Nucleophosmin is required for chromosome congression, proper mitotic spindle formation, and kinetochore-microtubule attachment in HeLa cells

Nucleophosmin (NPM) is an abundantly expressed multifunctional nucleolar phosphoprotein. Here we show that depletion of NPM by RNA interference causes defects in cell division, followed by an arrest of DNA synthesis due to activation of a p53-dependent checkpoint response in HeLa cells. Depletion of NPM leads to mitotic arrest due to spindle checkpoint activation. The mitotic cells arrested by NPM depletion have defects in chromosome congression, proper mitotic spindle and centrosome formation, as well as defects in kinetochore-microtubule attachments. Loss of NPM thus causes severe mitotic defects and delayed mitotic progression. These findings indicate that NPM is essential for mitotic progression and cell proliferation.     

A nucleolar protein RRS1 contributes to chromosome congression

We report here the functional analysis of human Regulator of Ribosome Synthesis 1 (RRS1) protein during mitosis. We demonstrate that RRS1 localizes in the nucleolus during interphase and is distributed at the chromosome periphery during mitosis. RNA interference experiments revealed that RRS1-depleted cells show abnormalities in chromosome alignment and spindle organization, which result in mitotic delay. RRS1 knockdown also perturbs the centromeric localization of Shugoshin 1 and results in premature separation of sister chromatids. Our results suggest that a nucleolar protein RRS1 contributes to chromosome congression.     

A Conserved Unfoldase Activity for the p97 AAA-ATPase in Proteasomal Degradation

The multifunctional AAA-ATPase p97 is one of the most abundant and conserved proteins in eukaryotic cells. The p97/Npl4/Ufd1 complex dislocates proteins that fail the protein quality control in the endoplasmic reticulum to the cytosol where they are subject to degradation by the ubiquitin/proteasome system. Substrate dislocation depends on the unfoldase activity of p97. Interestingly, p97 is also involved in the degradation of specific soluble proteasome substrates but the exact mode of action of p97 in this process is unclear. Here, we show that both the central pore and ATPase activity of p97 are necessary for the degradation of cytosolic ubiquitin-fusion substrates. Addition of a flexible extended C-terminal peptide to the substrate relieves the requirement for p97. Deletion mapping reveals a conserved length dependency of 20 residues for the peptide, which allows p97-independent degradation to occur. Our results suggest that initiation of unfolding may be more complex than previously anticipated and that the 19S regulatory complex of the proteasome can require preprocessing of highly folded, ubiquitylated substrates by the p97^Ufd1/Npl4 complex. Our data provide an explanation for the observation that p97 is only essential for a subpopulation of soluble substrates and predict that a common characteristic of soluble p97-dependent substrates is the lack of an initiation site to facilitate unfolding by the 26S proteasome.     

H1.X with different properties from other linker histones is required for mitotic progression

We report here the characterization of H1.X, a human histone H1 subtype. We demonstrate that H1.X accumulates in the nucleolus during interphase and is distributed at the chromosome periphery during mitosis. In addition, the results of fluorescence recovery after photobleaching indicate that the exchange of H1.X on and off chromatin is faster than that of the other H1 subtypes. Furthermore, RNA interference experiments reveal that H1.X is required for chromosome alignment and segregation. Our results suggest that H1.X has important functions in mitotic progression, which are different from those of the other H1 subtypes.     

多靶向RNA干扰技术在基因治疗中的应用与前景

RNA干扰(RNA interference,RNAi)通过转录后基因沉默效应特异性抑制靶基因的表达,其沉默机制的高效性、特异性及稳定性使这项技术成为生物医学领域研究基因治疗的重要工具。阐述RNAi技术的特点和RNAi疗法的现状,特别是多靶小干扰RNA(small interference RNA,siRNA)目前的发展态势及其各种结构性修饰,通过使用这些结构修饰的siRNA提高基因沉默的效率,将有助于提高疗效。但该技术在广泛应用于临床之前,仍存在一些亟待解决的问题与面临的挑战,需进一步研究。     

