, and defects in this region often lead to aneuploidy and cancer. We have previously reported purification of the interphase centromere complex (ICEN) from HeLa cells, and have demonstrated the presence of 40 proteins (ICEN1-40), along with CENP-A, -B, -C, -H and hMis6, by proteomic analysis. Here we report analysis of seven ICEN components with unknown function. Centromere localization of EGFP-tagged ICEN22, 24, 32, 33, 36, 37 and 39 was observed in transformant cells. Depletion of each of these proteins by short RNA interference produced abnormal metaphase cells carrying misaligned chromosomes and also produced cells containing aneuploid chromosomes, implying that these ICEN proteins take part in kinetochore functions. Interestingly, in the ICEN22, 32, 33, 37 or 39 siRNA-transfected cells, CENP-H and hMis6 signals disappeared from all the centromeres in abnormal mitotic cells containing misaligned chromosomes. These results suggest that the seven components of the ICEN complex are predominantly localized at the centromeres and are required for kinetochore function perhaps through or not through loading of CENP-H and hMis6 onto the centromere.     

The packaging of human immunodeficiency virus type 1 RNA is restricted by overexpression of an RNA helicase DHX30

RNA helicases are a large family of proteins that are able to unwind RNA duplex and remodel the structure of RNA-protein (RNP) complexes using energy derived from hydrolysis of nucleotide triphosphates (NTPs). Every step of cellular RNA metabolism involves the activity of RNA helicases. Not surprisingly, more and more RNA helicases are reported to participate in the replication of viruses including the human immunodeficiency virus type 1 (HIV-1). Here, we provide evidence that overexpression of an RNA helicase named DHX30 enhances HIV-1 gene expression, but leads to the generation of viruses that package significantly low levels of viral RNA and exhibit severely decreased infectivity. These data reveal the complex roles of DHX30 in HIV-1 replication and implicate an inhibitory activity of DHX30 in HIV-1 RNA packaging.     

The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast

Chromosome segregation depends on kinetochores, the structures that mediate chromosome attachment to the mitotic spindle. We isolated mutants in IPL1, which encodes a protein kinase, in a screen for budding yeast mutants that have defects in sister chromatid separation and segregation. Cytological tests show that ipl1 mutants can separate sister chromatids but are defective in chromosome segregation. Kinetochores assembled in extracts from ipl1 mutants show altered binding to microtubules. Ipl1p phosphorylates the kinetochore component Ndc10p in vitro and we propose that Ipl1p regulates kinetochore function via Ndc10p phosphorylation. Ipl1p localizes to the mitotic spindle and its levels are regulated during the cell cycle. This pattern of localization and regulation is similar to that of Ipl1p homologs in higher eukaryotes, such as the human aurora2 protein. Because aurora2 has been implicated in oncogenesis, defects in kinetochore function may contribute to genetic instability in human tumors.     

Schizosaccharomyces pombe centromere protein Mis19 links Mis16 and Mis18 to recruit CENP‐A through interacting with NMD factors and the SWI/SNF complex

CENP‐A is a centromere‐specific variant of histone H3 that is required for accurate chromosome segregation. The fission yeast Schizosaccharomyces pombe and mammalian Mis16 and Mis18 form a complex essential for CENP‐A recruitment to centromeres. It is unclear, however, how the Mis16‐Mis18 complex achieves this function. Here, we identified, by mass spectrometry, novel fission yeast centromere proteins Mis19 and Mis20 that directly interact with Mis16 and Mis18. Like Mis18, Mis19 and Mis20 are localized at the centromeres during interphase, but not in mitosis. Inactivation of Mis19 in a newly isolated temperature‐sensitive mutant resulted in CENP‐A delocalization and massive chromosome missegregation, whereas Mis20 was dispensable for proper chromosome segregation. Mis19 might be a bridge component for Mis16 and Mis18. We isolated extragenic suppressor mutants for temperature‐sensitive mis18 and mis19 mutants and used whole‐genome sequencing to determine the mutated sites. We identified two groups of loss‐of‐function suppressor mutations in non‐sense‐mediated mRNA decay factors (upf2 and ebs1), and in SWI/SNF chromatin‐remodeling components (snf5, snf22 and sol1). Our results suggest that the Mis16‐Mis18‐Mis19‐Mis20 CENP‐A‐recruiting complex, which is functional in the G1‐S phase, may be counteracted by the SWI/SNF chromatin‐remodeling complex and non‐sense‐mediated mRNA decay, which may prevent CENP‐A deposition at the centromere.     

Nuclear and mitochondrial localization of the putative RNA helicase DHX32

RNA helicases are involved in all aspects of RNA metabolism. We identified DHX32 as a novel gene with homology to the DHX family of RNA helicases. To gain insight into the role of DHX32 in RNA metabolism, we determined its subcellular localization. Electron microscopy immunocytochemistry detected DHX32 in the nucleus and mitochondria of the myeloid leukemia cell line HL-60. Confocal microscopy shows mitochondrial localization of DHX32 in HeLa cells. Double labeling showed close proximity of DHX32 to some of the newly synthesized RNA in the mitochondria. These results suggest that DHX32 is a putative RNA helicase that might be involved in regulating nuclear and mitochondrial gene expression.     

