RESUMO
Poly(A) tail shortening is a critical step in messenger RNA (mRNA) decay and control of gene expression. The carbon catabolite repressor 4 (CCR4)-associated factor 1 (CAF1) component of the CCR4-NOT deadenylase complex plays an essential role in mRNA deadenylation in most eukaryotes. However, while CAF1 has been extensively investigated in yeast and animals, its role in plants remains largely unknown. Here, we show that the Citrus sinensis CAF1 (CsCAF1) is a magnesium-dependent deadenylase implicated in resistance against the citrus canker bacteria Xanthomonas citri. CsCAF1 interacted with proteins of the CCR4-NOT complex, including CsVIP2, a NOT2 homologue, translin-associated factor X (CsTRAX) and the poly(A)-binding proteins CsPABPN and CsPABPC. CsCAF1 also interacted with PthA4, the main X. citri effector required for citrus canker elicitation. We also present evidence suggesting that PthA4 inhibits CsCAF1 deadenylase activity in vitro and stabilizes the mRNA encoded by the citrus canker susceptibility gene CsLOB1, which is transcriptionally activated by PthA4 during canker formation. Moreover, we show that an inhibitor of CsCAF1 deadenylase activity significantly enhanced canker development, despite causing a reduction in PthA4-dependent CsLOB1 transcription. These results thus link CsCAF1 with canker development and PthA4-dependent transcription in citrus plants.
Assuntos
Citrus sinensis/enzimologia , Citrus sinensis/microbiologia , Resistência à Doença/imunologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Ribonucleases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Citrus sinensis/genética , Citrus sinensis/imunologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Magnésio/farmacologia , Doenças das Plantas/genética , Proteínas de Plantas/genética , Poli A/metabolismo , Ligação Proteica/efeitos dos fármacos , Pirazóis/química , Pirazóis/farmacologia , Estabilidade de RNA/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Xanthomonas/efeitos dos fármacos , Xanthomonas/fisiologiaRESUMO
UNLABELLED: Oral cancer disease represents a significant fraction of all human cancer types and its poor early diagnosis contributes to reduced individual survival rate. The identification of proteins modulated in tumorigenic cells and its post-translational modifications may improve our understanding of tumor development in epithelial cells. We have analyzed the phosphoproteome of tumorigenic (SCC-9) and non-tumorigenic (HaCaT) cell lines using MS-based approach in order to identify phosphopeptides with differing patterns of modifications and/or abundance. Our results revealed the identity of 4,206 protein phosphorylation sites with sixty-two sites showing to be significantly modulated between the two cell lines. The phosphoproteome data showed an overrepresentation of proteins with a possible role in nuclear regulatory functions. Pathway analysis was further performed on the phosphoproteome dataset and differences and commonalities of the functional pathways present in tumorigenic and non-tumorigenic cells were identified. Phosphopeptides that belong to the proteins lamina-associated polypeptide 2 isoform alpha and serine-arginine repetitive matrix protein 2 were identified with differential abundance and they appear as promising tumor-related phosphopeptides. These two proteins may be related to the structural alterations generally found in the nucleus of tumorigenic cells. The identification of phosphorylation sites in tumorigenic cells may contribute to disclose novel signaling mechanisms associated with OSCC. SIGNIFICANCE: Oral Squamous Cell Carcinoma (OSCC) is an important cancer disease affecting thousands of people worldwide. Many cellular processes related to the development of oral cancer remain unknown; however, the studies performed in vitro with cancer cells have contributed to guide more specific research which may be further performed by using in vivo approaches or clinical samples. To our knowledge, only few studies have been published showing the results of phosphoproteome profiling of squamous cell carcinoma models, and many signaling proteins must be identified and functionally characterized in order to increase the knowledge available about the complexity of the signaling networks responsible for oral cancer development and its progression. Furthermore, our knowledge regarding proteins exclusive or very low abundant in cancer cells remains limited. A better understanding of the differences between signaling pathways present in epithelial cell lines may contribute to reveal the processes underlying the OSCC.