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...

Chromosomal puffs on the polytene chromosomes in the dipteran Chironomus tentans offer the possibility of comparing the appearance of RNA-binding proteins at different transcription sites. We raised a monoclonal antibody that recognized a 130 kDa protein, designated hrp130. Immunocytological analysis of isolated chromosomes showed that hrp130 is heavily accumulated in a specific puff, called Balbiani ring 3; only occasionally is hrp130 abundant in one or two additional puffs on other chromosomes. The immunolabeling was sensitive to RNase treatment, suggesting that hrp130 is associated with nascent ribonucleoproteins. As shown by immunoelectron microscopy hrp130 is distributed along the active BR3 genes. The full sequence of hrp130 was determined by cDNA cloning. The protein comprises 1028 amino acids and contains three WW domains in the N-terminal half and six FF domains in the C-terminal half of the molecule. The protein is conserved from Caenorhabditis elegans to mammals; the human homolog is known as the transcription elongation repressor CA150. We propose that the abundance of hrp130/CA150 in BR3 is connected with the exceptionally high level of splicing in this locus and that hrp130/CA150 adjusts the transcription rate to the numerous splicing events taking place along the gene to ensure proper splicing.

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The transducing activity of two different kinds of premature lysates of Ti-infected cells have been compared to normal lysates. The results show that Ti-transducing particles are made early in the maturation period. The average density of Ti-transducing particles is slightly greater than the density of plaque-forming Ti.

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