RESUMO
The 5' regions of eukaryotic mRNAs often contain upstream open reading frames (uORFs). The Neurospora crassa arg-2 uORF encodes the 24-residue arginine attenuator peptide (AAP). This regulatory uORF-encoded peptide, which is evolutionarily conserved in fungal transcripts specifying an arginine biosynthetic enzyme, functions as a nascent peptide within the ribosomal tunnel and negatively regulates gene expression. The nascent AAP causes ribosomes to stall at the uORF stop codon in response to arginine, thus, blocking ribosomes from reaching the ARG-2 initiation codon. Here scanning mutagenesis with alanine and proline was performed to systematically determine which AAP residues were important for conferring regulation. Changing many of the most highly conserved residues (Asp-12, Tyr-13, Lys-14, and Trp-19) abolished regulatory function. The minimal functional domain of the AAP was determined by positioning AAP sequences internally within a large polypeptide. Pulse-chase analyses revealed that residues 9-20 of the AAP composed the minimal domain that was sufficient to confer regulatory function. An extensive analysis of predicted fungal AAPs revealed that the minimal functional domain of the N. crassa AAP corresponded closely to the region that was most highly conserved among the fungi. We also observed that the tripeptide RGD could function similarly to arginine in triggering AAP-mediated ribosome stalling. These studies provide a better understanding of the elements required for a nascent peptide and a small regulatory molecule to control translational processes.
Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/metabolismo , Fragmentos de Peptídeos/metabolismo , Biossíntese de Proteínas/fisiologia , Ribossomos/metabolismo , Substituição de Aminoácidos , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Códon de Iniciação/genética , Códon de Iniciação/metabolismo , Proteínas Fúngicas/genética , Mutação de Sentido Incorreto , Neurospora crassa/genética , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína , RNA Fúngico/genética , RNA Fúngico/metabolismo , Ribossomos/genéticaRESUMO
Dysregulation of microRNAs is observed in many cancers, including breast cancer. In particular, miR-10b appears to play an important role in tumor cell invasion and breast cancer progression. In this study, we investigated hyaluronan (HA)-induced CD44 (a primary HA receptor) interaction with c-Src kinase and the transcriptional factor, Twist, in breast tumor cells (MDA-MB-231 cells). Our results indicate that HA binding to CD44 promotes c-Src kinase activation, which, in turn, increases Twist phosphorylation, leading to the nuclear translocation of Twist and transcriptional activation. Further analyses reveal that miR-10b is controlled by an upstream promoter containing the Twist binding site(s), whereas ChIP assays demonstrate that stimulation of miR-10b expression by HA/CD44-activated c-Src is Twist-dependent in breast tumor cells. This process results in the reduction of a tumor suppressor protein (HOXD10), RhoA/RhoC up-regulation, Rho-kinase (ROK) activation, and breast tumor cell invasion. Treatment of MDA-MB-231 cells with PP2 (a c-Src inhibitor) or Twist-specific siRNAs effectively blocks HA-mediated Twist signaling events, abrogates miR-10b production, and increases HOXD10 expression. Subsequently, this c-Src/Twist signaling inhibition causes down-regulation of RhoA/RhoC expression and impairment of ROK-regulated cytoskeleton function (e.g. tumor cell invasion). To further evaluate the role of miR-10b in RhoGTPase signaling, MDA-MB-231 cells were also transfected with a specific anti-miR-10b inhibitor in order to silence miR-10b expression and block its target functions. Our results demonstrate that anti-miR-10b inhibitor not only enhances HOXD10 expression but also abrogates HA/CD44-mediated tumor cell behaviors in breast tumor cells. Taken together, these findings indicate that the HA-induced CD44 interaction with c-Src-activated Twist plays a pivotal role in miR-10b production, leading to the down-regulation of tumor suppressor protein (HOXD10), RhoGTPase-ROK activation, and tumor cell invasion. All of these events are critical prerequisite steps for the acquisition of metastatic properties by human breast cancer cells.
