In vertebrates free messenger ribonucleoprotein (RNP) particles and polysomes contain an abundant Y-box protein called p50 (YB-1), which regulates translation, presumably by affecting the packaging of the RNA. Here, we have identified a p50-like protein in the dipteran Chironomus tentans and studied its relation with the biogenesis of mRNA in larval salivary glands. The salivary gland cells contain polytene chromosomes with the transcriptionally active regions blown up as puffs. A few giant puffs, called Balbiani rings (BRs), generate a transcription product, a large RNP particle,which can be visualised (with the electron microscope) during its assembly on the gene and during its transport to and through the nuclear pores. The p50-like protein studied, designated Ct-p40/50 (or p40/50 for short), was shown to contain a central cold-shock domain, an alanine- and proline-rich N-terminal domain, and a C-terminal domain with alternating acidic and basic regions, an organisation that is characteristic of p50 (YB-1). The p40/50 protein appears in two isoforms, p40 and p50, which contain 264 and 317 amino acids, respectively. The two isoforms share the first 258 amino acids and thus differ in amino-acid sequence only in the region close to the C-terminus. When a polyclonal antibody was raised against p40/50, western blot analysis and immunocytology showed that p40/50 is not only abundant in the cytoplasm but is also present in the nucleus. Immunolabelling of isolated polytene chromosomes showed that p40/50 appears in transcriptionally active regions, including the BRs. Using immunoelectron microscopy we revealed that p40/50 is added along the nascent transcripts and is also present in the released BR RNP particles in the nucleoplasm. Finally, by UV crosslinking in vivo we showed that p40/50 is bound to both nuclear and cytoplasmic poly(A) RNA. We conclude that p40/50 is being added cotranscriptionally along the growing BR pre-mRNA, is released with the processed mRNA into the nucleoplasm and probably remains associated with the mRNA both during nucleocytoplasmic transport and protein synthesis. Given that the p40/p50 protein, presumably with a role in translation, is loaded onto the primary transcript concomitant with transcription, an early programming of the cytoplasmic fate of mRNA is indicated.

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A specific messenger ribonucleoprotein (RNP) particle, Balbiani ring (BR) granules in the dipteran Chironomus tentans, can be visualized during passage through the nuclear pore complex (NPC). We have now examined the transport through the nuclear basket preceding the actual translocation through the NPC. The basket consists of eight fibrils anchored to the NPC core by nucleoprotein Nup153. On nuclear injection of anti-Nup153, the transport of BR granules is blocked. Many granules are retained on top of the nuclear basket, whereas no granules are seen in transit through NPC. Interestingly, the effect of Nup153 seems distant from the antibody-binding site at the base of the basket. We conclude that the entry into the basket is a two-step process: an mRMP first binds to the tip of the basket fibrils and only then is it transferred into the basket by a Nup153-dependent process. It is indicated that ribosomal subunits follow a similar pathway. INTRODUCTIONRNA molecules are exported from nucleus to cytoplasm as ribonucleoprotein (RNP) complexes (Daneholt, 2001;Dreyfuss et al., 2002;Lei and Silver, 2002). The translocation of RNPs through nuclear pores has been visualized in the electron microscope both for messenger RNPs (Stevens and Swift 1966;Mehlin et al., 1992;Kiseleva et al., 1998) and for ribosomes/ribosomal subunits (Franke and Scheer, 1974). The pores contain a specific supramolecular assembly, the nuclear pore complex (NPC), and the passage occurs through the central channel of the complex (Dworetzky and Feldherr, 1988). During the last decade, it has been revealed that the translocation apparently takes place in discrete steps (Daneholt, 1997). The molecular mechanisms involved are currently being revealed.The NPC is a three-layered structure with a central spoke assembly sandwiched between a nuclear and cytoplasmic ring (Allen et al., 2000;Vasu and Forbes, 2001;Fahrenkrog and Aebi, 2003;Suntharalingam and Wente, 2003). In the center, there is a 25-nm-wide channel filled with a meshwork of very thin fibrils, often designated as the central plug. Additional auxiliary components are attached to the NPC periphery. On the nuclear side, eight longer fibrils emanate from the nuclear ring. At their distal ends, these fibrils seem to bifurcate and laterally interdigitate with their neighboring fibrils, thereby forming another ringlike structure referred to as the terminal ring. Together, nuclear fibrils and terminal ring are considered a structural and functional entity, called the nuclear basket. On the cytoplasmic side, eight short fibrils extend from the cytoplasmic ring into cytoplasm.The NPC contains ϳ30 proteins designated nucleoporins, the majority of which are symmetrically organized in the core of the NPC (Rout et al., 2000;Cronshaw et al., 2002). The central plug material is composed of a group of nucleoporins containing multiple repeats of the dipeptide sequence FG. These repeats are involved in the actual translocation of macromolecules and supramolecular assemblies through the pore (see further below i...

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.

Y‐box proteins constitute an evolutionarily conserved family of DNA‐ and RNA‐binding proteins involved in the regulation of transcription and translation. In the dipteran Chironomus tentans, a homologue to the vertebrate Y‐box protein YB‐1 was recently characterized and designated ctYB‐1. It is transferred from nucleus to cytoplasm bound to mRNA and is likely to affect translation. It appears in two size variants, p40 and p50. We further analysed the two size variants and their interaction with mRNA. Southern blot analysis, in situ hybridization and RT‐PCR analysis suggested that there is just one YB‐1 gene, and that the two size variants represent splicing isoforms. In a C. tentans epithelial cell line, only p40 is present, whereas both variants appear together in eight tissues from fourth‐instar larvae, although in somewhat different proportions. Furthermore, the appearance of the two isoforms was studied in relation to a specific 35–40 kb mRNA transcript in the salivary glands, the Balbiani ring mRNA. Because of their exceptional size, Balbiani ring messenger ribonucleoprotein particles in nucleoplasm and Balbiani ring polysomes in cytoplasm could be identified and selectively studied. We were able to establish that both isoforms are associated with both nuclear and cytoplasmic Balbiani ring mRNA. In addition, a p50‐specific antibody coimmunoprecipitated p40 from Balbiani ring polysomes, suggesting that the two splicing isoforms are located along the same Balbiani ring mRNA molecule. The functional significance of the two isoforms is being discussed.