RESUMO
Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.
Assuntos
Coffea/genética , MicroRNAs/genética , Nitrogênio/deficiência , RNA Mensageiro/genética , RNA de Plantas/genética , Pequeno RNA não Traduzido/genética , Aminoácidos/isolamento & purificação , Aminoácidos/metabolismo , Compostos de Amônio/metabolismo , Coffea/metabolismo , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/classificação , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Nitratos/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Poli A/genética , Poli A/metabolismo , RNA Mensageiro/classificação , RNA Mensageiro/metabolismo , RNA de Plantas/classificação , RNA de Plantas/metabolismo , Pequeno RNA não Traduzido/classificação , Pequeno RNA não Traduzido/metabolismo , Sementes/genética , Sementes/metabolismo , Estresse Fisiológico , TranscriptomaRESUMO
The CFIm25 subunit of the heterotetrameric cleavage factor Im (CFIm) is a critical factor in the formation of the poly(A) tail at mRNA 3' end, regulating the recruitment of polyadenylation factors, poly(A) site selection, and cleavage/polyadenylation reactions. We previously reported the homologous protein (EhCFIm25) in Entamoeba histolytica, the protozoan causing human amoebiasis, and showed the relevance of conserved Leu135 and Tyr236 residues for RNA binding. We also identified the GUUG sequence as the recognition site of EhCFIm25. To understand the interactions network that allows the EhCFIm25 to maintain its three-dimensional structure and function, here we performed molecular dynamics simulations of wild-type (WT) and mutant proteins, alone or interacting with the GUUG molecule. Our results indicated that in the presence of the GUUG sequence, WT converged more quickly to lower RMSD values in comparison with mutant proteins. However, RMSF values showed that movements of amino acids of WT and EhCFIm25*L135 T were almost identical, interacting or not with the GUUG molecule. Interestingly, EhCFIm25*L135 T, which is the only mutant with a slight RNA binding activity experimentally, presents the same stabilization of bend structures and alpha helices as WT, notably in the C-terminus. Moreover, WT and EhCFIm25*L135 T presented almost the same number of contacts that mainly involve lysine residues interacting with the G4 nucleotide. Overall, our data proposed a clear description of the structural and mechanistic data that govern the RNA binding capacity of EhCFIm25.
Assuntos
Entamoeba histolytica/química , Leucina/química , Proteínas de Protozoários/química , RNA Bacteriano/química , RNA Mensageiro/química , Tirosina/química , Fatores de Poliadenilação e Clivagem de mRNA/química , Substituição de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Leucina/metabolismo , Simulação de Dinâmica Molecular , Mutação , Poli A/química , Poli A/genética , Poli A/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Termodinâmica , Tirosina/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25) from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X) domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25) was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A) polymerase (EhPAP) that is responsible for the synthesis of the poly(A) tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A) polymerase, another member of the pre-mRNA 3' end processing machinery in this protozoan parasite.
Assuntos
Entamoeba histolytica/genética , Exorribonucleases/genética , Poli A/genética , Subunidades Proteicas/genética , Proteínas de Ligação a RNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Sequência de Aminoácidos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Entamoeba histolytica/metabolismo , Exorribonucleases/metabolismo , Dados de Sequência Molecular , Poli A/metabolismo , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
In Trypanosoma cruzi gene expression regulation mainly relays on post-transcriptional events. Nevertheless, little is known about the signals which control mRNA abundance and functionality. We have previously found that CA repeated tracts (polyCA) are abundant in the vicinity of open reading frames and constitute specific targets for single stranded binding proteins from T. cruzi epimastigote. Given the reported examples of the involvement of polyCA motifs in gene expression regulation, we decided to further study their role in T. cruzi. Using an in silico genome-wide analysis, we identify the genes that contain polyCA within their predicted UTRs. We found that about 10% of T. cruzi genes carry polyCA therein. Strikingly, they are frequently concurrent with GT repeated tracts (polyGT), favoring the formation of a secondary structure exhibiting the complementary polydinucleotides in a double stranded helix. This feature is found in the species-specific family of genes coding for mucine associated proteins (MASPs) and other genes. For those polyCA-containing UTRs that lack polyGT, the polyCA is mainly predicted to adopt a single stranded structure. We further analyzed the functional role of such element using a reporter approach in T. cruzi. We found out that the insertion of polyCA at the 3' UTR of a reporter gene in the pTEX vector modulates its expression along the parasite's life cycle. While no significant change of the mRNA steady state of the reporter gene could be detected at the trypomastigote stage, significant increase in the epimastigote and reduction in the amastigote stage were observed. Altogether, these results suggest the involvement of polyCA as a signal in gene expression regulation in T. cruzi.
