Isolation and characterization of the heterogeneous nuclear RNA-ribonucleoprotein complex

Thesis/Dissertation · Tue Jan 01 04:00:00 EST 1985

OSTI ID:6704204

Choi, Y D

Exposure of cells to UV light of sufficient intensity brings about crosslinking of RNA to proteins which are in direct contact with it in vivo. The major (/sup 35/S)methionine-labeled proteins which become crosslinked to poly(A)/sup +/hnRNA in HeLa cells are of 120K, 68K, 53K, 43K, 41K, 38K, and 36K (K = kilodaltons). By immunizing mice with UV crosslinked complexes two monoclonal antibodies (2B12 and 4F4) against the C proteins (41K and 43K) and one (3G6) against the 120K protein of the hnRNP complex were obtained. Immunofluorescence microscopy demonstrates that the C proteins and 120K are segregated to the nucleus and are not associated with nucleoli or chromatin. The two C proteins are highly related to each other antigenically. Monoclonal antibody 4F4 identifies the C proteins of the hnRNP complex in widely divergent species from human to lizard. The C proteins are phosphorylated and are in contact with hnRNA in vivo. The hnRNP complex was isolated from vertebrate cell nuclei by immunoprecipitation with these monoclonal antibodies. This complex contains proteins and hnRNA of up to approx.10 kb. The major steady state labeled (/sup 35/S)methionine labeled proteins of the isolated complex from HeLa cells are of 34K, 36K, 36K (A1 and A2), 37K, 38K (B1 and B2), 41K, 43K (C1 and C2) and doublets at 68K and at 120K. These proteins are organized into a 30S particle. Large hnRNP complexes are composed of multiples of 30S particles which are connected by highly nuclease sensitive stretches of hnRNA. It it concluded that the hnRNP structure is an integral component of the mRNA formation pathway in the eukaryotic cell.

• Research Organization: Northwestern Univ., Evanston, IL (USA)
• OSTI ID: 6704204
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
BIOLOGICAL LOCALIZATION
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL CULTURES
CELL NUCLEI
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTROMAGNETIC RADIATION
EVEN-ODD NUCLEI
FLUORESCENCE
HELA CELLS
IMMUNOASSAY
IMMUNOLOGY
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LIPOTROPIC FACTORS
LUMINESCENCE
MESSENGER-RNA
METHIONINE
MOLECULAR STRUCTURE
NUCLEI
NUCLEIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORYLATION
PROTEINS
RADIATIONS
RADIOASSAY
RADIOIMMUNOASSAY
RADIOIMMUNOLOGY
RADIOISOTOPES
RNA
SULFUR 35
SULFUR ISOTOPES
TRACER TECHNIQUES
ULTRAVIOLET RADIATION