State of nucleolar proteins B23/nucleophosmin and UBF in HeLa cells during apoptosis induced by tumor necrosis factor

The structural state of two major nucleolar proteins, UBF and B23/nucleophosmin (both monomeric and oligomeric forms), was for the first time established in HeLa cells treated with apoptosis inducers: tumor necrosis factor (TNF-alpha), emetine, and their combination. The treatment of the cells with either TNF-alpha or emetine did not induce apoptosis and affect the state of UBF and nucleophosmin (both monomers and oligomers). Apoptosis was rather pronounced only if HeLa cells were treated with a mixture of TNF-alpha and emetine. States of the UBF and B23 proteins were analyzed in samples containing 25, 45, and 100% of cells with apoptotic nuclei. It was shown by immunoblotting that TNF-alpha-induced apoptosis of HeLa cells was associated with proteolysis of UBF and production of a 76-kD fragment, the content of which increased in correlation with the fraction of apoptotically changed cells. The N- and C-terminal amino acid sequences of UBF and its 76-kD fragment were characterized, and the site of the apoptosis-induced specific proteolysis was identified. As differentiated from UBF, protein B23 did not undergo proteolytic degradation during the TNF-alpha-induced apoptosis of HeLa cells and its content was unchanged even in the cell fraction with fragmentation of virtually all nuclei. However, the ratio between the monomeric and oligomeric states of B23 protein was changed in apoptotic cells, and apoptosis-specific forms of nucleophosmin were detected.     

Nuclear DNA helicase II (RNA helicase A) binds to an F-actin containing shell that surrounds the nucleolus

Nuclear DNA helicase II (NDH II), alternatively named RNA helicase A (RHA), is an F-actin binding protein that is particularly enriched in the nucleolus of mouse cells. Here, we show that the nucleolar localization of NDH II of murine 3T3 cells depended on an ongoing rRNA synthesis. NDH II migrated out of the nucleolus after administration of 0.05 microg/ml actinomycin D, while nucleolin and the upstream binding factor (UBF) remained there. In S phase-arrested mouse cells, NDH II was frequently found at the nucleolar periphery, where it was accompanied by newly synthesized nucleolar RNA. Human NDH II was mainly distributed through the whole nucleoplasm and not enriched in the nucleoli. However, in the human breast carcinoma cell line MCF-7, NDH II was also found at the nucleolar periphery, together with the tumor suppressor protein p53. Both NDH II and p53 were apparently attached to the F-actin-based filamentous network that surrounded the nucleoli. Accordingly, this subnuclear structure was sensitive to F-actin depolymerizing agents. Depolymerization with gelsolin led to a striking accumulation of NDH II in the nucleoli of MCF-7 cells. This effect was abolished by RNase, which extensively released nucleolus-bound NDH II when added together with gelsolin. Taken together, these results support the idea that an actin-based filamentous network may anchor NDH II at the nucleolar periphery for pre-ribosomal RNA processing, ribosome assembly, and/or transport.     

Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses

p53 protects against cancer through its capacity to induce cell cycle arrest or apoptosis under a large variety of cellular stresses. It is not known how such diversity of signals can be integrated by a single molecule. However, the literature reveals that a common denominator in all p53-inducing stresses is nucleolar disruption. We thus postulated that the impairment of nucleolar function might stabilize p53 by preventing its degradation. Using micropore irradiation, we demonstrate that large amounts of nuclear DNA damage fail to stabilize p53 unless the nucleolus is also disrupted. Forcing nucleolar disruption by anti-upstream binding factor (UBF) microinjection (in the absence of DNA damage) also causes p53 stabilization. We propose that the nucleolus is a stress sensor responsible for maintenance of low levels of p53, which are automatically elevated as soon as nucleolar function is impaired in response to stress. Our model integrates all known p53-inducing agents and also explains cell cycle-related variations in p53 levels which correlate with established phases of nucleolar assembly/disassembly through the cell cycle.     

Nucleolus: the fascinating nuclear body

Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs are synthesized, processed and assembled with ribosomal proteins. RNA polymerase I synthesizes the ribosomal RNAs and this activity is cell cycle regulated. The nucleolus reveals the functional organization of the nucleus in which the compartmentation of the different steps of ribosome biogenesis is observed whereas the nucleolar machineries are in permanent exchange with the nucleoplasm and other nuclear bodies. After mitosis, nucleolar assembly is a time and space regulated process controlled by the cell cycle. In addition, by generating a large volume in the nucleus with apparently no RNA polymerase II activity, the nucleolus creates a domain of retention/sequestration of molecules normally active outside the nucleolus. Viruses interact with the nucleolus and recruit nucleolar proteins to facilitate virus replication. The nucleolus is also a sensor of stress due to the redistribution of the ribosomal proteins in the nucleoplasm by nucleolus disruption. The nucleolus plays several crucial functions in the nucleus: in addition to its function as ribosome factory of the cells it is a multifunctional nuclear domain, and nucleolar activity is linked with several pathologies. Perspectives on the evolution of this research area are proposed.     

