Some Properties of RNA:Protein Complexes from the Nucleus of Eukaryotic Cells

Martin, Terence E.; Billings, Peter B.; Levey, Aĺlan I.; Ozarslan, S.; Quinlan, Thomas J.; Swift, Hewson; Urbas, L.

doi:10.1101/sqb.1974.038.01.094

Cited by 101 publications

(89 citation statements)

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“…servations (Samarina et al 1968;Martin et al 1974Martin et al , 1978Pederson 1974;Beyer et al 1977;Karn et al 1977;Maundrell and Scherrer 1979;Walker et al 1980;Le Stourgeon et al 1981;Steitz and Kamen 1981;Lothstein et al 1985;Wilk et al 1985), hnRNA sediments in sucrose gradients as heterogeneous material of greater than 30S and, after mild digestion with nuclease, as more homogeneous material corresponding to monoparticles at about 30S. Heparin converts the hnRNP particles to slightly slower sedimenting material, but it is evident that particles sedimenting only slightly slower than the control remain.…”

Section: Heparin-resistan T Hnrnp Particlesmentioning

confidence: 99%

Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins.

Piñol-Roma¹,

Choi²,

Matunis³

et al. 1988

Genes Dev.

409

405

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Heterogeneous nuclear RNA-ribonucleoprotein (hnRNP) particles can be efficiently purifed by a specific, rapid, and mild procedure using monoclonal antibodies to hnRNP proteins. We report here on the detailed analysis of the protein composition of immunopurified hnRNP particles from human HeLa cells. By two-dimensional gel electrophoresis, immunopurified hnRNP particles contain at least 24 polypeptides in the range of 34,000-120,000 daltons. The abundant 30,000-40,000 dalton proteins, A, B, and C, described previously, are a subset of these polypeptides. The protein compositions of hnRNP particles found in the nucleoplasm fraction and in the chromatin-nucleolar fraction are very similar. Upon addition of the polyanion heparin, most of the major proteins remain associated in heparin-resistant particles, and only several, mostly minor, proteins dissociate. This provides an aid in the classification of the proteins and an additional criterion for the definition of hnRNP particle components. Chromatography on single-stranded DNA (ssDNA)-agarose in a heparin-and moderate or high salt (higher than 300 mM NaCl)-resistant manner suggests that most, if not all, of these proteins are single-stranded nucleic acid-binding proteins. We describe a general method for the large-scale purification of hnRNP proteins by affinity chromatography on ssDNA columns and its use for the production of new monoclonal antibodies to hnRNP proteins.

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Section: Heparin-resistan T Hnrnp Particlesmentioning

confidence: 99%

Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins.

Piñol-Roma¹,

Choi²,

Matunis³

et al. 1988

Genes Dev.

409

405

View full text Add to dashboard Cite

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“…For example, in actively growing mammalian cells there is 20 to 30% as much individual core particle hnRNP as individual core particle histone (24). (ii) The majority of nuclear pre-mRNA sequences are recovered from nuclear extracts as 40 to 500S complexes which yield 40S monoparticles upon endogenous or exogenous ribonuclease activity (31,32,37,38,41 (35). Transcripts of these genes reveal 25-nm RNP complexes at the 5' and 3' splice junctions of the single intron (36).…”

mentioning

confidence: 99%

Ribonucleoproteins package 700 nucleotides of pre-mRNA into a repeating array of regular particles.

Conway¹,

Wooley²,

Bibring³

et al. 1988

Mol. Cell. Biol.

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An assay for the in vitro assembly of HeLa cell 40S nuclear ribonucleoprotein particles (hnRNP particles) has been developed. The substrates were single-stranded nucleic acid polymers of defined length and sequence prepared in vitro and the six major core particle proteins from isolated 40S hnRNP. The fidelity of in vitro assembly was evaluated on various physical parameters, including sedimentation, salt dissociation, polypeptide stoichiometry, UV-activated protein-RNA cross-linking, and overall morphology. Correct particle assembly depended on RNA length and on the input protein/RNA ratio but not on the concentration of the reactant mixture nor on the presence or absence of internal RNA processing signals, a 5'-cap structure, a 3'-poly(A) moiety, or ATP as energy source. RNA lengths between 685 and 726 nucleotides supported correct particle assembly. Dimers and oligomeric complexes that possessed the same polypeptide stoichiometry as native hnRNP assembled on RNA chains that were integral multiples of 700 nucleotides. Intermediate-length RNA supported the assembly of nonstoichiometric complexes lacking structural homogeneity. An analysis of these complexes indicates that proteins Al and A2 may be the first proteins to bind RNA during particle assembly. We conclude that the major proteins of 40S hnRNP particles contain the necessary information for packaging nascent transcripts into a repeating "ribonucleosomal" structure possessing a defined RNA length and protein composition but do not themselves contain the information for modulating packaging that may be required for RNA splicing.The biochemical events of pre-mRNA processing are known largely through studies on the RNA intermediates produced during splicing and studies on the nucleotide sequences required for RNA maturation (for reviews, see references 17 and 42). Nascent transcripts, however, are packaged by a specific subset of major nuclear proteins during transcription to form a ribonucleoprotein (RNP) complex in which the events of RNA splicing occur (see references 8 and 10 for reviews). Historically, these complexes were termed heterogeneous nuclear ribonucleoprotein particles (hnRNP) because the packaged moiety was heterogeneous nuclear RNA and not because the individual particles were known to be heterogeneous in composition and structure (10,38,41). Several observations indicate that nascent transcripts are in fact packaged into a regular repeating structure composed of a contiguous array of 40S hnRNP complexes. These observations suggest that the complexes may play only a passive role in RNA splicing. Other observations suggest that packaging may be transcript specific and thus imply that hnRNP may play a more direct role in the events of RNA processing.Observations in support of a fundamental packaging function for hnRNP are the following. (i) The intranuclear concentration of the major hnRNP is high. For example, in actively growing mammalian cells there is 20 to 30% as much individual core particle hnRNP as individual core particle histone (...

