Communicated by J.A.SteitzWe have previously shown that the yeast PRP19 protein is a spliceosomal component, but is not tightly associated with small nuclear RNAs. It appears to associate with the spliceosome concomitant with or just after dissociation of the U4 small nuclear RNA during spliceosome assembly. We have found that PRP19 is associated with a protein complex in the splicing extract and that at least one of the associated components is essential for splicing. Taking advantage of the epitope tagging technique, we have isolated the PRP19-associated complex by affmity chromatography. The isolated complex is functional for complementation for the heat-inactivated prpl9 mutant extract, and consists of at least seven polypeptides in addition to PRP19. At least three of these can interact directly with the PRP19 protein. We also show that the PRP19 protein itself is in an oligomeric form, which might be a prerequisite for its interaction with these proteins.

The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction. We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components. By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p. Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction. We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and proteinprotein interactions. Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e. concomitant with or immediately after dissociation of U4. The antiCef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex.The eukaryotic spliceosome is a multicomponent ribonucleoprotein particle composed of five small nuclear RNAs, U1, U2, U4/U6, and U5, and a number of protein factors (for reviews, see Refs. 1-6). Spliceosome assembly is a stepwise process involving sequential binding of small nuclear RNAs and protein factors (7-13). During spliceosome assembly, U1 first binds to the 5Ј splice site followed by binding of U2 to the branch site through base pair interactions between the small nuclear RNAs and the intron sequences. U4/U6 and U5 are then added to the spliceosome as a preformed three-small nuclear RNP particle. This triggers a conformational rearrangement of the spliceosome in which base pairing of U1 with the 5Ј splice site is replaced by U6, and base paired U4/U6 unwinds to form new base pairings between U6 and U2 (14 -17). U1 and U4 thus become only loosely associated with the spliceosome, which is now activated and ready for catalytic reactions. It is believed that such structural rearrangements of the spliceosome are mediated by protein factors. Although several proteins containing the DEX(D/H) box motif have been shown RNA unwindase activity (6, 18 -21), no substrate specificity could be demonstrated. It remains a question what dictates the substrate specificity and mediates conformational rearrangement of the spliceosome during spliceosome assembly.We have previously shown that the yeast Saccharomyces cerevisiae Prp19p protein is essential for pre-mRNA splicing and is required before the first step of the splicing reaction. Prp19p is not tightly associated with small nuclear RNAs but is associated with the spliceosome immediately after or concomitant with dissociation of U4 from the spliceosome, s...

The Prp19p-associated complex is essential for the yeast pre-mRNA splicing reaction. The complex consists of at least eight protein components, but is not tightly associated with spliceosomal snRNAs. By a combination of genetic and biochemical methods we previously identified four components of this complex, Ntc25p, Ntc85p, Ntc30p and Ntc20p, all of them being novel splicing factors. We have now identified three other components of the complex, Ntc90p, Ntc77p and Ntc31p. These three proteins were also associated with the spliceosome during the splicing reaction in the same manner as Prp19p, concurrently with or immediately after dissociation of U4 snRNA. Two-hybrid analysis revealed that none of these proteins interacted with Prp19p or Ntc25p, but all interacted with Ntc85p. An interaction network between the identified components of the Prp19p-associated complex is demonstrated. Biochemical analysis revealed that Ntc90p, Ntc31p, Ntc30p and Ntc20p form a subcomplex, which, through interacting with Ntc85p and Ntc77p, can associate with Prp19p and Ntc25p to form the Prp19p-associated complex. Genetic analysis suggests that Ntc31p, Ntc30p and Ntc20p may play roles in modulating the function of Ntc90p.

Positive-strand RNA virus genome replication occurs in membrane-associated RNA replication complexes, whose assembly remains poorly understood. Here we show that prior to RNA replication, the multifunctional, transmembrane RNA replication protein A of the nodavirus flock house virus (FHV) recruits FHV genomic RNA1 to a membrane-associated state in both Drosophila melanogaster and Saccharomyces cerevisiae cells. Protein A has mitochondrial membrane-targeting, self-interaction, RNA-dependent RNA polymerase (RdRp), and RNA capping domains. In the absence of RdRp activity due to an active site mutation (A D692E ), protein A stimulated RNA1 accumulation by increasing RNA1 stability. Protein A D692E stimulated RNA1 accumulation in wild-type cells and in xrn1 ؊ yeast defective in decapped RNA decay, showing that increased RNA1 stability was not due to protein A-mediated RNA1 recapping. Increased RNA1 stability was closely linked with protein A-induced membrane association of the stabilized RNA and was highly selective for RNA1. Substantial N-and C-proximal regions of protein A were dispensable for these activities. However, increased RNA1 accumulation was eliminated by deleting protein A amino acids (aa) 1 to 370 but was restored completely by adding back the transmembrane domain (aa 1 to 35) and partially by adding back peripheral membrane association sequences in aa 36 to 370. Moreover, although RNA polymerase activity was not required, even small deletions in or around the RdRp domain abolished increased RNA1 accumulation. These and other results show that prior to negative-strand RNA synthesis, multiple domains of mitochondrially targeted protein A cooperate to selectively recruit FHV genomic RNA to membranes where RNA replication complexes form.All positive-strand RNA viruses replicate in association with intracellular membranes. The particular membrane(s) used during RNA replication varies, with many positive-strand RNA viruses replicating in association with the endoplasmic reticulum, while others use lysosomal, mitochondrial, peroxisomal, or other membranes (16,17,26,28,36,42,43,45,55,56,60). Some viral RNA replication proteins carry membranetargeting signals and interact with other viral proteins to localize these to the sites of RNA replication complex formation (18,56,58,62). In a few cases, interactions contributing to recruitment of viral RNA replication templates have also been identified. Brome mosaic virus (BMV) helicase-like protein 1a, e.g., induces formation of spherular endoplasmic reticulum membrane invaginations and selectively recruits BMV 2a polymerase and BMV genomic RNAs to these structures to form RNA replication complexes (1,11,12,32,62,66). Similarly, tomato bushy stunt virus replication protein p33 binds defective interfering RNA (DI-RNA) in vitro, and the related cucumber necrosis virus p33 protein recruits such RNAs to replication complex sites in vivo (47,48,51,54).Flock house virus (FHV) is the best-studied member of the alphanodaviruses in the Nodaviridae family. The bipartite FHV gen...

BackgroundThe effective therapies for oral cancer patients of stage III and IV are generally surgical excision and radiation combined with adjuvant chemotherapy using 5-Fu and Cisplatin. However, the five-year survival rate is still less than 30% in Taiwan. Therefore, evaluation of effective drugs for oral cancer treatment is an important issue. Many studies indicated that aurora kinases (A, B and C) were potential targets for cancer therapies. Reversine was proved to be a novel aurora kinases inhibitor with lower toxicity recently. In this study, the potentiality for reversine as an anticancer agent in oral squamous cell carcinoma (OSCC) was evaluated.MethodsEffects of reversine on cell growth, cell cycle progress, apoptosis, and autophagy were evaluated mainly by cell counting, flow cytometry, immunoblot, and immunofluorescence.ResultsThe results demonstrated that reversine significantly suppressed the proliferation of two OSCC cell lines (OC2 and OCSL) and markedly rendered cell cycle arrest at G2/M stage. Reversine also induced cell death via both caspase-dependent and -independent apoptosis. In addition, reversine could inhibit Akt/mTORC1 signaling pathway, accounting for its ability to induce autophagy.ConclusionsTaken together, reversine suppresses growth of OSCC via multiple mechanisms, which may be a unique advantage for developing novel therapeutic regimens for treatment of oral cancer in the future.