Y D Choi

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

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-beta-D-ribofuranosyl 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.

Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.

Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 +/- 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell II:127-138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix--a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase.

Ultraviolet-induced cross-linking of RNA to proteins in vivo

Publisher Summary This chapter describes methods for RNA–protein cross-linking in living cells as a means of identification of RNA-binding proteins in vivo . The chapter focuses on heterogeneous nuclear RNA (hnRNA)- and messenger RNA (mRNA)-binding proteins. The most stringent definition of genuine ribonucleoproteins (RNPs) is that these are proteins, which are bound directly to the RNA of interest in the living cell. UV cross-linking of RNP complexes takes advantage of the fact that UV light of sufficient intensity generates highly reactive species of RNA, which react virtually indiscriminately with molecules, including proteins, with which the RNA is in stable, direct contact. The cross-linked proteins can be released from the RNA by exhaustive RNase digestion and can be analyzed by gel-electrophoretic techniques. This method provides a powerful tool for the identification of bona fide RNP proteins. The fractionation of cells into nuclear and cytoplasmic components prior to the chromatographic step allows the distinction between hnRNA-containing RNP complexes (hnRNPs), from the nuclear fraction, and mRNAcontaining RNP complexes (mRNPs), from the cytoplasmic fraction.

Immunological methods for purification and characterization of heterogeneous nuclear ribonucleoprotein particles.

Publisher Summary Heterogeneous nuclear RNAs (hnRNAs) are associated in the cell with specific proteins to form hnRNA-ribonucleoprotein (hnRNP) complexes, also referred to as hnRNP particles. The immunopurification procedure has a number of advantages over methods traditionally used for studying hnRNP complexes such as sucrose gradient sedimentation of 30 S particles and photochemical cross-linking of proteins to RNA. The purity of the immunopurified hnRNP complexes is obvious by the absence of abundant nuclear proteins such as histones. Under the conditions described here, there are no significant amounts of other RNP proteins, such as those of small nuclear RNPs (snRNPs). The initial step in the hnRNP immunopurification procedure involves separation of the nuclear and cytoplasmic fractions, and the subsequent removal of nucleoli and insoluble chromatin from the nuclear fraction in order to generate what is operationally defined as the nucleoplasm. It is also essential to always include a control immunopurification with preimmune serum or immunoglobulins from the parent myeloma cell line used for the production of the hybridomas.

The Ribonucleoprotein Structures Along the Pathway of mRNA Formation

Heterogeneous nuclear RNAs (hnRNAs), some of which are mRNA precursors, and the mature mRNAs are associated in eukaryotic cells with specific proteins to form ribonucleoprotein complexes (RNP). The RNP proteins are likely to play a major role in the formation, packaging, processing, and function of mRNA. The major proteins that interact with hnRNA and with mRNA were identified by photochemical RNA-protein cross-linking in intact cells and monoclonal antibodies to several of these proteins were produced. Using these antibodies the hnRNP proteins were characterized and the hnRNP complex was isolated from vertebrate cell nuclei. The hnRNP complex is a unitary structure of consistent, defined and conserved components. The proteins of the hnRNP complex were identified and the general organization of hnRNA and proteins in the hRNP complex were studied.