RESUMO

mRNA translation is a fundamental process for life. Selection of the translation initiation site (TIS) is crucial, as it establishes the correct open reading frame for mRNA decoding. Studies in vertebrate mRNAs discovered that a purine at -3 and a G at +4 (where A of the AUG initiator codon is numbered + 1), promote TIS recognition. However, the TIS context in other eukaryotes has been poorly experimentally analyzed. We analyzed in vitro the influence of the -3, -2, -1 and + 4 positions of the TIS context in rabbit, Drosophila, wheat, and yeast. We observed that -3A conferred the best translational efficiency across these species. However, we found variability at the + 4 position for optimal translation. In addition, the Kozak motif that was defined from mammalian cells was only weakly predictive for wheat and essentially non-predictive for yeast. We discovered eight conserved sequences that significantly disfavored translation. Due to the big differences in translational efficiency observed among weak TIS context sequences, we define a novel category that we termed 'barren AUG context sequences (BACS)', which represent sequences disfavoring translation. Analysis of mRNA-ribosomal complexes structures provided insights into the function of BACS. The gene ontology of the BACS-containing mRNAs is presented.

Assuntos

Códon de Iniciação , Sequência Conservada , Biossíntese de Proteínas , Animais , Coelhos , Códon de Iniciação/genética , Mamíferos/genética , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Leveduras , Eucariotos/genética , Eucariotos/metabolismo

RESUMO

Cervical cancer is the fourth most common cause of cancer in women worldwide in terms of both incidence and mortality. Persistent infection with high-risk types of human papillomavirus (HPV), namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, constitute a necessary cause for the development of cervical cancer. Viral oncoproteins E6 and E7 play central roles in the carcinogenic process by virtue of their interactions with cell master proteins such as p53, retinoblastoma (Rb), mammalian target of rapamycin (mTOR), and c-MYC. For the synthesis of E6 and E7, HPVs use a bicistronic messenger RNA (mRNA) that has been studied in cultured cells. Here, we report that in cervical tumors, HPV-18, -39, and -45 transcribe E6/E7 mRNAs with extremely short 5' untranslated regions (UTRs) or even lacking a 5' UTR (i.e., zero to three nucleotides long) to express E6. We show that the translation of HPV-18 E6 cistron is regulated by the motif ACCaugGCGCG(C/A)UUU surrounding the AUG start codon, which we term Translation Initiation of Leaderless mRNAs (TILM). This motif is conserved in all HPV types of the phylogenetically coherent group forming genus alpha, species 7, which infect mucosal epithelia. We further show that the translation of HPV-18 E6 largely relies on the cap structure and eIF4E and eIF4AI, two key translation initiation factors linking translation and cancer but does not involve scanning. Our results support the notion that E6 forms the center of the positive oncogenic feedback loop node involving eIF4E, the mTOR cascade, and p53.

Assuntos

Proteínas de Ligação a DNA/genética , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/genética , Papillomavirus Humano 18/genética , Proteínas Oncogênicas Virais/genética , RNA Mensageiro/genética , Regiões 5' não Traduzidas/genética , Linhagem Celular Tumoral , Códon de Iniciação/genética , Proteínas de Ligação a DNA/biossíntese , Feminino , Regulação Viral da Expressão Gênica/genética , Células HEK293 , Células HaCaT , Células HeLa , Papillomavirus Humano 18/metabolismo , Humanos , Proteínas Oncogênicas Virais/biossíntese , Iniciação Traducional da Cadeia Peptídica/genética , RNA Viral/genética , Serina-Treonina Quinases TOR/genética , Proteína Supressora de Tumor p53/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia

