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Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress. INTRODUCTIONThe nucleus is a dynamic organelle consisting of interacting chromosomal and protein compartments. One of the major pathways of nuclear translocation is the movement of preribosomal particles from the nucleolus into the cytoplasm for the assembly of functional ribosomes. The main nucleolar functions involve RNA polymerase (pol) I transcription, posttranscriptional maturation of pre-rRNA transcripts and their subsequent assembly with ribosomal proteins into preribosomal particles. Other functions have been attributed to the nucleolus (for reviews, see Carmo-Fonseca et al., 2000;Olson, 2004b) and include the processing of RNA pol III transcripts, RNA editing, sequestration of cell cycle components in yeast, and Mdm2 protein in mammalian cells. The localization of telomere proteins and telomerase RNA in nucleoli suggests a role for the nucleolus in aging.Nucleolar components are found in all cells and tissues although the size, shape, and number of nucleoli may change depending on the species, cell type, and functional state. Transmission electron microscopy (TEM) has revealed three major structures within nucleoli: fibrillar centers (FC), dense fibrillar components (DFC), and the granular component (GC; for reviews, see Busch and Smetana, 1970;Goessens, 1984;Shaw and Jordan, 1995;Scheer and Hock, 1999). rDNA transcription units are found in the FC and consist of tandem repeats of these genes. rRNAs are harbored within the DFC and are processed there. It is therefore thought that rRNA transcription occurs at the interface betw...

Covalent attachment of ubiquitin-like proteins (Ubls) is a predominant mechanism for regulating protein function in eukaryotes. Several structurally related Ubls, such as ubiquitin, SUMO, NEDD8, and ISG15, modify a vast number of proteins, altering their functions in a variety of ways. Ubl modifications can affect the target's half-life, subcellular localization, enzymatic activity, or ability to interact with protein or DNA partners. Generally, these diverse Ubls are covalently attached via their C termini to their targets by parallel, but specific, cascades involving three classes of enzymes known as E1, E2, and E3. Structures are now available for many protein complexes in E1-E2-E3 cascades, revealing a series of modular building blocks and providing mechanistic insights into their functions.

The anaphase-promoting complex/cyclosome bound to CDC20 (APC/CCDC20) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) which inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which has been proposed to require CDC20 auto-ubiquitylation. Here we characterized APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C’s MCC binding site, is required for APC/CMCC-dependent CDC20 auto-ubiquitylation and degradation, and for timely anaphase initiation, but is dispensable for substrate ubiquitylation by APC/CCDC20 and APC/CCDH1. Our results support the view that MCC is continuously assembled and disassembled to enable rapid activation of APC/CCDC20 and that CDC20 auto-ubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators, and report the first generation of recombinant human APC/C.

PTB-associated splicing factor (PSF) has been implicated in both early and late steps of pre-mRNA splicing, but its exact role in this process remains unclear. Here we show that PSF interacts with p54 nrb , a highly related protein first identified based on cross-reactivity to antibodies against the yeast second-step splicing factor Prp18. We performed RNA-binding experiments to determine the preferred RNA-binding sequences for PSF and p54 nrb , both individually and in combination. In all cases, iterative selection assays identified a purine-rich sequence located on the 39 side of U5 snRNA stem 1b. Filter-binding assays and RNA affinity selection experiments demonstrated that PSF and p54 nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity. Sedimentation analyses show that both proteins associate with spliceosomes and with U4/U6.U5 tri-snPNP.

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A universal feature of positive-strand RNA viruses is the involvement of host intracellular membranes in RNA replication complex formation and function. This conclusion is based primarily on four observations. First, immunofluorescence and immunoelectron microscopy have localized viral replicase proteins and nascent viral RNA synthesis to intracellular membranes (16,18,24,28,29,33,42,47,51,53,56). Second, in vitro viral RNA-dependent RNA polymerase (RdRp) activity cofractionates with cellular membranes (6,8,12,51,58). Third, detergents suppress, and in some instances, phospholipids enhance the in vitro activities of viral replicase proteins (2,8,57,58). Fourth, lipid synthesis inhibitors (20,30,36) and mutations in lipid synthesis genes (26) inhibit viral RNA replication. Recent results also show that at least some positive-strand RNA viruses use membrane rearrangements to create virusspecific, membrane-bounded compartments in which RNA replication occurs (51).Despite these observations, many fundamental questions remain about the interaction of viral replication factors with host intracellular membranes and the specific roles of membranes in viral RNA replication complex formation and function.Moreover, while most viruses assemble their replication complexes on a specific membrane or membranes, different viruses use different membranes. For various positive-strand RNA viruses, membranes derived from the endoplasmic reticulum (ER) (26,29,35,42,(46)(47)(48)51), Golgi apparatus (47), lysosomes (18,24,28,47), endosomes (18, 24), vacuoles (52), mitochondria (10,15,32,33), perixosomes (9, 10), and chloroplasts (14) have all been implicated in viral RNA replication complex formation and function. The significance of this diversity of intracellular membranes used by different viruses is unknown.The wide variety of intracellular membrane compartments used by different positive-strand RNA viruses, and their specific targeting, suggests that individual viruses may have unique host factor requirements supplied by specific intracellular membranes. Alternatively, many or all host intracellular membranes could provide the functions necessary for viral RNA replication complex formation and function, and the specific intracellular localization of individual viruses may be related to other steps in the viral life cycle, such as viral protein translation or encapsidation. Results that help distinguish between these competing hypotheses potentially have significant therapeutic implications, as antiviral drugs designed to block viral RNA replication complex formation or function on a specific intracellular membrane compartment will be ineffective if alternative membranes can be used efficiently.To study intracellular viral RNA replication complex targeting, we use Flock House virus (FHV), an alphanodavirus that has been used as a model to investigate viral capsid formation and RNA packaging (49,50,59), viral RNA replication and

The multi-subunit Anaphase Promoting Complex (APC) is an essential cell cycle regulator. Although CDC26 is known to play a role in APC assembly, its molecular function has remained unclear. Biophysical, structural, and genetic studies presented here reveal that CDC26 stabilizes the structure of APC6, a core TPR protein required for APC integrity. Interestingly, CDC26–APC6 association involves an intermolecular TPR mimic composed of one helix from each protein.