RESUMO
M1 RNA, the gene product of rnpB, is the catalytic subunit of RNase P in Escherichia coli. M1 RNA is transcribed from a proximal promoter as pM1 RNA, a precursor M1 RNA, and then is processed at its 3' end by RNase E. In addition to pM1 RNA, large rnpB-containing transcripts are produced from unknown upstream promoters. However, it is not known yet how these large transcripts contribute to M1 RNA biosynthesis. To examine their biological relevance to M1 RNA biosynthesis, we constructed a model upstream transcript, upRNA, and analyzed its cellular metabolism. We found that upRNA was primarily degraded rather than processed to M1 RNA in the cell and that this degradation occurred in RNase E-dependent manner. The in vitro cleavage assay with the N-terminal catalytic fraction of RNase E showed that the M1 RNA structural sequence in upRNA was much more vulnerable to the enzyme than the sequence in pM1 RNA. Considering that RNase E is a processing enzyme involved in 3' end formation of M1 RNA, our results imply that this enzyme plays a dual role in processing and degradation to achieve tight control of M1 RNA biosynthesis.
Assuntos
Endorribonucleases/metabolismo , Proteínas de Escherichia coli/biossíntese , Escherichia coli/enzimologia , Ribonuclease P/biossíntese , Escherichia coli/genética , Proteínas de Escherichia coli/química , Regulação Bacteriana da Expressão Gênica , Modelos Biológicos , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonuclease P/química , Especificidade por SubstratoRESUMO
Prostate-specific-membrane-antigen (PSMA) is a marker protein expressed primarily in prostate epithelium. Its prostate-specific expression is conferred by PSMA enhancer (PSME), localized within the third intron of PSMA-encoding gene FOLH1. We recently reported that the 5'-end 90 bp of PSME harbored crucial enhancer elements for high PSMA expression. Deletion of this 90 bp sequence, called PSME(del3), significantly diminished PSME activity. We have further analyzed the regulatory elements in this 90 bp by transient transfection of linker scanning mutants. Two mutants, LN17 and 18, which harbored an AP-1 site and an AP-3 site, respectively, exhibited significantly lower enhancer activity. Subsequent site-directed mutagenesis changing the AP-3 site abolished the enhancer activity of PSME but not AP-1, indicating that AP-3 was the key cis-element enabling high PSMA expression. In addition, a 12 bp AP-3 site was able to enhance PSME(del3) activity by almost 40% higher compared to full-length PSME. However, AP-3 alone retained just the basal level of activity, indicating that the action by AP-3 was mediated by cooperation with other transcription factors binding to the PSME(del3) region. Transcription factor NFATc1 isoforms in nuclear extract were co-precipitated with the biotinylated AP-3 site by immobilized agarose beads and the genomic DNA containing PSME was precipitated by antibodies reactive to NFATc1, demonstrating that NFATc1 isoforms bound to the AP-3 site in PSME in vivo. Furthermore, ionomycin (calcium ionophore) and TPA augmented the enhancer activity of PSME, implying that calcium is an important regulator for PSMA expression in prostate cancer cell.
Assuntos
Antígenos de Superfície , Carboxipeptidases/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Biotinilação , Western Blotting , Cálcio/metabolismo , Carboxipeptidases/genética , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/química , Eletroforese em Gel de Poliacrilamida , Elementos Facilitadores Genéticos , Deleção de Genes , Glutamato Carboxipeptidase II , Humanos , Células Jurkat , Luciferases/metabolismo , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese , Mutagênese Sítio-Dirigida , Mutação , Fatores de Transcrição NFATC , Plasmídeos/metabolismo , Testes de Precipitina , Neoplasias da Próstata/química , Ligação Proteica , Isoformas de Proteínas , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/química , Transfecção , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
The expression of prostate-specific membrane antigen (PSMA) and prostate-specific antigen (PSA), two well characterized marker proteins, remains highly active in the hormone refractory stage of prostate cancer. In this study, an artificial chimeric enhancer (PSES) composed of two modified regulatory elements controlling the expression of PSA and PSMA genes was tested for its promoter activity and tissue specificity using the reporter system. As a result, this novel PSES promoter remained silent in PSA- and PSMA-negative prostate and non-prostate cancer cell lines, but mediated high levels of luciferase in PSA- and PSMA-expressing prostate cancer cell lines in the presence and absence of androgen. To determine whether PSES could be used for in vivo gene therapy of prostate cancer, a recombinant adenovirus, Ad-PSES-luc, was constructed. Luciferase activity in prostate cancer cell lines mediated by Ad-PSES-luc was 400- to 1000-fold higher than in several other non-prostate cell lines, suggesting the high tissue-specificity of the PSES promoter in an adenoviral vector. Finally, recombinant virus Ad-PSES-luc was injected into mice to evaluate the tissue-discriminatory promoter activity in an experimental animal. Unlike Ad-CMV-luc, the luciferase activity from systemic injection of Ad-PSES-luc was fairly low in all major organs. However, when injected into prostate, Ad-PSES-luc drove high luciferase activity almost exclusively in prostate and not in other tissues. Our results demonstrated the potential use of PSES for the treatment of androgen-independent prostate cancer patients.
