Bertil Daneholt

The Murine SCP3 Gene Is Required for Synaptonemal Complex Assembly, Chromosome Synapsis, and Male Fertility

During meiosis, the homologous chromosomes pair and recombine. An evolutionarily conserved protein structure, the synaptonemal complex (SC), is located along the paired meiotic chromosomes. We have studied the function of a structural component in the axial/lateral element of the SC, the synaptonemal complex protein 3 (SCP3). A null mutation in the SCP3 gene was generated, and we noted that homozygous mutant males were sterile due to massive apoptotic cell death during meiotic prophase. The SCP3-deficient male mice failed to form axial/lateral elements and SCs, and the chromosomes in the mutant spermatocytes did not synapse. While the absence of SCP3 affected the nuclear distribution of DNA repair and recombination proteins (Rad51 and RPA), as well as synaptonemal complex protein 1 (SCP1), a residual chromatin organization remained in the mutant meiotic cells.

A Pre-mRNA-Binding Protein Accompanies the RNA from the Gene through the Nuclear Pores and into Polysomes

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.

A Look at Messenger RNP Moving through the Nuclear Pore

but nevertheless most of them are still unknown. The After the processing of pre–messenger RNA (pre-mRNA) nucleoproteins examined usually display characteristic, in the cell nucleus, the mature messsenger RNA (mRNA) often repeated, structural motifs, the significance of is transferred to the cytoplasm. Over the years, research which are not yet fully understood. Several of the in this area has focused more on the net effect of this nucleoporins have been recovered in defined supramonucleocytoplasmic transport than on the translocation lecular complexes, and some of the nucleoporins have event per se. It is true that early studies using electron been approximately located to the cytoplasmic or numicroscopy demonstrated that high molecular weight clear fibers or to the core of the NPC. It is, however, RNA, associated with proteins in ribonucleoprotein evident that we still have only a very fragmented view (RNP) complexes, leaves the nucleus through the nuof the molecular organization of the NPC. Better knowlclear pores; endogenous messenger RNP (mRNP) partiedge of the NPC structure is essential not only to elucicles as well as ribosomal subunits were directly visualdate direct molecular interactions between the transport ized in transit through the pore (Franke and Scheer, substrate and the NPC but also to understand major 1974). Furthermore, when injected into amphibian nuconformational changes in the NPC related to transport, clei, colloidal gold particles covered with transfer RNA, such as the expansion of the central channel. 5S RNA, or poly(A) could be seen passing through the Immediately upon synthesis, pre-mRNA becomes aspores into the cytoplasm (Dworetzky and Feldherr, sociated with proteins to form the RNP particles, desig1988). However, the actual mechanisms involved could nated pre-mRNP or hnRNP (heterogeneous nuclear rinot be elucidated in these early studies. Today, the posbonucleoprotein; for review, see Dreyfuss et al., 1993). sibilities of exploring the translocation process are conThese particles or complexes appear in the cell as perisiderably more favorable because of recent progress chromatin fibrils or, when the fibril is packed into higher on both the nuclear pore complex (NPC) and pre-mRNP order structures,as perichromatin granules. The populaparticles. Furthermore, specific structural elements tion of hnRNP proteins is complex; in humans, for examwithin the pre-mRNP complexes have been shown to ple, there are about 20 major (named A–U) and a large be important for export, and these will be crucial starting number of minor protein species. The hnRNP proteins tools for uncovering the translocation process. contain a modular structure with one or more RNAThe NPC is a cylindrical assembly with 8-fold rotabinding domains and at least one auxiliary domain that tional symmetry (Franke and Scheer, 1974; Davis, 1995; is probably involved in protein–protein interactions. The Panté and Aebi, 1996). The core of the NPC can be various hnRNP proteins bind preferentially to distinct described as a tripartite structure: a spoke–central plug RNA sequences, and each type of pre-mRNP particle assembly framed by two thin coaxial rings, a nuclear is likely to contain a specific subset of the proteins. ring facing the nucleoplasm, and a cytoplasmic ring The sequence-dependent binding of hnRNP proteins is facing the cytoplasm (Figure 1). The central plug, also compatible with specific roles for the proteins in, for designated the transporter, attains the shape of a cylinexample, splicing and transport. It was originally beder with a tapered center. Cell physiology experiments lieved that the RNA-binding proteins are confined to the have shown that the NPC contains constantly open 9 cell nucleus, butsome of them (e.g., A1, E, and K) shuttle; nm aqueous channels whose position within the NPC that is, they appear transiently in the cytoplasm. is not clear. These tiny channels allow diffusion of ions Early electron microscopy studies demonstrated that and smaller molecules, but not even the smallest RNA RNP particles bind to (or diffuse along) the nuclear fibers molecules, such as transfer RNA, can move through a of the NPC and enter the central channel of the pore 9 nm channel. The central plug, however, contains a (Franke and Scheer, 1974). However, most of the RNP channel that can expand to a diameter of about 25 nm particles could not be identified properly, nor were they and permit large objects such as RNA (and RNP) to sufficiently large to be studied in further detail. Recently, be exported. Because the large channel is open only however, it has been possible to investigate thoroughly transiently, it is looked upon as a gated transport chanthe translocation of a specific pre-mRNP particle of giant nel, although there is no strong direct evidence for a size: the Balbiani ring (BR) granules in the salivary glands discrete gate. On both sides of the NPC core are fibers of the dipteran Chironomus tentans (Mehlin and Daneconnected to the rings and extending out into the surholt, 1993). The BR genes, 35–40 kb in size, encode rounding medium for the putative binding of compolarge secretory proteins. The genes contain four small nents to be transported (Franke and Scheer, 1974). The introns, three at the 59 end and one at the 39 end. Becytoplasmic fibers are rather short, whereas the nuclear cause more than 30 kb of the primary transcript consists

