Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription.

Abstract

Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 (38K) become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-p-Dribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared. Immunofluorescence microscopy shows mostly cytoplasmic and some nuclear staining. These observations demonstrate that commonly used inhibitors of transcription affect the physical state of messenger ribonucleoproteins in vivo.The complexes of proteins with heterogeneous nuclear RNA (hnRNA) (heterogeneous nuclear ribonucleoproteins [hnRNPs]) and with mRNA (messenger ribonucleoproteins [mRNPs]) are considered to be the functional assemblies which are involved in the synthesis and function of these polynucleotides in the cell (for a review, see reference 33). The proteins which form these complexes are, therefore, of key interest and are the focus of much research. The major difficulties in identifying RNPs by conventional isolation techniques stem from the fact that the criteria of copurification of certain proteins with polynucleotides are, in general, not sufficiently stringent to ascertain whether these are genuine RNP components in vivo. This is due to the fact that nonspecific interactions between RNA and proteins are likely to occur in vitro (2, 14, 38). UV cross-linking of RNA to protein in intact cells overcomes these difficulties and allows the identification of proteins which are in direct contact with hnRNA and mRNA in vivo (10,34,35,50,51,53).The in vivo UV cross-linking approach relies on the fact that UV light of sufficient intensity generates photoreactive species of RNA which react virtually indiscriminately with molecu...

Monoclonal antibody characterization of the C proteins of heterogeneous nuclear ribonucleoprotein complexes in vertebrate cells.

“…The poly(A) + material was pelleted by centrifugation at 12,500 g and resuspendcd in 75/zl of 10 mM Tris-HO, pH 7.4, containing 1 mM CaCI2, and digestion with RNAse was carried out with 25 ug/ml pancreatic RNase A (Worthington Biochemical Corp.), and 400 U/ml micrococcal nuclease (P-L Bicobemicals, Inc., Milwaukee, WI) for 60 min at 37°C (11,12). To inhibit possible traces of proteasc, the pancreatic RNase was preboiled and aprotinin (0.5%), pepstatin A ( 1 ~g/ml), and leupeptin ( l eg/ml) (Sigma Chemical Co.) were included in the digestion mixture.…”

“…Protein samples were electrophoresed on an SDS-contain-1998 THE JOURNAL OF CELL BtOLOGV-VOLUME 99, 1984 ing discontinuous PAGE system (SDS PAGE) (11). The separating gel had a final acrylamide concentration of 12.5%.…”

“…RNPs: Procedures were as described recently (11,12). Briefly, poly(A) ÷ material from UV-irradiated HeLa cells (nuclei and cytoplasm), which eluted from the oligo(dT)-cellulose column after two cycles of chromatography, was used for preparation of monoclonal antibodies in mice (12).…”

Purification and characterization of the Ner repressor of bacteriophage Mu

“…Protein concentrations were determined as described by Lowry et al [13]. Proteins were subjected to electrophoresis in 15% SDS-polyacrylamide gels [14] and stained with silver as described by Morrissey [15]. Crude extracts of strain LF123 were prepared as previously described [4].…”

“…The position of sample elution was monitored by: (a) protein gel electrophoresis [14] followed by silver staining of the gels [15]; (b) measurement of the A280 with an ISCO UA-5 absorbance monitor; and (c) the band competition assay [lo].…”