The release of 40S hnRNP particles by brief digestion of HeLa nuclei with micrococcal nuclease

Walker, Barbara; Lothstein, Leonard; Baker, Christopher L.; LeStourgeon, Wallace M.

doi:10.1093/nar/8.16.3639

Cited by 39 publications

(16 citation statements)

References 23 publications

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“…The choice of these transcript lengths was based on our previous studies showing that the RNA fragments recovered from sucrose gradient-isolated 40S monoparticles ranged from 500 to 800 nucleotides, with most fragments being about 700 nucleotides in length (3,23,47). All three transcript lengths gave the same results (Fig.…”

Section: Resultsmentioning

confidence: 93%

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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Section: Resultsmentioning

confidence: 93%

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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“…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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“…Nuclear purity was monitored by phasecontrast microscopy. The nuclear pellet, containing 1.2-1.5 x lo9 nuclei, was washed in 50 ml STM buffer (90 mM NaCl, 1 .O mM MgClz, 10 mM Tris-HCl, pH 8.0), resuspended in 3.0 ml of the same buffer and ribonucleoprotein complexes were released by sonic disruption on ice [12]. The nuclear sonicate was incubated at 37°C for 10 min to increase the yield of 40 S monoparticles via the action of endogenous nucleases.…”

Section: Isolation Of 40 S Hnrnp Particlesmentioning

confidence: 99%

Identification of proliferation‐sensitive human proteins amongst components of the 40 S hnRNP particles

Celis¹,

Bravo²,

Arenstorf³

et al. 1986

FEBS Letters

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The core proteins of HeLa 40 S hnRNP monoparticles have been identified in the HeLa protein catalogue. Human proteins previously indentified as proliferation-sensitive [NEPHGE 21 and 17; Bravo, R. and Celis, J.E. (1982) Clin. Chem. 28, 7661, as well as two proteins characterized in this study (NEPHGE 16 W and 16 Wl), are shown to be components of these. particles. These basic nuclear polypeptides correspond to core proteins A,, B,,, Bz and Cq, respectively. The significance of these results in terms of composition and function of hnRNP particles is discussed. Prolverating cell Resting cellNuclear protein hnRNP function mRNA transport

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The release of 40S hnRNP particles by brief digestion of HeLa nuclei with micrococcal nuclease

Cited by 39 publications

References 23 publications

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

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

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

Identification of proliferation‐sensitive human proteins amongst components of the 40 S hnRNP particles

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