Assuntos
Repetições de Dinucleotídeos/fisiologia , Regulação da Expressão Gênica/fisiologia , Poli A/genética , Poli C/genética , RNA de Protozoário/química , Trypanosoma cruzi/metabolismo , Análise por Conglomerados , Biologia Computacional , Repetições de Dinucleotídeos/genética , Regulação da Expressão Gênica/genética , Genes Reporter , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/fisiologia , Trypanosoma cruzi/genética , Regiões não Traduzidas/genética , Regiões não Traduzidas/fisiologiaRESUMO
Our previous studies have shown that tandem Alu repeats inhibit green fluorescent protein (GFP) gene expression when inserted downstream of the GFP gene in the pEGFP-C1 vector. We found that the 22R sequence (5'-GTGAAAAAAATGCTTTATTTGT-3') from the antisense PolyA (240 bp polyadenylation signal) of simian virus 40, eliminated repression of GFP gene expression when inserted between the GFP gene and the Alu repeats. The 22R sequence contains an imperfect palindrome; based on RNA structure software prediction, it forms an unstable stem-loop structure, including a loop, a first stem, a bulge, and a second stem. Analysis of mutations of the loop length of the 22R sequence showed that the three-nucleotide loop (wild-type, 22R) induced much stronger GFP expression than did other loop lengths. Two mutations, 4TMI (A7âT, A17âT) and 5AMI (A6âT, T18âA), which caused the base type changes in the bulge and in the second stem in the 22R sequence, induced stronger GFP gene expression than 22R itself. Mutation of the bulge base (A17âT), leading to complete complementation of the stem, caused weaker GFP gene expression. Sequences without a palindrome (7pieA, 5'-GTGAAAAAAATG CAAAAAAAGT-3', 7pieT, 5'-GTGTTTTTTTTGCTTTTTTTGT-3') did not activate GFP gene expression. We conclude that an imperfect palindrome affects and can increase GFP gene expression.
Assuntos
Regulação Viral da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Sinais de Poliadenilação na Ponta 3' do RNA , Vírus 40 dos Símios/genética , Proteínas Virais/genética , Elementos Alu/genética , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/biossíntese , Células HeLa , Humanos , Sequências Repetidas Invertidas , Plasmídeos , Poli A/genética , RNA Viral/genética , RNA Viral/metabolismo , Análise de Sequência de DNA , Vírus 40 dos Símios/metabolismoRESUMO
Antibodies to specific nucleic acid conformations are amongst the methods that have allowed the study of non-canonical (Watson-Crick) DNA structures in higher organisms. In this work, the structural limitations for the immunological detection of DNA.RNA hybrid duplexes were examined using specific RNA homopolymers as probes for homopolymer polydeoxyadenylic acid (poly(dA)).polydeoxythymidylic acid (poly(dT))-rich regions of Rhynchosciara americana (Diptera: Sciaridae) chromosomes. Anti-DNA.RNA duplexes did not react with the complex formed between chromosomal poly(dA) and exogenous polyuridylic acid (poly(rU)). Additionally, poly(rU) prevented the detection of polyadenylic acid.poly(dT) hybrid duplexes preformed in situ. These results raised the possibility that three-stranded structures rather than duplexes were formed in chromosomal sites. To test this hypothesis, the specificity of antibodies to triple-helical nucleic acids was reassessed employing distinct nucleic acid configurations. These antibodies were raised to the poly(dA).poly(rU).poly(rU) complex and have been used here for the first time in immunocytochemistry. Anti-triplex antibodies recognised the complex poly(dA).poly(rU).poly(rU) assembled with poly(rU) in poly(dA).poly(dT)-rich homopolymer regions of R. americana chromosomes. The antibodies could not detect short triplex stretches, suggesting the existence of constraints for triple-helix detection, probably related to triplex tract length. In addition, anti-poly(dA).poly(rU).poly(rU) antibodies reacted with the pericentric heterochromatin of RNase-treated polytene chromosomes of R. americana and Drosophila melanogaster. In apparent agreement with data obtained in cell types from other organisms, the results of this work suggest that significant triple-helix DNA extensions can be formed in pericentric regions of these species.
Assuntos
DNA/química , Dípteros/genética , Drosophila melanogaster/genética , RNA/química , Animais , Anticorpos Monoclonais , Cromossomos de Mamíferos/genética , Ensaio de Imunoadsorção Enzimática , Técnicas Imunoenzimáticas , Hibridização In Situ , Conformação de Ácido Nucleico , Poli A/química , Poli A/genética , Poli A/imunologia , Poli U/química , Poli U/genética , Poli U/imunologiaRESUMO
BACKGROUND: Paracoccidioides brasiliensis is a thermo-dimorphic fungus that causes paracoccidiodomycosis (PCM). Glycoprotein gp43 is the fungal main diagnostic antigen, which can also protect against murine PCM and interact with extracellular matrix proteins. It is structurally related to glucanases, however not active, and whose expression varies considerably. We have presently studied polymorphisms in the PbGP43 flanking regions to help understand such variations. RESULTS: we tested the protein-binding capacity of oligonucleotides covering the PbGP43 proximal 5' flanking region, including overlap and mutated probes. We used electrophoretic mobility shift assays and found DNA binding regions between positions -134 to -103 and -255 to -215. Only mutation at -230, characteristic of P. brasiliensis phylogenetic species PS2, altered binding affinity. Next, we cloned and sequenced the 5' intergenic region up to position -2,047 from P. brasiliensis Pb339 and observed that it is composed of three tandem repetitive regions of about 500 bp preceded upstream by 442 bp. Correspondent PCR fragments of about 2,000 bp were found in eight out of fourteen isolates; in PS2 samples they were 1,500-bp long due to the absence of one repetitive region, as detected in Pb3. We also compared fifty-six PbGP43 3' UTR sequences from ten isolates and have not observed polymorphisms; however we detected two main poly(A) clusters (1,420 to 1,441 and 1,451 to 1,457) of multiple cleavage sites. In a single isolate we found one to seven sites. CONCLUSIONS: We observed that the amount of PbGP43 transcripts accumulated in P. brasiliensis Pb339 grown in defined medium was about 1,000-fold higher than in Pb18 and 120-fold higher than in Pb3. We have described a series of features in the gene flanking regions and differences among isolates, including DNA-binding sequences, which might impact gene regulation. Little is known about regulatory sequences in thermo-dimorphic fungi. The peculiar structure of tandem repetitive fragments in the 5' intergenic region of PbGP43, their characteristic sequences, besides the presence of multiple poly(A) cleavage sites in the 3' UTR will certainly guide future studies.