RESUMO

Survivin (BIRC5) is an anti-apoptotic protein that is important in cancer. Mechanisms responsible for controlling Survivin levels in cells include transcriptional regulation and modulation of protein stability via post-translational modifications; however to date, translational control has been poorly studied. Here, we focused particularly on the primary control elements present in the Survivin 5' untranslated region (5'UTR). Bioinformatic analysis of ribosome occupancy on the Survivin 5'UTR revealed the presence of elongating ribosomes upstream of the canonical initiator AUG, suggesting an alternative upstream initiator AUG (uAUG) might exist. This uAUG was found out-of-frame at position -71 and appeared as a conserved element in mammals. RACE analysis revealed different transcriptional start sites for BIRC5, which indicated that translational control by this uAUG is restricted to longer 5'UTR variants. We studied the activity of the uAUG in different cell types by cloning the Survivin 5'UTR DNA sequence (wild-type and mutated variants) upstream of renilla luciferase (RLuc) into a pcDNA3 plasmid. Changes in RLuc activity were determined by luminescence assays and Western blotting. Results showed that when this uAUG was mutated to AUU or AGG in the cloned Survivin 5'UTR, RLuc activity was significantly increased. Similar results were obtained when uAUG was positioned inframe with the RLuc initiator AUG. Immunodetection of Renilla (35 kDa) by Western blotting revealed the presence of a second band (37 kDa approximately) in cells transfected with the Inframe reporter constructs, indicating that the uAUG was functional in our experimental conditions. In conclusion, our experimental data demonstrate the presence of an alternative and inhibitory initiator uAUG in the Survivin 5' UTR. This inhibitory uAUG may help understanding how Survivin expression is downregulated under physiological or pathological conditions.

Assuntos

Regiões 5' não Traduzidas/genética , Códon de Iniciação/genética , Survivina/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Sequência Conservada/genética , Células HEK293 , Humanos , Luciferases/metabolismo , Mamíferos/genética , Fases de Leitura Aberta/genética , Biossíntese de Proteínas/genética

RESUMO

Roegneria kamoji Ohwi is an excellent forage grass due to its high feeding value and high resistance to some biotic and abiotic stresses. However, the start codon targeted (SCoT) polymorphism has not been conducted on R. kamoji. In this study, an orthogonal L16 (45) design was employed to investigate the effects of five factors (Mg2+, dNTPs, Taq DNA polymerase, primer, and template DNA) on the polymerase chain reaction (PCR) to determine the optimal SCoT-PCR system for R. kamoji. The results showed that the most suitable conditions for SCoT-PCR in R. kamoji included 1.5 mM Mg2+, 0.15 mM dNTPs, 1.0 U Taq DNA polymerase, 0.4 pM primer, and 40 ng template DNA. SCoT primers 39 and 41 were used to verify the stability of the optimal reaction system, and amplification bands obtained from diverse samples were found to be clear, rich, and stable in polymorphisms, indicating that this reaction system can be used for SCoT-PCR analysis of R. kamoji. We have developed a simple and rapid way to study the mutual effects of factors and to obtain positive results through the use of an orthogonal design L16 (45) to optimize the SCoT-PCR system. This method may provide basic information for molecular marker-assisted breeding and analyses of genetic diversity in R. kamoji.

Assuntos

Códon de Iniciação/genética , Poaceae/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético , Primers do DNA/genética , Variação Genética , Poaceae/crescimento & desenvolvimento

RESUMO

Eukaryotic translation initiation factor 5A (eIF5A), a protein containing the amino acid residue hypusine required for its activity, is involved in a number of physiological and pathological cellular processes. In humans, several EIF5A1 transcript variants encode the canonical eIF5A1 isoform B, whereas the hitherto uncharacterized variant A is expected to code for a hypothetical eIF5A1 isoform, referred to as isoform A, which has an additional N-terminal extension. Herein, we validate the existence of eIF5A1 isoform A and its production from transcript variant A. In fact, variant A was shown to encode both eIF5A1 isoforms A and B. Mutagenic assays revealed different efficiencies in the start codons present in variant A, contributing to the production of isoform B at higher levels than isoform A. Immunoblotting and mass spectrometric analyses showed that isoform A can undergo hypusination and acetylation at specific lysine residues, as observed for isoform B. Examination of the N-terminal extension suggested that it might confer mitochondrial targeting. Correspondingly, we found that isoform A, but not isoform B, co-purified with mitochondria when the proteins were overproduced. These findings suggest that eIF5A1 isoform A has a role in mitochondrial function. J. Cell. Physiol. 231: 2682-2689, 2016. © 2016 Wiley Periodicals, Inc.