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“…When either endogenous or exogenous nucleases are allowed to act on the hnRNP, particles between 30 and 50S are released. These core particles are considered to be the basic structural units of hnRNP (13,16), and although there may be as many as 40 protein components of these particles, there are usually only six major proteins, with molecular weights between 30,000 and 45,000 (3,10,16,26,33). These major proteins can be cross-linked by UV light to RNA in situ (5); therefore, the interaction between hnRNA and protein is apparently close.…”

mentioning

confidence: 99%

Specific regions of beta-globin RNA are resistant to nuclease digestion in RNA-protein complexes in chicken reticulocyte nuclei.

R¹,

Ross²,

Chae³

1985

Mol. Cell. Biol.

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. Chem. 258:3991-3995, 1983). We found that I-globin RNA is assembled into heterogenous nuclear RNA-protein complexes in a specific manner. There are several regions of nuclease resistance in the first and third exons interrupted at regular intervals by sensitive regions. The second exon has only one nuclease-resistant region. The resistant regions range in size from 20 to 50 nucleotides. This organization may reflect a specific mode of assembly for heterogenous nuclear RNA-protein complexes.Concomitant with the transcription of heterogenous nuclear RNA (hnRNA), the transcript is bound to proteins (14,20) to form heterogenous nuclear RNA-protein complexes (hnRNP). When precautions are taken to limit the action of endogenous nucleases, the isolated hnRNP can be quite large (200S) and have a beaded appearance in electron micrographs (12,21,31). When either endogenous or exogenous nucleases are allowed to act on the hnRNP, particles between 30 and 50S are released. These core particles are considered to be the basic structural units of hnRNP (13,16), and although there may be as many as 40 protein components of these particles, there are usually only six major proteins, with molecular weights between 30,000 and 45,000 (3,10,16,26,33). These major proteins can be cross-linked by UV light to RNA in situ (5); therefore, the interaction between hnRNA and protein is apparently close.Since the assembly of hnRNA into hnRNP takes place while transcription is occurring, the subsequent steps in the maturation of the RNA must take place with the proteins attached to the transcript. These proteins might serve to protect the RNA from digestion by nucleases. Alternatively, these proteins could stabilize (1) or actually induce (35) an RNA structure which is favorable for processing or transport. A central question regarding the structure of hnRNP is whether the proteins are bound to the RNA in a specific manner. Beyer et al. (4) provided visual evidence, in the form of electron micrographs, that hnRNP are assembled nonrandomly. There is biochemical evidence supporting the specific assembly of hnRNP (23,28,32,34). In all of the related biochemical studies, nuclease digestion combined with analysis of nuclease-resistant RNA has been the basic approach, with the assumption that interaction with proteins affords resistance to nuclease digestion. Solution hybridization (28) and Southern blot hybridization (22,23,32) ever, sensitivity of these methods is low and at best can only define a general area that is resistant to nuclease.We found that 3-globin RNA is assembled into hnRNP in a specific manner in chicken reticulocytes. Nuclease-resistant regions have been mapped, at the resolution of nucleotides, by the new RNA mapping method we have recently developed (25). There are extensive regions of nuclease resistance in the first and third exons, interrupted at regular intervals by sensitive regions. The second exon has only one nuclease-resistant region. We discuss how this organization could reflect a specific mode of assembly for hnR...

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Some Properties of RNA:Protein Complexes from the Nucleus of Eukaryotic Cells

Cited by 101 publications

References 0 publications

Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins.

Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins.

Ribonucleoproteins package 700 nucleotides of pre-mRNA into a repeating array of regular particles.

Specific regions of beta-globin RNA are resistant to nuclease digestion in RNA-protein complexes in chicken reticulocyte nuclei.

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