Inhibition of Protein Synthesis: A Mechanism of Amebicide Action of Emetine and Other Structurally Related Compounds*

New amebicides usually have been discovered by empirical screening procedures while other drugs, such as emetine, were adopted after their clinical efficacy was demonstrated. Emetine, puromycin, and cycloheximide are amebicides recently shown to act as inhibitors of protein synthesis in animal cells; the present study was designed to determine the general relationship of this mode of action to amebicidal activity. Our results indicate that amebicides structurally related to emetine inhibit protein synthesis in Entamoeba histolytica. It seems likely that the lethality of many amebicides may be attributed to specific effects on macromolecular synthesis in E. histolytica.     

Delocalization of nucleolar poly(ADP-ribose) polymerase-1 to the nucleoplasm and its novel link to cellular sensitivity to DNA damage

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme activated by binding to DNA breaks, which causes PARP-1 automodification. PARP-1 activation is required for regulating various cellular processes, including DNA repair and cell death induction. PARP-1 involved in these regulations is localized in the nucleoplasm, but approximately 40% of PARP-1 can be found in the nucleolus. Previously, we have reported that nucleolar PARP-1 is delocalized to the nucleoplasm in cells exposed to DNA-damaging agents. However, the functional roles of this delocalization in cellular response to DNA damage is not well understood, since this approach simultaneously induces the delocalization of PARP-1 and its automodification. We therefore devised an approach for separating these processes. Unmodified PARP-1 was first delocalized from the nucleolus using camptothecin. Then, PARP-1 was activated by exposure of cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). In contrast to treatment with MNNG alone, delocalization of PARP-1 by CPT, prior to its activation by MNNG, induced extensive automodification of PARP-1. DNA repair activity and consumption of intracellular NAD⁺ were not affected by this activation. On the other hand, activation led to an increased formation of apoptotic cells, and this effect was suppressed by inhibition of PARP-1 activity. These results suggest that delocalization of PARP-1 from the nucleolus to the nucleoplasm sensitizes cells to DNA damage-induced apoptosis. As it has been suggested that the nucleolus has a role in stress sensing, nucleolar PARP-1 could participate in a process involved in nucleolus-mediated stress sensing.     

Human PinX1 Mediates TRF1 Accumulation in Nucleolus and Enhances TRF1 Binding to Telomeres

Human PinX1 (hPinX1) is known to interact with telomere repeat binding factor 1 (TRF1) and telomerase. Here, we report that hPinX1 regulates the nucleolar accumulation and telomeric association of TRF1. In HeLa, HA-hPinX1 was co-localized with fibrillarin, a nucleolar protein, in 51% of the transfected cells and was present in the nucleoplasm of the remaining 48%. Mutant analysis showed that the C-terminal region was important for nucleolar localization, while the N-terminus exhibited an inhibitory effect on nucleolar localization. Unlike HA- and Myc-hPinX1, GFP-hPinX1 resided predominantly in the nucleolus. Nuclear hPinX1 bound to telomeres and other repeat sequences as well but, despite its interaction with TRF1, nucleolar hPinX1 did not bind to telomeres. Nucleolar hPinX1 forced endogenous TRF1 accumulation in the nucleolus. Furthermore, TRF1 binding to telomeres was upregulated in cells over-expressing hPinX1. In an ALT cell line, WI-38 VA-13, TRF1 did not co-localize with hPinX1 in the nucleoli. In summary, hPinX1 likely interacts with TRF1 in both the nucleolus and the nucleoplasm, and excess hPinX1 results in increased telomere binding of TRF1. The PinX1 function of mediating TRF1 nucleolar accumulation is absent from ALT cells, suggesting that it might be telomerase-dependent.     

低氧对小麦根端分生细胞核仁结构和功能的影响

为了探讨低氧对小麦根端分生细胞核仁结构和功能的影响,本实验以普通小麦为材料,用低氧水处理其根尖,按常规细胞制片、银染、电镜观察、间接免疫荧光染色和半定量PCR分析等手段开展研究.观察发现:(1)低氧水处理后小麦核仁结构发生膨胀、突出、进而凝集、内部出现空泡、细微结构消失、核仁通道结构异常、甚至解体等一系列变异现象.(2)间接免疫荧光染色技术观察看到,低氧水处理后小麦核仁内的核磷蛋白B23向核质甚至胞质扩散.(3)半定量PCR分析显示,低氧处理后rRNA基因的表达量较对照明显降低,而且C23的表达信号几乎检测不到,表明核糖体RNA和核仁蛋白C23基因的表达均显著下调,低氧严重抑制它们的转录.研究证明,低氧除了对小麦根端分生细胞核仁结构有破坏作用外,还严重抑制核仁的功能.     

扁蓿豆冷诱导基因差异表达分析

以直立型扁蓿豆幼苗为试验材料,采用cDNA-AFLP技术分析扁蓿豆在低温胁迫诱导下的基因表达差异.结果显示,利用筛选的64对引物组合,对0℃低温处理3～5叶期扁蓿豆幼苗的叶片cDNA进行扩增,共获得549条差异表达的转录衍生片段(TDFs).选取上调表达较好的43条片段进行克隆、测序、Blastx比对和功能分析,其中32个TDFs的蛋白序列与基础代谢、信号转导、转录因子、防御等功能有关,11个TDFs为假设蛋白、未知蛋白或没有找到一致序列.利用荧光定量PCR对3种不同上调表达差异片段进行验证,结果可从数值上更准确地显示差异片段在低温胁迫过程中的相对表达量.