The vertebrate nuclear pore complex (NPC) is a macromolecular assembly of protein subcomplexes forming a structure of eightfold radial symmetry. The NPC core consists of globular subunits sandwiched between two coaxial ring-like structures of which the ring facing the nuclear interior is capped by a fibrous structure called the nuclear basket. By postembedding immunoelectron microscopy, we have mapped the positions of several human NPC proteins relative to the NPC core and its associated basket, including Nup93, Nup96, Nup98, Nup107, Nup153, Nup205, and the coiled coil-dominated 267-kDa protein Tpr. To further assess their contributions to NPC and basket architecture, the genes encoding Nup93, Nup96, Nup107, and Nup205 were posttranscriptionally silenced by RNA interference (RNAi) in HeLa cells, complementing recent RNAi experiments on Nup153 and Tpr. We show that Nup96 and Nup107 are core elements of the NPC proper that are essential for NPC assembly and docking of Nup153 and Tpr to the NPC. Nup93 and Nup205 are other NPC core elements that are important for long-term maintenance of NPCs but initially dispensable for the anchoring of Nup153 and Tpr. Immunogold-labeling for Nup98 also results in preferential labeling of NPC core regions, whereas Nup153 is shown to bind via its amino-terminal domain to the nuclear coaxial ring linking the NPC core structures and Tpr. The position of Tpr in turn is shown to coincide with that of the nuclear basket, with different Tpr protein domains corresponding to distinct basket segments. We propose a model in which Tpr constitutes the central architectural element that forms the scaffold of the nuclear basket.

In the larval salivary glands of C. tentans, it is possible to visualize by electron microscopy how Balbiani ring (BR) pre-mRNA associates with proteins to form pre-mRNP particles, how these particles move to and through the nuclear pore, and how the BR RNA is engaged in the formation of giant polysomes in the cytoplasm. Here, we study C. tentans hrp36, an abundant protein in the BR particles, and establish that it is similar to the mammalian hnRNP A1. By immuno-electron microscopy it is demonstrated that hrp36 is added to BR RNA concomitant with transcription, remains in nucleoplasmic BR particles, and is translocated through the nuclear pore still associated with BR RNA. It appears in the giant BR RNA-containing polysomes, where it remains as an abundant protein in spite of ongoing translation.

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The synaptonemal complex protein SCP3 is part of the lateral element of the synaptonemal complex, a meiosis-specific protein structure essential for synapsis of homologous chromosomes. We have investigated the fiber-forming properties of SCP3 to elucidate its role in the synaptonemal complex. By synthesis of SCP3 in cultured somatic cells, it has been shown that SCP3 can self-assemble into thick fibers and that this process requires the COOH-terminal coiled coil domain of SCP3, as well as the NH2-terminal nonhelical domain. We have further analyzed the thick SCP3 fibers by transmission electron microscopy and immunoelectron microscopy. We found that the fibers display a transversal striation with a periodicity of ∼20 nm and consist of a large number of closely associated, thin fibers, 5–10 nm in diameter. These features suggest that the SCP3 fibers are structurally related to intermediate filaments. It is known that in some species the lateral elements of the synaptonemal complex show a highly ordered striated structure resembling that of the SCP3 fibers. We propose that SCP3 fibers constitute the core of the lateral elements of the synaptonemal complex and function as a molecular framework to which other proteins attach, regulating DNA binding to the chromatid axis, sister chromatid cohesion, synapsis, and recombination.

To determine the function of actin in the cell nucleus, we sought to identify nuclear actin-binding proteins in the dipteran Chironomus tentans using DNase I-affinity chromatography. We identified the RNA-binding protein hrp65 as an actin-binding protein and showed that the C-terminal sequence of the hrp65-2 isoform is able to interact directly with actin in vitro. In vivo crosslinking and coimmunoprecipitation experiments indicated that hrp65 and actin are also associated in the living cell. Moreover, in vivo administration of a competing peptide corresponding to the C-terminal sequence of hrp65-2 disrupted the actin-hrp65-2 interaction and caused a specific and drastic reduction of transcription as judged by puff regression and diminished bromo-UTP incorporation. Our results indicate that an actin-based mechanism is implicated in the transcription of most if not all RNA polymerase II genes and suggest that an actin-hrp65-2 interaction is required to maintain the normal transcriptional activity of the cell. Furthermore, immunoelectron microscopy experiments and nuclear run-on assays suggest that the actin-hrp65-2 complex plays a role in transcription elongation.A ctin and myosin I are present not only in the cytoplasm but also in the cell nucleus (1, 2), where they have been implicated in transcription of protein-coding genes (3-5) and nuclear export (6). Many other cytoskeletal components have also been found in the cell nucleus, and some of them have been tentatively implicated in gene expression (reviewed in ref. 1).Actin has been found associated with (pre)-messenger ribonucleoprotein (pre-mRNP) complexes in a variety of organisms, and we recently revealed that actin is a bona fide component of the Balbiani ring (BR) pre-mRNP particles in the salivary gland cells of the dipteran Chironomus tentans (7). The BR genes code for large secretory proteins that are expressed in the salivary gland cells of C. tentans (reviewed in ref. 8). The BR pre-mRNAs are assembled into large RNP particles, the BR pre-mRNPs, that can be visualized directly by transmission electron microscopy (reviewed in ref. 9). Actin is incorporated cotranscriptionally into the newly synthesized BR pre-mRNPs by binding to a subset of heterogeneous nuclear RNP (hnRNP) proteins such as the hnRNP A1-like protein hrp36 (7). Remarkably, the presence of actin in RNP complexes is not restricted to the BR particles of C. tentans, because actin has also been found associated with certain hnRNP proteins of the A͞B group in mammalian pre-mRNPs (10).The presence of actin in the cell nucleus is well documented, but the precise role of nuclear actin is still not understood. To obtain further insight into the function(s) of nuclear actin, we sought to identify additional proteins of C. tentans that bind to actin in the cell nucleus. Materials and MethodsDetailed descriptions of the materials and methods can be found in Supporting Materials and Methods, which is published as supporting information on the PNAS web site, www.pnas.org.DNase I-Sepharose Pull-Down...

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