Assuntos

Códon de Iniciação/genética , Mitocôndrias/metabolismo , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Sequência de Aminoácidos , Sequência de Bases , Simulação por Computador , Células HeLa , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Fator de Iniciação de Tradução Eucariótico 5A

RESUMO

Colossoma macropomum (Cuvier, 1816) is the largest characin of South America. This species and its congeners mainly feed on zooplankton, insects, snails and decaying plants. In this paper, we sequenced and annotated the complete mitogenome of C. macropomum. The total length is 16,703 bp, and it typically consist of 37 genes, including 13 protein-coding genes, two rRNAs, 22 tRNA, a light-strand replication origin (OL) and a large control region (D-loop). The overall base composition is 29.9%, 24.6%, 29.5% and 15.9% for A, T, C and G, respectively, with a slight bias on AT content (54.6%). All protein-coding genes share the start codon ATG, except for COI, which begins with GTG. Most of them have TAA or TAG as the stop codon, except COII, ND4 use AGA and COI, Cytb use an incomplete stop codon T. This information could provide useful molecular data and contribute to further phylogenetic studies of Characiformes and Serrasalmidae.

Assuntos

Caraciformes/genética , Genoma Mitocondrial/genética , Mitocôndrias/genética , Animais , Composição de Bases/genética , Códon de Iniciação/genética , Códon de Terminação/genética , Genes Mitocondriais/genética , Filogenia , RNA Ribossômico/genética , RNA de Transferência/genética , Análise de Sequência de DNA/métodos , América do Sul , Sequenciamento Completo do Genoma/métodos

RESUMO

Lonicera macranthoides is an important traditional Chinese herb. The lack of information regarding the genetic structure and genetic relationships among its cultivars has hindered the conservation and utilization of this resource. This study used start codon targeted markers to assess the genetic diversity and other genetic characteristics of five single-variety L. macranthoides populations in China. Using 22 primers produced a total of 266 bands, of which 227 were polymorphic, indicating a high level of polymorphism. At the species level, genetic diversity was high: percentage of polymorphic loci (PPB) = 85.34%, effective number of alleles (NE) = 1.3479, Nei's gene diversity (H) = 0.2075, and Shannon's information index (Hsp, species level) = 0.3198. However, at the varietal population level, genetic diversity was lower, with averages of: PPB = 19.74%, NE = 1.0946, H = 0.0561, Hpop = 0.0850 (population level). Nei's genetic differentiation coefficient was 0.7319, which is consistent with Shannon's population genetic differentiation coefficient (0.7324). This indicates that most of the genetic variation in this species exists among the varietal populations. The differentiation among varieties may have been caused by artificial selection, mode of reproduction, and barriers to gene flow (0.1831). The genetic similarity coefficient ranged from 0.7222 to 0.9419. Phylogenetic analysis showed the five varieties to form two major clades. Results suggest that cultivar breeders should strengthen the exchange of germplasm and increase the mutual penetration of useful genes, which would broaden the hereditary basis of L. macranthoides.

Assuntos

Códon de Iniciação/genética , Variação Genética , Lonicera/genética , Polimorfismo Genético , China , Análise por Conglomerados , Fluxo Gênico , Marcadores Genéticos , Filogenia , Reação em Cadeia da Polimerase

RESUMO

Thirty six start codon targeted (SCoT) primers were used for characterization of 48 accessions of Jatropha curcas from different countries and include material with genetic variation for levels of phorbol esters, yield, seed oil content, test weight and plant type. SCoT analysis revealed high polymorphism and 74% of the primers generated polymorphic profiles. The SCoT6 primer discriminated edible and toxic accessions in a single reaction while the SCoT26 and 27 primers produced amplicons specific to toxic and non-toxic accessions, respectively. The polymorphic SCoT markers obtained with these three primers were converted to sequence characterized amplicon regions (SCARs) which resulted in codominant SCARs with SCoT6 primer and dominant SCARs with SCoT 26 and 27 primers. The codominant nature of SCoT6 primer and the resultant SCAR6 primer were validated on intraspecific hybrids derived from a cross between non-toxic and toxic accessions. The accession JP38 from Madagascar was found to be distinct and showed accession specific bands with 9 different SCoT primers. Sequence analysis of polymorphic amplicons obtained with SCoT6 primer showed a 65 bp deletion in accessions with low/zero phorbol esters. Diversity analysis separated the toxic and non-toxic accessions into two groups and the accessions JP29 and JP48 from Mexico formed a third cluster.

