Isolation and Structure of the Ribonucleoprotein Fibrils Containing Heterogeneous Nuclear RNA

Jacob, Monique; Devilliers, Ginette; Fuchs, Jean-Paul; Gallinaro, Hélène; Gattoni, Renata; Judes, C.; Stévenin, James

doi:10.1016/b978-0-12-147608-3.50011-2

Cited by 16 publications

(28 citation statements)

References 90 publications

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“…Anti-mouse an-VOL. 16,1996 RNA-BINDING PROTEINS IN BR PRE-mRNPstibodies coupled to colloidal gold were applied, the immunosignal was silver enhanced, and the specimens were studied by bright-field and phase-contrast microscopy. Two of the hybridoma clones, 4F9 and 2E4, were chosen for the study of BR RNA-binding proteins.…”

Section: Resultsmentioning

confidence: 99%

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Identification of Two RNA-Binding Proteins in Balbiani Ring Premessenger Ribonucleoprotein Granules and Presence of These Proteins in Specific Subsets of Heterogeneous Nuclear Ribonucleoprotein Particles

Wurtz-T

Kiseleva

Nacheva

et al. 1996

Molecular and Cellular Biology

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Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in giant chromosomal puffs, the BRs, in the larval salivary glands of the dipteran Chironomus tentans. Monoclonal antibodies were raised against nuclear proteins collected on a single-stranded-DNA-agarose affinity column, and two of them were used to identify RNA-binding proteins in BR granules. First, in Western blots (immunoblots), one of the antibodies recognized a 36-kDa protein and the other recognized a 45-kDa protein. Second, both antibodies bound to the BRs in immunocytological experiments. It was shown in cross-linking experiments that the two proteins are associated with heterogeneous nuclear RNP (hnRNP) complexes extracted from C. tentans nuclei. By immunoelectron microscopy of isolated and partly unfolded BR RNPs, it was specifically demonstrated that the BR granules contain the two proteins and, in addition, that both proteins are distributed frequently along the RNP fiber of the particles. Thus, the 36-and 45-kDa proteins are likely to be abundant, RNA-binding proteins in the BR particles. To elucidate to what extent the two proteins are also present in other hnRNPs, we studied the binding of the antibodies to chromosomal puffs in general. It was observed that many puffs in addition to the BRs harbor the two proteins, but there are also puffs containing only one of the components, either the 36-or the 45-kDa protein. We conclude that the two proteins are not randomly bound to all hnRNPs but that each of them seems to be linked to a specific subset of the particles.The nuclear precursors of mRNA, often designated hnRNA (heterogeneous nuclear RNA), are associated with proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs) (8, 10, 11). The hnRNP proteins constitute a major fraction of the nuclear proteins; in growing cells, they are as abundant as the histone proteins (18). They bind to the RNA concomitantly with transcription (12, 27) and are generally assumed to be confined to the cell nucleus (17, 35), although some of them have recently been shown to shuttle between the nucleus and the cytoplasm (34). The hnRNP population is complex; for example, there are about 20 major and a large number of minor hnRNP proteins in humans (33; see also reference 16). The hnRNP proteins contain a modular structure with one or more RNA-binding domains and at least one auxiliary domain that is probably involved in protein-protein interactions (for a recent review, see reference 11).hnRNP proteins have a general RNA-binding ability, but more and more information indicating that the proteins bind preferentially to distinct RNA sequences is accumulating. For example, the binding of several hnRNP proteins to different ribohomopolymers was studied, and it was found that various hnRNP proteins show different relative affinities for the ribonucleotide homopolymers (42). The hnRNP C proteins recognize a binding site consisting of five uridines in pre-mRNA polyadenylation substrates (28,46). The polypyrimidine tract...

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Section: Resultsmentioning

confidence: 99%

“…16,1996 RNA-BINDING PROTEINS IN BR PRE-mRNPs 1429 ticle, and thus both the 36-and the 45-kDa proteins are likely to appear in the particles in a large number of copies. Only a few grains were detected when the negative-control antibody of the same subclass was used (Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification of Two RNA-Binding Proteins in Balbiani Ring Premessenger Ribonucleoprotein Granules and Presence of These Proteins in Specific Subsets of Heterogeneous Nuclear Ribonucleoprotein Particles

Wurtz-T

Kiseleva

Nacheva

et al. 1996

Molecular and Cellular Biology

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“…As well known, when fractionation of nuclear extracts proceeds in the absence of exogenously added RNase inhibitors, the hnRNP complexes are cleaved to yield smaller structures and a population of 40-50 S hnRNPs referred to as monoparticles is highly enriched (review [19]). Sucrose gradient fractionation of the rat liver nuclear extracts is shown in fig.2A where a typical profile (ODz54) of such a gradient is presented.…”