Jab1 mediates protein degradation of the Rad9-Rad1-Hus1 checkpoint complex

The Rad1-Rad9-Hus1 (9-1-1) complex serves a dual role as a DNA-damage sensor in checkpoint signaling and as a mediator in the DNA repair pathway. However, the intercellular mechanisms that regulate the 9-1-1 complex are poorly understood. Jab1, the fifth component of the COP9 signalosome complex, has a central role in the degradation of multiple proteins and is emerging as an important regulator in cancer development. Here, we tested the hypothesis that Jab1 controls the protein stability of the 9-1-1 complex via the proteosome pathway. We provide evidence that Jab1 physically associates with the 9-1-1 complex, and show that this association is mediated through direct interaction between Jab1 and Rad1, one of the subunits of the 9-1-1 complex. Importantly, Jab1 causes translocation of the 9-1-1 complex from the nucleus to the cytoplasm, mediating rapid degradation of the 9-1-1 complex via the 26 S proteasome. Furthermore, Jab1 significantly suppresses checkpoint signaling activation, DNA synthesis recovery from blockage and cell viability after replication stresses such as UV exposure, gamma radiation and treatment with hydroxyurea. These results suggest that Jab1 is an important regulator for the stability of protein 9-1-1 control in cells, which may provide novel information on the involvement of Jab1 in the checkpoint and DNA repair signaling in response to DNA damage.     

RNAi in X inactivation: contrasting findings on the role of interference

X inactivation is the process that brings about the dosage equivalence of X‐linked genes in females to that of males. This complex process initiated at a very early stage of female embryonic development is orchestrated by long non‐coding RNAs transcribed in both sense and antisense orientation. Recent studies present contradicting evidence for the role of small RNAs and RNase III enzyme Dicer in the X inactivation process. In this review, I discuss these results in the overall perspective of X inactivation and gene silencing.     

Uncovering RNAi mechanisms in plants: biochemistry enters the foray

In plants, the RNA interference (RNAi) machinery responds to a variety of triggers including viral infection, transgenes, repeated elements and transposons. All of these triggers lead to silencing outcomes ranging from mRNA degradation to translational repression to chromatin remodeling. Thus, plants offer us a potentially unique opportunity to understand the full range of RNAi effector mechanisms. In this review, we discuss the recent developments in our understanding of plant RNAi mechanisms from a biochemical perspective.     

Both the p33 and p55 Subunits of the Helicobacter pylori VacA Toxin Are Targeted to Mammalian Mitochondria

Helicobacter pylori infection causes peptic ulcers and gastric cancer. A major toxin secreted by H. pylori is the bipartite vacuolating cytotoxin A, VacA. The toxin is believed to enter host cells as two subunits: the p55 subunit (55 kDa) and the p33 subunit (33 kDa). At the biochemical level, it has been shown that VacA forms through the assembly of large multimeric pores composed of both the p33 subunit and the p55 subunit in biological membranes. One of the major target organelles of VacA is the mitochondria. Since only the p33 subunit has been reported to be translocated into mitochondria and the p55 subunit is not imported, it has been contentious as to whether VacA assembles into pores in a mitochondrial membrane. Here we show the p55 protein is imported into the mitochondria along with the p33 protein subunit. The p33 subunit integrally associates with the mitochondrial inner membrane, and both the p33 subunit and the p55 subunit are exposed to the mitochondrial intermembrane space. Their colocalization suggests that they could reassemble and form a pore in the inner mitochondrial membrane.     

Subcellular localization of the interaction of bipolar landmarks Bud8p and Bud9p with Rax2p in Saccharomyces cerevisiae diploid cells

In Saccharomyces cerevisiae, the bud site selection of diploid cells is regulated by at least four persistent landmarks, Bud8p, Bud9p, Rax1p, and Rax2p. Bud8p and Bud9p are essential for the establishment of bipolar budding and localize mainly to the distal and the proximal poles, respectively. Their subcellular localizations are regulated through interaction with Rax1p/Rax2p. We investigated when and where Bud8p and Bud9p physically interact with Rax2p in vivo using a split-GFP method. GFP fluorescence showed that Bud8p physically interacted with Rax2p at the proximal or distal pole in unbudded cells; a physical interaction was also observed at the opposite pole to the growing bud in mother cells with a large-size bud. Bud9p physically interacted with Rax2p at the birth scar in budded mother cells. These observations suggest that the interaction of Rax2p with Bud8p and Bud9p may contribute to the translocation of bipolar landmarks to the correct sites.