Assuntos
Antígenos de Fungos/genética , Proteínas Fúngicas/genética , Glicoproteínas/genética , Paracoccidioides/genética , Polimorfismo Genético , Regiões 3' não Traduzidas , DNA Fúngico/genética , Poli A/genética , Ligação Proteica , Análise de Sequência de DNA , Sequências de Repetição em TandemRESUMO
Gene expression in Trypanosomatids requires processing of polycistronic transcripts to generate monocistronic mRNAs by cleavage events that are coupled to the addition of a Spliced Leader sequence (SL) at the 5'-end and a poly(A) tail at the 3'-end of each mRNA. Here we investigate the sequence requirements involved in Trypanosoma cruzi mRNA processing by mapping all available expressed sequence tags and cDNAs containing poly(A) tail and/or SL to genomic intergenic regions. Amongst other parameters, we determined that the median lengths of 5' untranslated region (UTR) and 3'UTR sequences are 35 and 264 nucleotides, respectively; and that the median distance between SL addition sites and a polypyrimidine motif is 18 nucleotides, whereas the median distance between poly(A) addition sites and the closest polypyrimidine-rich sequence is 40 nucleotides.
Assuntos
Regulação da Expressão Gênica , RNA Mensageiro/genética , Trypanosoma cruzi/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Poli A/genética , RNA Líder para Processamento/genética , RNA Líder para Processamento/metabolismo , Trans-Splicing , Trypanosoma cruzi/metabolismoRESUMO
In most eukaryotic cells, the poly(A) tail at the 3'-end of messenger RNA (mRNA) is essential for nuclear export, translatability, stability and transcription termination. Poly(A) tail formation involves multi-protein complexes that interact with specific sequences in 3'-untranslated region (3'-UTR) of precursor mRNA (pre-mRNA). Here we have performed a computational analysis of a large EST and genomic sequences collection from Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, to identify conserved elements that could be involved in pre-mRNA polyadenylation. Results evidenced the presence of an AU-rich domain corresponding to the consensus UA(A/U)UU polyadenylation signal or variants, the cleavage and polyadenylation site that is generally denoted by U residue and flanked by two U-rich tracts, and a novel A-rich element. This predicted array was validated through the analysis of genomic sequences and predicted mRNA folding of genes with known polyadenylation site. The molecular organization of pre-mRNA 3'-UTR cis-regulatory elements appears to be roughly conserved through evolutionary scale, whereas the polyadenylation signal seems to be species-specific in protozoan parasites and the novel A-rich element is unique for the primitive eukaryote E. histolytica. To our knowledge, this paper is the first work about the identification of potential pre-mRNA 3'-UTR cis-regulatory sequences through in silico analysis of large sets of cDNA and genomic sequences in a protozoan parasite.
Assuntos
Biologia Computacional , Entamoeba histolytica/genética , Etiquetas de Sequências Expressas , Genoma/genética , Poliadenilação/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Bases de Dados Factuais , Humanos , Dados de Sequência Molecular , Poli A/genética , Poli A/metabolismo , Valor Preditivo dos Testes , Processamento de Terminações 3' de RNA/genética , Precursores de RNA/genética , RNA Mensageiro/genética , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Transport mechanisms involved in pH homeostasis are relevant for the survival of Leishmania parasites. The presence of chloride conductive pathways in Leishmania has been anticipated since anion channel inhibitors limit the proton extrusion mediated by the H+ATPase, which is the major regulator of intracellular pH in amastigotes. In this study, we used Xenopus laevis oocytes as a heterologous expression system in which to study the expression of ion channels upon microinjection of polyA mRNA from Leishmania amazonensis. After injection of polyA mRNA into the oocytes, we measured three different types of currents. We discuss the possible origin of each, and propose that Type 3 currents could be the result of the heterologous expression of proteins from Leishmania since they show different pharmacological and biophysical properties as compared to endogenous oocyte currents.