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

S. Piñol-Roma, Y. D. Choi, Michael J Matunis, G. Dreyfuss

Research output: Contribution to journalArticle

Abstract

Heterogeneous nuclear RNA-ribonucleoprotein (hnRNP) particles can be efficiently purified 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.

Original languageEnglish (US)
Pages (from-to)215-227
Number of pages13
JournalGenes & development
Volume2
Issue number2
StatePublished - Feb 1988
Externally publishedYes

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Heterogeneous Nuclear RNA
Heterogeneous-Nuclear Ribonucleoproteins
RNA-Binding Proteins
Proteins
Heparin
Single-Stranded DNA
Monoclonal Antibodies
Peptides
Electrophoresis, Gel, Two-Dimensional
Staphylococcal Protein A
Protein C
Affinity Chromatography
HeLa Cells
Sepharose
Nucleic Acids
Chromatin
Chromatography
Carrier Proteins
Salts

ASJC Scopus subject areas

  • Developmental Biology
  • Genetics

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Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins. / Piñol-Roma, S.; Choi, Y. D.; Matunis, Michael J; Dreyfuss, G.

In: Genes & development, Vol. 2, No. 2, 02.1988, p. 215-227.

Research output: Contribution to journalArticle

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