Tpr directly binds to Mad1 and Mad2 and is important for the Mad1-Mad2-mediated mitotic spindle checkpoint

The mitotic arrest-deficient protein Mad1 forms a complex with Mad2, which is required for imposing mitotic arrest on cells in which the spindle assembly is perturbed. By mass spectrometry of affinity-purified Mad2-associated factors, we identified the translocated promoter region (Tpr), a component of the nuclear pore complex (NPC), as a novel Mad2-interacting protein. Tpr directly binds to Mad1 and Mad2. Depletion of Tpr in HeLa cells disrupts the NPC localization of Mad1 and Mad2 during interphase and decreases the levels of Mad1-bound Mad2. Furthermore, depletion of Tpr decreases the levels of Mad1 at kinetochores during prometaphase, correlating with the inability of Mad1 to activate Mad2, which is required for inhibiting APC^Cdc20. These findings reveal an important role for Tpr in which Mad1–Mad2 proteins are regulated during the cell cycle and mitotic spindle checkpoint signaling.     

Centrosomes in spindle organization and chromosome segregation: a mechanistic view

Centrosomes are complex structures, which are embedded into the opposite poles of the mitotic spindle of most animals, acting as microtubule organizing centres. Surprisingly, in several biological systems, such as flies, chicken, or human cells, centrosomes are not essential for cell division. Nonetheless, they ensure faithful chromosome segregation. Moreover, mis-functioning centrosomes can act in a dominant-negative manner, resulting in erroneous mitotic progression. Here, I review the mechanisms by which centrosomes contribute to proper spindle organization and faithful chromosome segregation under physiological conditions and discuss how errors in centrosome function impair transmission of the genomic material in a pathological setting.     

非编码RNA在肿瘤中对自噬的调控

非编码RNA（ncRNA）是不参与蛋白质编码的RNA的总称,包括微小RNA（miRNA）、siRNA、piRNA、长链非编码RNA（lncRNA）、转运RNA（tRNA）、核糖体RNA（rRNA）等。其在蛋白质转录与后转录过程中有重要的调节作用。自噬是细胞在异常环境中维持生存的一种方式。大量研究表明自噬与肿瘤也存在着密切的关系,而自噬对于肿瘤的影响具有“两面性”。许多研究探讨了非编码RNA在肿瘤细胞中对于自噬过程的调控．文章综述了miRNA与lncRNA这两种研究深入的非编码RNA。     

Modulation of Alp4 function in Schizosaccharomyces pombe induces novel phenotypes that imply distinct functions for nuclear and cytoplasmic gamma-tubulin complexes

The gamma-tubulin complex acts as a nucleation unit for microtubule assembly. It remains unknown, however, how spatial and temporal regulation of the complex activity affects microtubule-mediated cellular processes. Alp4 is one of the essential components of the S. pombe gamma-tubulin complex. We show here that overproduction of a carboxy-terminal form of Alp4 (Alp4C) and its derivatives tagged to a nuclear localization signal or to a nuclear export signal affect localization of gamma-tubulin complexes and induces novel phenotypes that reflect distinct functions of nuclear and cytoplasmic gamma-tubulin complexes. Nuclear Alp4C induces a Wee1-dependent G2 delay, reduces the levels of the gamma-tubulin complex at the spindle pole body, and results in defects in mitotic progression including spindle assembly, cytoplasmic microtubule disassembly, and chromosome segregation. In contrast, cytoplasmic Alp4C induces oscillatory nuclear movement and affects levels of cell polarity markers, Bud6 and Tip1, at the cell ends. These results demonstrate that regulation of nuclear gamma-tubulin complex activity is essential for cell cycle progression through the G2/M boundary and M phase, whereas regulation of cytoplasmic gamma-tubulin complex activity is important for nuclear positioning and cell polarity control during interphase.     

Chromosomal instability upregulates interferon in acute myeloid leukemia

Chromosome instability (CIN) generates genetic and karyotypic diversity that is common in hematological malignancies. Low to moderate levels of CIN are well tolerated and can promote cancer proliferation. However, high levels of CIN are lethal. Thus, CIN may serve both as a prognostic factor to predict clinical outcome and as a predictive biomarker. A retrospective study was performed to evaluate CIN in acute myeloid leukemia (AML). Chromosome mis‐segregation frequency was correlated with clinical outcome in bone marrow core biopsy specimens from 17 AML cases. Additionally, we induced chromosome segregation errors in AML cell lines with AZ3146, an inhibitor of the Mps1 mitotic checkpoint kinase, to quantify the phenotypic effects of high CIN. We observed a broad distribution of chromosome mis‐segregation frequency in AML bone marrow core specimens. High CIN correlated with complex karyotype in AML, as expected, although there was no clear survival effect. In addition to CIN, experimentally inducing chromosome segregation errors by Mps1 inhibition in AML cell lines causes DNA damage, micronuclei formation, and upregulation of interferon stimulated genes. High levels of CIN appear to be immunostimulatory, suggesting an opportunity to combine mitotic checkpoint inhibitors with immunotherapy in treatment of AML.     

非编码RNA在肝癌中的作用

近年来,国内外掀起RNA研究的热潮,焦点即为非编码RNA的生物作用机制。众多研究提示,非编码RNA （ non?coding RNA, ncRNA）在体内组成了复杂的分子网络,与蛋白质调控网络相对应,协同参与着肿瘤的发生发展、侵袭、转移和预后治疗。肝癌是最常见的恶性肿瘤疾病之一,在我国发病率极高,目前仍缺乏有效的治疗方式,严重威胁着人类健康。众多ncRNA在肝癌中表达失调,这些ncRNA作为肝癌基因治疗的潜在靶点,可推动肝癌基因靶向治疗的发展,研究意义重大。