Assuntos

Códon de Iniciação/genética , Jatropha/genética , Polimorfismo Genético , África , Ásia , Sequência de Bases , Códon de Iniciação/metabolismo , Primers do DNA/genética , Primers do DNA/metabolismo , Marcadores Genéticos , Jatropha/metabolismo , México , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência

RESUMO

A 630 bp cDNA encoding an L35 ribosomal protein of Paracoccidioides brasiliensis, designated as Pbl35, was cloned from a yeast expression library. Pbl35 encodes a polypeptide of 125 amino acids, with a predicted molecular mass of 14.5 kDa and a pI of 11.0. The deduced PbL35 shows significant conservation in respect to other described ribosomal L35 proteins from eukaryotes and prokaryotes. Motifs of ribosomal proteins are present in PbL35, including a bipartite nuclear localization signal (NLS) that could be related to the protein addressing to the nucleolus for the ribosomal assembly. The mRNA for PbL35, about 700 nucleotides in length, is expressed at a high level in P. brasiliensis. The PbL35 and the deduced amino acid sequence constitute the first description of a ribosomal protein in P. brasiliensis. The cDNA was deposited in GenBank under accession number AF416509.

Assuntos

DNA Complementar/genética , Paracoccidioides/genética , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon de Iniciação/genética , Códon de Terminação/genética , Sequência Conservada/genética , DNA Complementar/química , DNA Complementar/isolamento & purificação , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Genes Fúngicos/genética , Genes Fúngicos/fisiologia , Ponto Isoelétrico , Dados de Sequência Molecular , Peso Molecular , Sinais de Localização Nuclear/genética , Fases de Leitura Aberta/genética , Sinais de Poliadenilação na Ponta 3' do RNA/genética , RNA Fúngico/análise , RNA Fúngico/isolamento & purificação , RNA Mensageiro/análise , RNA Mensageiro/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência , Transcrição Gênica/fisiologia

RESUMO

A cDNA encoding the N-acetyl-beta-D-glucosaminidase (NAG) protein of Paracoccidioides brasiliensis, Pb NAG1, was cloned and characterized. The 2663-nucleotide sequence of the cDNA consisted of a single open reading frame encoding a protein with a predicted molecular mass of 64.73 kDa and an isoeletric point of 6.35. The predicted protein includes a putative 30-amino-acid signal peptide. The protein as a whole shares considerable sequence similarity with 'classic' NAG. The primary sequence of Pb NAG1 was used to infer phylogenetic relationships. The amino acid sequence of Pb NAG1 has 45, 31 and 30% identity, respectively, with homologous sequences from Trichoderma harzianum, Aspergillus nidulans and Candida albicans. In particular, striking homology was observed with the active site regions of the glycosyl hydrolase group of proteins (family 20). The expected active site consensus motif G X D E and catalytic Asp and Glu residues at positions 373 and 374 were found, reinforcing that Pb NAG1 belongs to glycosyl hydrolase family 20. The nucleotide sequence of Pb nag1 and its flanking regions have been deposited, along with the amino acid sequence of the deduced protein, in GenBank under accession number AF419158.

Assuntos

Acetilglucosaminidase/genética , Paracoccidioides/enzimologia , Paracoccidioides/genética , Regiões 5' não Traduzidas/genética , Acetilglucosaminidase/isolamento & purificação , Sequência de Aminoácidos , Aspergillus nidulans/genética , Sequência de Bases , Sítios de Ligação/genética , Candida albicans/genética , Domínio Catalítico/genética , Clonagem Molecular , Códon de Iniciação/genética , Códon de Terminação/genética , Sequência Conservada/genética , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Genes Fúngicos/genética , Genes Fúngicos/fisiologia , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta/genética , Filogenia , Sinais Direcionadores de Proteínas/genética , Sinais de Poliadenilação na Ponta 3' do RNA/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Trichoderma/genética