Assembly and transport of a premessenger RNP particle

Salivary gland cells in the larvae of the dipteran Chironomus tentans offer unique possibilities to visualize the assembly and nucleocytoplasmic transport of a specific transcription product. Each nucleus harbors four giant polytene chromosomes, whose transcription sites are expanded, or puffed. On chromosome IV, there are two puffs of exceptional size, Balbiani ring (BR) 1 and BR 2. A BR gene is 35–40 kb, contains four short introns, and encodes a 1-MDa salivary polypeptide. The BR transcript is packed with proteins into a ribonucleoprotein (RNP) fibril that is folded into a compact ring-like structure. The completed RNP particle is released into the nucleoplasm and transported to the nuclear pore, where the RNP fibril is gradually unfolded and passes through the pore. On the cytoplasmic side, the exiting extended RNP fibril becomes engaged in protein synthesis and the ensuing polysome is anchored to the endoplasmic reticulum. Several of the BR particle proteins have been characterized, and their fate during the assembly and transport of the BR particle has been elucidated. The proteins studied are all added cotranscriptionally to the pre-mRNA molecule. The various proteins behave differently during RNA transport, and the flow pattern of each protein is related to the particular function of the protein. Because the cotranscriptional assembly of the pre-mRNP particle involves proteins functioning in the nucleus as well as proteins functioning in the cytoplasm, it is concluded that the fate of the mRNA molecule is determined to a considerable extent already at the gene level.

Sedimentation properties of the newly synthesized RNA from isolated nuclear components of Chironomus tentans salivary gland cells

Sedimentation analyses of labelled RNA from isolated nuclear components of Chironomus tentans salivary gland cells are presented. It is shown that nucleoli form a 38 s component which is converted to one 30 s and one 20 s component. The 30 s fraction is probably composed of the heavy ribosomal RNA fraction and an intermediate precursor, slightly larger than 30 s. The finished light ribosomal 17 s RNA molecule, to which the 20 s component is believed to be a specific intermediate precursor, was never observed in the nucleolus. Label due to preribosomal and ribosomal components was absent from chromosomes and nuclear sap. The nucleoli do not form a labelled 4 s RNA peak, in contrast to chromosomes and nuclear sap. The distribution of 4 s RNA label among the chromosomes suggests its formation from a large number of loci. The chromosomes also produce polydisperse RNA in the sedimentation range of 10 to 90 s. The polydispersity is not a function of heterogeneity in band origin but is found in the RNA of the single Balbiani ring. The polydisperse RNA has a sedimentation range that is higher than that reported for messenger RNA.

Characterization of active transcription units in Balbiani rings of chironomus tentans

Specific active transcription units on chromosome IV in the salivary glands of Chironomus tentans have been visualized by the Miller spreading technique and in situ by conventional electron microscopy. These units are likely to be located in the two most conspicuous puffs on chromosome IV, Balbiani ring 1 (BR 1) and Balbiani ring 2 (BR 2). The transcription units in these Balbiani rings generate 75S RNA molecules constituting putative messenger RNA species for the predominant cellular product, the salivary polypeptides. Solitary active transcription units with a mean length of 7.7 micron were observed most frequently. The lateral ribonucleoprotein (RNP) fibers of each unit formed a single length gradient. The number of fibers per unit was 123 (+/- 24), or about 16 growing RNP fibers per micron of chromosome fiber. The considerable variation in the number of RNP fibers per unit suggests that transcription can be modulated at the level of the individual gene. The modulation is probably achieved via the initiation event and/or via an early pretermination step, but a change in the elongation rate could not be excluded. The number of polymerases starting to traverse the whole gene was estimated to be six per min and transcription unit, and the rate of RNA chain elongation was calculated to be 31 nucleotides per second at 18 degrees C. The properties of the chromosome fiber within the active 75S RNA units and also within their vicinity were studied in the Miller spreads. The inactive chromosome fiber exhibited a uniform beaded conformation, while the active fiber was sparsely and irregularly beaded. Furthermore, the chromosome fiber was more extended in the active 75S RNA unit than in inactive regions (DNA packing ratios of 1.6 and 1.9, respectively). By comparing the properties of the active 75S RNA gene with those of active genes in other systems, it was inferred that the loss of beads and the extension of the fiber in the active unit is probably directly related to the level of transcriptive activity. Finally, a smooth nonbeaded segment of 0.18 micron in length was found to precede the RNP fiber gradient. This segment may have a role in the process of transcriptional regulation. On the basis of comparison with the active transcription units in spread preparations. It was possible to identify active units in the Balbiani rings in sectioned material using conventional electron microscopy. In both BR 1 and BR 2 an active unit appeared as a loop, consisting of a fiber axis and having RNP granules attached to the loop axis by stalks. The growing RNP fibers therefore seem to be organized into granular structures during the transcription process, and the final products in BR 1 and BR2 are granules, 500 A in diameter, each containing a 75S RNA molecule.