Assuntos
Neoplasias da Mama/patologia , Citoesqueleto/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Adjuvantes Imunológicos/metabolismo , Apoptose , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteína Tirosina Quinase CSK , Adesão Celular , Movimento Celular , Proliferação de Células , Imunoprecipitação da Cromatina , Feminino , Imunofluorescência , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Receptores de Hialuronatos/genética , Técnicas Imunoenzimáticas , MicroRNAs/genética , Invasividade Neoplásica , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Tirosina Quinases/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Proteína 1 Relacionada a Twist/antagonistas & inibidores , Proteína 1 Relacionada a Twist/genética , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína de Ligação a GTP rhoC , Quinases da Família srcRESUMO
The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.
Assuntos
Aspergillus fumigatus/genética , Aspergillus nidulans/genética , Aspergillus oryzae/genética , Genoma Fúngico/genética , Genômica , Aspergillus fumigatus/fisiologia , Aspergillus nidulans/fisiologia , Aspergillus oryzae/fisiologia , Sequência de Bases , Sequência Consenso/genética , Sequência Conservada/genética , Evolução Molecular , Genes Fúngicos Tipo Acasalamento/genética , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Proteoma/genética , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de DNA , Sintenia/genéticaRESUMO
Multidrug resistance and disease relapse is a challenging clinical problem in the treatment of breast cancer. In this study, we investigated the hyaluronan (HA)-induced interaction between CD44 (a primary HA receptor) and protein kinase Cepsilon (PKCepsilon), which regulates a number of human breast tumor cell functions. Our results indicate that HA binding to CD44 promotes PKCepsilon activation, which, in turn, increases the phosphorylation of the stem cell marker, Nanog, in the breast tumor cell line MCF-7. Phosphorylated Nanog is then translocated from the cytosol to the nucleus and becomes associated with RNase III DROSHA and the RNA helicase p68. This process leads to microRNA-21 (miR-21) production and a tumor suppressor protein (e.g. PDCD4 (program cell death 4)) reduction. All of these events contribute to up-regulation of inhibitors of apoptosis proteins (IAPs) and MDR1 (multidrug-resistant protein), resulting in anti-apoptosis and chemotherapy resistance. Transfection of MCF-7 cells with PKCepsilon or Nanog-specific small interfering RNAs effectively blocks HA-mediated PKCepsilon-Nanog signaling events, abrogates miR-21 production, and increases PDCD4 expression/eIF4A binding. Subsequently, this PKCepsilon-Nanog signaling inhibition causes IAP/MDR1 down-regulation, apoptosis, and chemosensitivity. To further evaluate the role of miR-21 in oncogenesis and chemoresistance, MCF-7 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and inhibit its target functions. Our results indicate that anti-miR-21 inhibitor not only enhances PDCD4 expression/eIF4A binding but also blocks HA-CD44-mediated tumor cell behaviors. Thus, this newly discovered HA-CD44 signaling pathway should provide important drug targets for sensitizing tumor cell apoptosis and overcoming chemotherapy resistance in breast cancer cells.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Homeodomínio/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , MicroRNAs/metabolismo , Proteína Quinase C-épsilon/metabolismo , Proteínas de Ligação a RNA/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Northern Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , RNA Helicases DEAD-box/metabolismo , Resistencia a Medicamentos Antineoplásicos , Imunofluorescência , Proteínas de Homeodomínio/genética , Humanos , Immunoblotting , Imunoprecipitação , MicroRNAs/genética , Proteína Homeobox Nanog , Fosforilação , Ligação Proteica , Proteína Quinase C-épsilon/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/metabolismo , Transdução de Sinais , Transfecção , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Hematopoietic stem cells (HSCs) require highly regulated rates of protein synthesis, but it is unclear if they or lineage-committed progenitors preferentially recruit transcripts to translating ribosomes. We utilized polysome profiling, RNA sequencing, and whole-proteomic approaches to examine the translatome in LSK (Lin-Sca-1+c-Kit+) and myeloid progenitor (MP; Lin-Sca-1-c-Kit+) cells. Our studies show that LSKs exhibit low global translation but high translational efficiencies (TEs) of mRNAs required for HSC maintenance. In contrast, MPs activate translation in an mTOR-independent manner due, at least in part, to proteasomal degradation of mTOR by the E3 ubiquitin ligase c-Cbl. In the near absence of mTOR, CDK1 activates eIF4E-dependent translation in MPs through phosphorylation of 4E-BP1. Aberrant activation of mTOR expression and signaling in c-Cbl-deficient MPs results in increased mature myeloid lineage output. Overall, our data demonstrate that hematopoietic stem and progenitor cells (HSPCs) undergo translational reprogramming mediated by previously uncharacterized mechanisms of translational regulation.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Proteômica , Células-Tronco Hematopoéticas , Transdução de Sinais , Serina-Treonina Quinases TORRESUMO
