The hrp23 Protein in the Balbiani Ring Pre-mRNP Particles Is Released Just before or at the Binding of the Particles to the Nuclear Pore Complex

Sun, Xin; Alzhanova-Ericsson, Alla T.; Visa, Neus; Aissouni, Youssef; Zhao, Jian; Daneholt, Bertil

doi:10.1083/jcb.142.5.1181

Cited by 34 publications

(45 citation statements)

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“…The antibody against synaptonemal complex protein 3 (SCP3) used in negative controls was kindly provided by C. Höög (Karolinska Institute, Stockholm). Antibodies against hrp65 (11), hrp23 (12), and hrp45 (13) have been described in earlier studies.…”

Section: Methodsmentioning

confidence: 99%

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

Percipalle

Fomproix

Kylberg

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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129

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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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Section: Methodsmentioning

confidence: 99%

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

Percipalle

Fomproix

Kylberg

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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129

107

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“…The isolation of polytene chromosomes from salivary glands, the RNase treatment of the chromosomes and the immunocytological analysis have been previously described (Sun et al, 1998). The affinity-purified anti-p40/50 antibody was used diluted 1:50.…”

Section: Immunocytology Of Isolated Polytene Chromosomesmentioning

confidence: 99%

“…Western blot analysis was performed as described previously by Sun et al (Sun et al, 1998). Both the anti-p40/50 serum and pre-immune serum were used in a 1:10,000 dilution.…”

Section: Sds-page and Western Blot Analysismentioning

confidence: 99%

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

Soop

Nashchekin

Zhao

et al. 2003

Journal of Cell Science

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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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“…The data led a scenario that pre-mRNA associated with both NES-containing and NRS-containing proteins is retained in the nucleus until the NRS-containing proteins are released from the complex. As a matter of fact, hnRNP C1 is known to preferably bind intron-like sequences (for a review see and some insect hnRNP proteins, likely the functional homologs of nonshuttling human hnRNP proteins, are released from mRNP complexes just before mRNA export (Sun et al, 1998). This may be one of the mechanism that ensures only fully matured mRNA molecules are exported.…”

Section: Hnrnp Proteinsmentioning

confidence: 99%

The Molecular Mechanisms of Messenger RNA Nuclear Export.

Siomi

2000

Cell Struct. Funct.

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ABSTRACT. In eukaryotic cells, the nuclear membrane creates a barrier between the nucleus and the cytoplasm. Whereas RNA synthesis occurs in the nucleus, they mostly function in the cytoplasm; thus export of RNA molecules from the nucleus to the cytoplasm is indispensable for normal function of the cells. The molecular mechanisms involved in each kind of cellular RNA export is gradually understood. The focus of this review will be mRNA export. mRNAs are multiformed. In order to ensure that this variety of mRNA molecules are all exported, cells are probably equipped with multiple export pathways. A number of proteins is predicted to be involved in mRNA export. Ascertaining which proteins play crucial roles in the pathways is the key point in the study of mRNA export.Key words: nuclear transport/RNA export/WRNA/RNA-binding protein soluble nuclear export factors/RNA processing In eukaryotic cells, genomes, the templates for general transcription, are sequestered within the nucleus and this compartment is the only place in cells devoted to the synthesis of RNA molecules. After transcription, the RNA molecules undergo a variety of posttranscriptional processing steps. The majority of RNAs including transfer RNA, rinsbosomal RNA, small nuclear RNA and messenger RNA, are exported to the cytoplasm. Nuclear transport occurs through nuclear pore complexes (NPCs) located across the nuclear envelope. A nuclear pore complex is an assembly of up to one hundred different proteins termed nucleoporins (Nups) (Ohno, 1998;Yang, 1998;Stoffler, 1999). While relatively small molecules such as ions and proteins smaller than 40 kDa can diffuse through the nuclear pores, macromolecules including RNAs traverse the NPC by an active, temperature-dependent manner.Active transport both in and out of the nucleus requires soluble nuclear transport receptors, which first bind specific sequences (nuclear import or export signals) within the proteins to be transported (cargos) and then transport them in concert with Nups (for reviews see Izaurralde and Adam, 1998;Mattaj and Englmeier, 1998;Nakielny and Dreyfuss, 1999). Directionality of nuclear transport is provided by a small GTPase Ran (Dahlberg and Lund, 1998). In the nucleus Ran exists mostly as the GTP-bound form and causes dissociation of cargos from the nuclear import factors upon translocation across the nuclear membrane (Görlich et al., 1996;Rexach and Blobel, 1995). In the case of nuclear export, however, RanGTP is required in efficient binding of nuclear export factors to the molecules to be exported (Arts et al., 1998a;Fornerod et al., 1997;Kutay et al., 1997). Cytoplasmically localized RanGAP and RanBP1 facilitate hydrolysis of Ran-bound GTP to GDP. The exchange of RanGTP to RanGDP causes dissociation of the exported cargos from the nuclear export factors upon translocation to the cytoplasm (Görlich, 1998;Kehlenbach et al., 1999;Kutay et al., 1997). Whether hydrolysis of RanGTP is the energy source for the nuclear transport is still questionable (Englmeier et al., 1999;Izaurralde et...

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The hrp23 Protein in the Balbiani Ring Pre-mRNP Particles Is Released Just before or at the Binding of the Particles to the Nuclear Pore Complex

Cited by 34 publications

References 84 publications

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

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

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

The Molecular Mechanisms of Messenger RNA Nuclear Export.

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