RESUMO
In this study we have investigated the role of Epr, a minor extracellular serine protease, in the swarming motility of Bacillus subtilis 168. We identified that the protease activity of Epr was dispensable for swarming. Since the protease activity of Epr was confined to its N-terminal domain, we hypothesized instead that its C-terminal domain (CTD) could be critical for swarming. Our study showed that not only the expression of Epr-CTD was necessary, but also its secretion was crucial for the swarming motility of B. subtilis 168.
Assuntos
Bacillus subtilis/fisiologia , Regulação Bacteriana da Expressão Gênica , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Movimento , Mutagênese Sítio-Dirigida , Serina Endopeptidases/genéticaRESUMO
Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology with the alternative pre-mRNA processing of caspase 9 as one example. In this study, we delve into the underlying molecular mechanisms that regulate the alternative splicing of caspase 9. Specifically, the pre-mRNA sequence of caspase 9 was analyzed for RNA cis-elements known to interact with SRSF1, a required enhancer for caspase 9 RNA splicing. This analysis revealed 13 possible RNA cis-elements for interaction with SRSF1 with mutagenesis of these RNA cis-elements identifying a strong intronic splicing enhancer located in intron 6 (C9-I6/ISE). SRSF1 specifically interacted with this sequence, which was required for SRSF1 to act as a splicing enhancer of the inclusion of the 4 exon cassette. To further determine the biological importance of this mechanism, we employed RNA oligonucleotides to redirect caspase 9 pre-mRNA splicing in favor of caspase 9b expression, which resulted in an increase in the IC(50) of non-small cell lung cancer (NSCLC) cells to daunorubicin, cisplatinum, and paclitaxel. In contrast, downregulation of caspase 9b induced a decrease in the IC(50) of these chemotherapeutic drugs. Finally, these studies showed that caspase 9 RNA splicing was a major mechanism for the synergistic effects of combination therapy with daunorubicin and erlotinib. Overall, we have identified a novel intronic splicing enhancer that regulates caspase 9 RNA splicing and specifically interacts with SRSF1. Furthermore, we showed that the alternative splicing of caspase 9 is an important molecular mechanism with therapeutic relevance to NSCLCs.
Assuntos
Processamento Alternativo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Caspase 9/genética , Daunorrubicina/uso terapêutico , Pneumopatias/tratamento farmacológico , Proteínas Nucleares/metabolismo , Quinazolinas/uso terapêutico , Proteínas de Ligação a RNA/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Elementos Facilitadores Genéticos , Cloridrato de Erlotinib , Células HeLa , Humanos , Íntrons/genética , Pneumopatias/genética , Proteínas Nucleares/genética , Paclitaxel/uso terapêutico , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , RNA Antissenso/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-ArgininaRESUMO
Epr is a minor extracellular protease secreted by Bacillus subtilis 168. In this study, we show that epr is transcribed by E sigma(D), the RNA polymerase associated with transcription of genes involved in chemotaxis and motility. Disruption of epr abolished swarming of Bacillus subtilis, suggesting its involvement in motility.