Section: 2mentioning

confidence: 99%

Autoantibodies to the core proteins of hnRNPs

et al. 1988

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A novel autoantibody reacting with the core polypeptides of hnRNP particles has been detected in the serum of a patient with systemic lupus eythematosus (SLE) and Sjogren's syndrome manifestations. Immunoblot analysis, using either rat liver or HeLa nuclear extracts as the antigen source, demonstrated that the autoantibody interacts with a specific subgroup of the core polypeptides of hnRNP particles, namely 4, B, and B,, but not with A,, C, and C,.

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“…Apart from the general problem of RNA and protein degradation (for discussion, see, e.g., ref. 7), there are several biological reasons for this difficulty. First, most premessenger RNP species are present in low amounts, usually at less than 1% of all hnRNP (8).…”

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confidence: 99%

Isolation and initial characterization of a specific premessenger ribonucleoprotein particle.

Wurtz

Lönnroth²,

Ovchinnikov³

et al. 1990

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

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A specific type of premessenger ribonucleoprotein (RNP) particle, Balbiani ring (BR) granules, has been isolated from heterogeneous nuclear RNP (hnRNP) in the salivary glands of the dipteran Chironomus tentans. A BR granule contains a single 75S RNA molecule coding for a large secretory protein (Spl). The isolation procedure is based on the abundance and exceptional size of the BR granules: in EDTAcontaining sucrose gradients they sediment as a sharp 300S peak ahead of the remainder of the hnRNP population. The isolated BR granules were identified on the basis of both ultrastructural and biochemical criteria: large spherical particles that contain 75S RNA and BR sequences. A threedimensional reconstruction of isolated particles by electron microscope tomography further supported the identification of the isolated particles as BR granules. In contrast to the entire hnRNP population, the BR granules exhibited a sharp peak in CsCl gradients with a buoyant density of 1.45 g/cm3. This result indicates that a BR granule consists of 40% RNA and 60% protein by weight, corresponding to a 75S RNA molecule of 12 megadaltons and a total protein content of 18 megadaltons, or about 500 average-sized protein molecules.During synthesis, growing premessenger RNA molecules are immediately coupled to proteins and form ribonucleoprotein (RNP) complexes (1, 2). The structure of the RNP particles can to a large extent be understood as a chain of beads, a polyparticle (ref. 3; for recent reviews, see refs. 4 and 5). The beads, the monoparticles, contain a simple set of 5-10 proteins, which constitute the so-called core proteins of the polyparticles. Ultrastructural and biochemical studies indicate, however, that premessenger RNP particles also exhibit gene-specific features (for references, see ref. 6). Therefore, for full understanding of the functional behavior of the particles during processing and transport, specific premessenger particles have to be studied as individual entities.It has proven very difficult to isolate specific RNP particles from the complex premessenger RNP population [heterogeneous nuclear (hn) RNP] in the cell nucleus. Apart from the general problem of RNA and protein degradation (for discussion, see, e.g., ref. 7), there are several biological reasons for this difficulty. First, most premessenger RNP species are present in low amounts, usually at less than 1% of all hnRNP (8). Second, a large proportion ofthe particles are in a nascent state and therefore constitute a heterogeneous population as to size (5). Third, most of the premessenger RNP particles are being spliced and dramatically reduced in size, often in many steps and not necessarily in one and the same order (9). It is, therefore, not surprising that the isolation of a well-defined premessenger RNP particle has not yet, to our knowledge, been accomplished.In the present paper we report on the isolation of a specific type of premessenger RNP particle, the Balbiani ring (BR) granules in the salivary glands of the dipteran Chironomus tentans. ...

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Isolation and Structure of the Ribonucleoprotein Fibrils Containing Heterogeneous Nuclear RNA

Cited by 16 publications

References 90 publications

Identification of Two RNA-Binding Proteins in Balbiani Ring Premessenger Ribonucleoprotein Granules and Presence of These Proteins in Specific Subsets of Heterogeneous Nuclear Ribonucleoprotein Particles

Identification of Two RNA-Binding Proteins in Balbiani Ring Premessenger Ribonucleoprotein Granules and Presence of These Proteins in Specific Subsets of Heterogeneous Nuclear Ribonucleoprotein Particles

Autoantibodies to the core proteins of hnRNPs

Isolation and initial characterization of a specific premessenger ribonucleoprotein particle.

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