The Neurospora crassa arg-2 and the Saccharomyces cerevisiae ortholog CPA1 encode the arginine-specific carbamoyl-phosphate synthetase (CPS-A) small subunit. Arginine decreases synthesis of this subunit through the action of a 5' upstream open reading frame in the mRNA that encodes a cis-regulatory element, the arginine attenuator peptide (AAP), which stalls ribosomes in response to arginine. We performed a comparative analysis of the genomic structure and predicted peptide sequence of the AAP and CPS-A small subunit across many fungi. Differences at the genomic level included variation in intron number and position within the AAP and CPS-A coding regions and differences in known regulatory motifs. Although differences exist in AAP sequence, there were three absolutely conserved amino acid residues in the predicted peptide, including an aspartic acid crucial for arginine-dependent regulation of arg-2 and CPA1. A diverged Basidiomycete AAP was shown to retain function as an Arg-specific negative regulator of translation.
Assuntos
Ascomicetos/genética , Basidiomycota/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Evolução Molecular , Fases de Leitura Aberta , Motivos de Aminoácidos , Sequência de Aminoácidos , Ascomicetos/enzimologia , Basidiomycota/enzimologia , Dados de Sequência Molecular , Filogenia , Subunidades Proteicas , Alinhamento de SequênciaRESUMO
The her-2 (neu, erbB-2) oncogene encodes a 185-kDa transmembrane receptor tyrosine kinase. HER2 overexpression occurs in numerous primary human tumors and contributes to 25-30% of breast and ovarian carcinomas. Synthesis of HER2 is controlled in part by an upstream open reading frame (uORF) present in the transcript. We used synthetic capped and polyadenylated mRNAs containing sequences derived from the 5' region of the her-2 transcript fused to a firefly luciferase (LUC) reporter to examine this uORF's effect on translation in cell-free systems derived from reticulocytes, wheat germ and Neurospora crassa, and in RNA-transfected HeLa cells. The uORF reduced translation of the downstream cistron in all systems. [(35)S]Met labeling of in vitro translation products obtained indicated that the uORF also affected downstream start-site selection. Primer extension inhibition (toeprint) assays of ribosomes loaded at initiation codons in reticulocyte lysates indicated that the uORF affected the interaction of ribosomes with the primary her-2 AUG codon.
Assuntos
Códon de Iniciação/genética , Fases de Leitura Aberta/genética , Biossíntese de Proteínas/genética , Receptor ErbB-2/genética , Reticulócitos/metabolismo , Animais , Células Cultivadas , CoelhosRESUMO
The evolutionarily conserved fungal arginine attenuator peptide (AAP), as a nascent peptide, stalls the translating ribosome in response to the presence of a high concentration of the amino acid arginine. Here we examine whether the AAP maintains regulatory function in fungal, plant, and animal cell-free translation systems when placed as a domain near the N terminus or internally within a large polypeptide. Pulse-chase analyses of the radiolabeled polypeptides synthesized in these systems indicated that wild-type AAP functions at either position to stall polypeptide synthesis in response to arginine. Toeprint analyses performed to map the positions of stalled ribosomes on transcripts introduced into the fungal system revealed that ribosome stalling required translation of the AAP coding sequence. The positions of the stalled ribosomes were consistent with the sizes of the radiolabeled polypeptide intermediates. These findings demonstrate that an internal polypeptide domain in a nascent chain can regulate eukaryotic translational elongation in response to a small molecule. Apparently the peptide-sensing features are conserved in fungal, plant, and animal ribosomes. These data provide precedents for translational strategies that would allow domains within nascent polypeptide chains to modulate gene expression.