Birgitta Björkroth

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.

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.

Presence of histone H1 on an active Balbiani ring gene

We have investigated whether histone H1 is present on active Balbiani ring genes in the salivary glands of Chironomus tentans using immunoelectron microscopy. The genes were studied in two activity states: maximally activated genes with a fully extended template and repressed genes in a 30 nm fiber conformation. Histone H1 was recorded on the gene in both conformations; the immunosignal was considerably stronger in the transcriptionally active state, probably reflecting the increased accessibility of histone H1 to the antibody in unfolded versus compacted chromatin. We conclude that during transcription the DNA template is extended and the nucleosomes are disrupted at the RNA polymerases, but histone H1, and most likely also the core histones, remains bound to the template.

Structure of the chromatin axis during transcription

A new isolation procedure for polytene chromosomes has been developed which permits visualization of the native chromatin template of transcriptionally active genes. The Balbiani ring genes in the salivary glands of Chironomus tentans have been analyzed specifically: these genes are exceptionally long (37 kb) and very active in transcription. The most abundant configuration of the template is an extended fiber, approximately 5 nm in diameter. When the distance between adjacent RNA polymerases is unusually long, the template is packed into a 10 nm fiber. Occasionally, the fiber can further fold into a loose coil forming a more or less distinct 30 nm fiber. It is concluded that a large part of the chromatin axis is in a fully extended form during transcription of the Balbiani ring genes. However, if a given segment of the template is not continuously occupied by RNA polymerases it can be packed into a single nucleosome, into a string of nucleosomes (the thin fiber) or even into a supercoiled string of nucleosomes (the thick fiber). A comprehensive model based on the opposing topological effects of nucleosome disassembly and DNA melting caused by the RNA polymerase, is presented to account for the observed dynamic behavior of the chromatin template.

Rapid reformation of the thick chromosome fiber upon completion of RNA synthesis at the Balbiani ring genes in Chironomus tentans

We have studied the ultrastructure of the Balbiani ring genes in Chironomus tentans during treatment with the RNA synthesis inhibitor DRB (5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole). This nucleoside analogue blocks transcription at or near the initiation site but does not interfere with the elongation and termination processes. In the ordinary active state the Balbiani ring genes display a 5 nm chromosome fiber, carrying densely distributed, growing ribonucleoprotein particles (Andersson et al., 1980). When the transcriptional activity declines, a 10 nm fiber can be observed between sparsely distributed RNA polymerases. Furthermore, after passage of the last RNA polymerase the 10 nm fiber can be seen as well as its gradual packing into a 25 nm thick fiber. Thus, the active chromosome fiber is rapidly packed into higher order structures when the fiber is not directly involved in transcription. The formation of the thick fiber does not require that the gene along its entire length is devoid of active RNA polymerases. The thick fiber can again be mobilized for transcription, since in reversion experiments the BR genes appear as ordinary active genes with an extended nucleofilament and densely packed nascent transcription products. The dynamic behaviour of the chromosome fiber during transcription is discussed as well as the packing and unpacking of a gene into higher order structures.

The in situ structure of the active 75 S RNA genes in Balbiani rings of Chironomus tentans

Balbiani rings 1 and 2 in the salivary glands of Chironomus tentans are known to contain transcriptionally active 75S RNA genes. These have earlier been characterized in the electron microscope when spread on a surface according to Miller. In the present study the 75S RNA genes are studied as they appear as loops within the cell. Serial sections through Balbiani rings have been analysed, and both proximal and distal segments of active 75S RNA genes have been reconstructed. The growing ribonucleoprotein particles were visualized as well as the putative RNA polymerases at the bases of the ribonucleoprotein (RNP) fibres and a thin (5 nm) chromosomal axis. In the proximal portion of the active gene (about one-quarter of the gene) the RNP fibres appear as 20 nm thick fibers, gradually increasing in length. In the distal part of the gene no further increase in length of the 20 nm RNP fibres takes place. Instead the peripheral parts of the RNP fibres are packed into globular structures, which increase in size along the gene. In the terminal part of the active gene the globules attain about the same diameter (50 nm) as the large nuclear sap granules, which are likely to represent the completed and released RNP particles from the 75S RNA genes. Information on the density in situ of the growing RNP fibres (36 fibres/μm chromosome fibre axis) was used to estimate the DNA compaction of the chromosome fibre during transcription. Assuming the same fibre density as in the spread genes (10/μm DNA), we calculated that the DNA compaction of the chromosome fibre amounts to 3.6. The thin chromosome axis as well as the low DNA compaction figure imply that the nucleofilament is not supercoiled in the intensively transcribed 75S RNA genes. The chromosome fibre is in fact considerably more extended than a nucleofilament in non-transcribed chromatin (DNA compaction 5–7; micrococcal nuclease digestion experiments show a 189 base pair repeat for Chironomus polytene chromosomes). Our data on the properties of the chromosome fibre in situ are discussed in relation to the transcription process.

Nuclear poly(A)-binding protein PABPN1 is associated with RNA polymerase II during transcription and accompanies the released transcript to the nuclear pore.

The nuclear poly(A)-binding protein, PABPN1, has been previously shown to regulate mRNA poly(A) tail length and to interact with selected proteins involved in mRNA synthesis and trafficking. To further understand the role of PABPN1 in mRNA metabolism, we used cryo-immunoelectron microscopy to determine the fate of PABPN1 at various stages in the assembly and transport of the Chironomus tentans salivary gland Balbiani ring (BR) mRNA ribonucleoprotein (mRNP) complex. PABPN1 is found on BR mRNPs within the nucleoplasm as well as on mRNPs docked at the nuclear pore. Very little PABPN1 is detected on the cytoplasmic side of the nuclear envelope, suggesting that PABPN1 is displaced from mRNPs during or shortly after passage through the nuclear pore. Surprisingly, we also find PABPN1 associated with RNA polymerase II along the chromatin axis of the BR gene. Our results suggest that PABPN1 binds to the polymerase before, at, or shortly after the start of transcription, and that the assembly of PABPN1 onto the poly(A) tail may be coupled to transcription. Furthermore, PABPN1 remains associated with the released BR mRNP until the mRNP is translocated from the nucleus to the cytoplasm.

A p50-like Y-box protein with a putative translational role becomes associated with pre-mRNA concomitant with transcription

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.