Ro ribonucleoprotein (RNP) complexes are composed of one molecule of a small noncoding cytoplasmic RNA, termed Y RNA, and the two proteins Ro60 and La. Additional proteins such as hnRNP I, hnRNP K, or nucleolin have recently been shown to be associated with subpopulations of Y RNAs. Ro RNPs appear to be localized in the cytoplasm of all higher eukaryotic cells but their functions have remained elusive. To shed light on possible functions of Ro RNPs, we tested protein components of these complexes for RNA chaperone properties employing two in vitro chaperone assays and additionally an in vivo chaperone assay. In these assays the splicing activity of a group I intron is measured. La showed pronounced RNA chaperone activity in the cis-splicing assay in vitro and also in vivo, whereas no activity was seen in the trans-splicing assay in vitro. Both hnRNP I and hnRNP K exhibited strong chaperone activity in the two in vitro assays, however, proved to be cytotoxic in the in vivo assay. No chaperone activity was observed for Ro60 in vitro and a moderate activity was detected in vivo. In vitro chaperone activities of La and hnRNP I were completely inhibited upon binding of Y RNA. Taken together, these data suggest that the Ro RNP components La, hnRNP K, and hnRNP I possess RNA chaperone activity, while Ro60-Y RNA complexes might function as transporters, bringing other Y RNA binding proteins to their specific targets.

Polyomavirus large and small T antigens cooperate in the induction of S phase in serum-deprived Swiss 3T3 cells. While the large T antigen is able to induce S phase-specific enzymes, we have recently shown that both T antigens contribute to the production of the cyclins E and A and that the small T antigen is essential for the induction of cyclin A-dependent cdk2 activity (S. Schüchner and E. Wintersberger, J. Virol. 73:9266-9273, 1999). Here we present our attempts to elucidate the mechanisms by which the large and the small T antigens transactivate the murine cyclin A gene. Using Swiss 3T3 cells carrying the T antigens and various mutants thereof under the hormone-inducible mouse mammary tumor virus promoter, as well as transient-cotransfection experiments with the T antigens and cyclin A promoter-luciferase reporter constructs, we found the following. The large T antigen activates the cyclin A promoter via two transcription factor binding sites, a cyclic AMP responsive element (CRE), and the major negative regulatory site called CDE-CHR. While an intact binding site for pocket proteins is required for the function of this T antigen at the CDE-CHR, its activity at the CRE is largely independent thereof. In contrast, an intact J domain and an intact zinc finger are required at both sites. The small T antigen also appears to have an influence on the cyclin A promoter through the CRE as well as the CDE-CHR. For this an interaction with protein phosphatase 2A is essential; mutation of the J domain does not totally eliminate but greatly reduces the transactivating ability.The induction of S phase of the cell cycle is a complex reaction usually initiated at the cell surface through binding of ligands to receptors. Signal transduction pathways lead to the synthesis of S phase-specific enzymes and regulators, including the cyclins E and A. DNA tumor viruses require cells in S phase because they heavily depend on the cellular DNA synthesis machinery for the replication of their own DNA. Since they frequently infect differentiated cells, they have to interfere with the cellular mechanisms of growth regulation in order to drive cells out of the G 0 phase and into the S phase (reviewed in reference 19). This is accomplished by viral proteins which interact with various intermediates of the signal transduction pathway downstream of events taking place at the cell surface. One class of target for such viral proteins are the pocket proteins: pRB and its relatives p107 and p130. They negatively regulate members of the transcription factor family E2F (reviewed in reference 8). Adenovirus E1A, human papillomavirus (HPV) E7 protein, and the large T (LT) antigens of simian virus 40 (SV40), and polyomavirus (Py) bind to pocket proteins and cause a dissociation of the repressive complexes, which results in the transactivation of E2F responsive genes (19). Another region of the pleiotropic T antigens which is essential for this reaction is the J domain (reviewed in references 4 and 16), a sequence present in many chaperones capable ...