In mammalian selenoprotein mRNAs, the recognition of UGA as selenocysteine requires selenocysteine insertion sequence (SECIS) elements that are contained in a stable stem-loop structure in the 3 untranslated region (UTR). In this study, we investigated the SECIS elements and cellular proteins required for selenocysteine insertion in rat phospholipid hydroperoxide glutathione peroxidase (PhGPx). We developed a translational readthrough assay for selenoprotein biosynthesis by using the gene for luciferase as a reporter. Insertion of a UGA or UAA codon into the coding region of luciferase abolished luciferase activity. However, activity was restored to the UGA mutant, but not to the UAA mutant, upon insertion of the PhGPx 3 UTR. The 3 UTR of rat glutathione peroxidase (GPx) also allowed translational readthrough, whereas the PhGPx and GPx antisense 3 UTRs did not. Deletion of two conserved SECIS elements in the PhGPx 3 UTR (AUGA in the 5 stem or AAAAC in the terminal loop) abolished readthrough activity. UV cross-linking studies identified a 120-kDa protein in rat testis that binds specifically to the sense strands of the PhGPx and GPx 3 UTRs. Direct cross-linking and competition experiments with deletion mutant RNAs demonstrated that binding of the 120-kDa protein requires the AUGA SECIS element but not AAAAC. Point mutations in the AUGA motif that abolished protein binding also prevented readthrough of the UGA codon. Our results suggest that the 120-kDa protein is a significant component of the mechanism of selenocysteine incorporation in mammalian cells.
The splicing factor SPF45 (RBM17) is frequently overexpressed in many solid tumors, and stable expression in HeLa cells confers resistance to doxorubicin and vincristine. In this study, we characterized stable transfectants of A2780 ovarian carcinoma cells. In a 3-day cytotoxicity assay, human SPF45 overexpression conferred 3-to 21-fold resistance to carboplatin, vinorelbine, doxorubicin, etoposide, mitoxantrone, and vincristine. In addition, resistance to gemcitabine and pemetrexed was observed at the highest drug concentrations tested. Knockdown of SPF45 in parental A2780 cells using a hammerhead ribozyme sensitized A2780 cells to etoposide by f5-fold relative to a catalytically inactive ribozyme control and untransfected cells, suggesting a role for SPF45 in intrinsic resistance to some drugs. A2780-SPF45 cells accumulated similar levels of doxorubicin as vector-transfected and parental A2780 cells, indicating that drug resistance is not due to differences in drug accumulation. Efforts to identify small molecules that could block SPF45-mediated drug resistance revealed that the selective estrogen receptor (ER) modulators tamoxifen and LY117018 (a raloxifene analogue) partially reversed SPF45-mediated drug resistance to mitoxantrone in A2780-SPF45 cells from 21-fold to 8-and 5-fold, respectively, but did not significantly affect the mitoxantrone sensitivity of vector control cells. Quantitative PCR showed that ERB but not ERA was expressed in A2780 transfectants. Coimmunoprecipitation experiments suggest that SPF45 and ERB physically interact in vivo. Thus, SPF45-mediated drug resistance in A2780 cells may result in part from effects of SPF45 on the transcription or alternate splicing of ERBregulated genes. (Cancer Res 2005; 65(15): 6593-600)
Purified ribonucleoprotein complexes of human parainfluenza virus type 3 (HPIV-3) virions required, in addition to the viral proteins, soluble cytoplasmic proteins from uninfected cells for the synthesis of mRNAs in vitro. In contrast to Sendai virus transcription, in vitro RNA synthesis from HPIV-3 ribonucleoprotein complexes was not stimulated significantly by purified tubulin. Moreover, cytoplasmic extract depleted of tubulin by immunoprecipitation stimulated HPIV-3 transcription effectively, suggesting involvement of a host protein(s) other than tubulin in the HPIV-3 transcription process. The transcription stimulatory factor was purified from uninfected cell extract by conventional chromatography and was found to contain a major 43-kDa polypeptide. In Western blot (immunoblot) analysis, this protein reacted with antiactin antibody, suggesting that the 43-kDa polypeptide is actin. This possibility was further supported by its polymerization activity and properties of binding to blue-Sepharose and heparin-Sepharose columns. Furthermore, when the cell extract was depleted of actin by immunoprecipitation by antiactin antibody, the stimulatory activity was abolished, indicating an involvement of actin in the stimulation of HPIV-3 transcription. After purification from RNases, similar stimulatory activity associated with the 43-kDa protein was detected in other cell lines as well, including CV-1, HeLa, and BHK.
The M. tuberculosis rpoA gene, encoding the a subunit of RNA polymerase, was isolated by a polymerase chain reaction-based strategy using primers directed against regions of a that are highly conserved among Gram-positive and Gram-negative bacteria. The amplified DNA was utilized as a hybridization probe to recover the entire rpoA gene from a cosmid library of genomic DNA from virulent M. tuberculosis strain H37Rv. Nucleotide sequencing indicated that the 1044 bp M. tuberculosis rpoA open reading frame (ORF) encodes a protein of 347 amino acids which shows significant structural similarity to the a subunits of diverse bacterial species. Recombinant M. tuberculosis a subunit was overproduced in soluble form in E. coli as a hexahistidine-tagged fusion protein and purified to homogeneity by affinity chromatography.
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