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

Abstract

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.

Transcription on lampbrush chromosome loops in the absence of U2 snRNA.

“…One possibility is that the negatively charged oligos interact with basic proteins on the chromosome loops that are normally complexed with the pre-mRNA (DiMario et al, 1989). Likely candidates would be the A and B group hnRNPs, which are well-known to bind both RNA and single-stranded DNA (Kumar et al, 1986;Riva et al, 1986;Pifiol-Roma et al, 1988). Another possibility is that the oligos interact with metal ions such as Ca++ or Mg++, whose sequestering might lead to secondary effects on the chromosomes.…”

Regulation of tissue-specific alternative splicing: exon-specific cis-elements govern the splicing of leukocyte common antigen pre-mRNA.

“…As for the chemical nature of the trans-acting factor, a diffusible factor, such as a protein or nucleoprotein, is the most obvious candidate, although the relevance of differences in pH and/or salt concentration (Schmitt et al, 1987) cannot be excluded. In the nucleus, precursor mRNA forms heterogeneous nuclear ribonucleoprotein (hnRNP) by complexing with > 20 different proteins (Beyer et al, 1977;Pinol-Roma et al, 1988). Some hnRNP proteins have been implicated in premRNA splicing (Choi et al, 1986;Mayrand et al, 1986).…”

The hnRNP F protein: unique primary structure, nucleic acid-binding properties, and subcellular localization

“…It is within these hnRNP complexes that pre-mRNAs are processed to form mature mRNA transcripts before export from the nucleus (1). Immunopurified hnRNP complexes from human HeLa cells contain more than 20 individual proteins designated hnRNP Al (34 kD) through hnRNP U (120 kD) (2), and characterization of these proteins is important because they are likely to influence mRNA metabolism. Many hnRNP proteins have been characterized at the molecular level, including hnRNP A1 (3), A2/B1 (4), C1/C2 (5), I (6-9), K (10), L (11), M (12) and U (13).…”