An actin–ribonucleoprotein interaction is involved in transcription by RNA polymerase II

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.

The mRNA export factor Dbp5 is associated with Balbiani ring mRNP from gene to cytoplasm

The DEAD box RNA helicase Dbp5 is essential for nucleocytoplasmic transport of mRNA–protein (mRNP) complexes. Dbp5 is present mainly in the cytoplasm and is enriched at the cytoplasmic side of nuclear pore complexes (NPCs), suggesting that it acts in the late part of mRNP export. Here, we visualize the assembly and transport of a specific mRNP particle, the Balbiani ring mRNP in the dipteran Chironomus tentans, and show that a Dbp5 homologue in C.tentans, Ct‐Dbp5, binds to pre‐mRNP co‐transcriptionally and accompanies the mRNP to and through the nuclear pores and into the cytoplasm. We also demonstrate that Ct‐Dbp5 accumulates in the nucleus and partly disappears from the NPC when nuclear export of mRNA is inhibited. The fact that Ct‐Dbp5 is present along the exiting mRNP fibril extending from the nuclear pore into the cytoplasm supports the view that Ct‐Dbp5 is involved in restructuring the mRNP prior to translation. Finally, the addition of the export factor Dbp5 to the growing transcript highlights the importance of the co‐transcriptional loading process in determining the fate of mRNA.

Visualization of the formation and transport of a specific hnRNP particle

The growth and maturation of the transcription products on the Balbiani ring (BR) genes in Chironomus tentans has been characterized by electron microscopy. The BR transcript is packed into a series of well defined ribonucleoprotein structures of increasing complexity: a 10 nm fiber, a 19 nm fiber, a 26 nm fiber, and a 50 nm granule. The basic 10 nm element was revealed in Miller spreads. The in situ structure of the transcription products and RNA compaction estimates suggested that the 10 nm fiber is packed into the 19 nm fiber as a tight coil. The transition of the 19 nm fiber into the 26 nm fiber is accompanied by a major change of the basic 10 nm fold into a noncoiled structure. Finally, the 26 nm fiber makes a one and one-third left-handed turn forming the final product, the BR granule. Upon translocation through the nuclear pore the BR granule becomes rod-shaped, which most likely corresponds to a relaxation of the highest-order structure into a straight 26 nm fiber.

The size of the transcription unit in balbiani ring 2 of Chironomus tentans as derived from analysis of the primary transcript and 75 S RNA

The size of the transcription unit for 75 S RNA in Balbiani ring 2, a giant puff in the salivary glands of Chironomus tentans, has been derived from determinations of the size of salivary gland 75 S RNA as well as of the size of the primary transcript in BR2.§ Salivary gland 75 S RNA, predominantly located in the cytoplasm, was analyzed by electrophoresis in formaldehyde-containing agarose gels. The molecular weight was shown to be at least 12 × 106 but probably not exceeding 13 to 14 × 106. The size was also determined by contour length measurements in the electron microscope, which gave a molecular weight of 12.3 × 106. Since this value fell within the size range established by the electrophoretic method, it was accepted as an estimate of the size of 75 S RNA. The biochemical and electron microscopic evidence for considering 75 S RNA as a single, covalently linked polynucleotide is discussed. Balbiani ring 2 RNA, containing the finished primary transcript as well as growing RNA molecules, was analyzed in agarose/formaldehyde gels with salivary gland 75 S RNA as an internal size marker. From the distribution of the nascent RNA it was concluded that the finished primary transcript essentially migrated coincident with 75 S RNA. Thus, the molecular weight value of 12.3 × 106 was also adopted for the primary transcript. This would imply that the corresponding transcription unit in BR2 comprises about 37 × 103 base-pairs of DNA. The predicted size of the transcription unit in BR2 was related to the information available on the BR2 chromomere and the adjacent interchromomeres. It was concluded that only a minor part of the unit (less than 5× 103 base-pairs) can be accommodated in an interchromomere adjacent to the BR2 chromomere. Most, if not all, of the unit has to be present in the BR2 chromomere itself. On the other hand, there is more DNA in a BR2 chromomere (> 100 × 103 base-pairs) than can be accounted for by one single 75 S RNA transcription unit.