RESUMO
From synthesis to decay, mRNA associates with RNA-binding proteins (RBPs) establishing dynamic ribonucleoprotein particles (RNPs). Understanding the composition and function of RNPs is fundamental to understanding how eukaryotic mRNAs are controlled. This is especially relevant for trypanosomes and related kinetoplastid parasites, which mostly rely on post-transcriptional mechanisms to control gene expression. Crucial for trypanosome differentiation, development, or even response to heat shock, RBPs are known to be essential modulators of diverse molecular processes. The recent application of large-scale quantitative methods, such as Next-Generation Sequencing (NGS) and quantitative mass spectrometry, has revealed new exciting features about the parasite RNA-related metabolism. Novel proteins carrying RNA-binding activity, including many proteins without RNA-related ontology were discovered setting a necessary groundwork to get in insights into RNA biology. CONCLUSION: This review aims to give the reader an understanding of current trypanosome RNP research, highlighting the progress made using high-throughput approaches.
RESUMO
Control of gene expression at the post-transcriptional level is essential in all organisms, and RNA-binding proteins play critical roles from mRNA synthesis to decay. To fully understand this process, it is necessary to identify the complete set of RNA-binding proteins and the functional consequences of the protein-mRNA interactions. Here, we provide an overview of the proteins that bind to mRNAs and their functions in the pathogenic bloodstream form of Trypanosoma brucei. We describe the production of a small collection of open-reading frames encoding proteins potentially involved in mRNA metabolism. With this ORFeome collection, we used tethering to screen for proteins that play a role in post-transcriptional control. A yeast two-hybrid screen showed that several of the discovered repressors interact with components of the CAF1/NOT1 deadenylation complex. To identify the RNA-binding proteins, we obtained the mRNA-bound proteome. We identified 155 high-confidence candidates, including many not previously annotated as RNA-binding proteins. Twenty seven of these proteins affected reporter expression in the tethering screen. Our study provides novel insights into the potential trypanosome mRNPs composition, architecture and function.
Assuntos
Proteoma/genética , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA de Protozoário/genética , Proteínas de Ligação a RNA/genética , Trypanosoma brucei brucei/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Ensaios de Triagem em Larga Escala , Proteínas de Protozoários/análise , Proteínas de Protozoários/metabolismo , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/metabolismoRESUMO
In trypanosomatids, gene expression is regulated mainly by post-transcriptional mechanisms, which affect mRNA processing, translation and degradation. Currently, our understanding of factors that regulate either mRNA stability or translation is rather limited. We know that often, the regulators are proteins that bind to the 3'-untranslated region; they presumably interact with ribonucleases and translation factors. However, very few such proteins have been characterized in any detail. Here we describe a genome-wide screen to find proteins implicated in post-transcriptional regulation in Trypanosoma brucei. We made a library of random genomic fragments in a plasmid that was designed for expression of proteins fused to an RNA-binding domain, the lambda-N peptide. This was transfected into cells expressing mRNAs encoding a positive or negative selectable marker, and bearing the "boxB" lambda-N recognition element in the 3'-untranslated region. The screen identified about 300 proteins that could be implicated in post-transcriptional mRNA regulation. These included known regulators, degradative enzymes and translation factors, many canonical RNA-binding proteins, and proteins that act via multi-protein complexes. However there were also nearly 150 potential regulators with no previously annotated function, or functions unrelated to mRNA metabolism. Almost 50 novel regulators were shown to bind RNA using a targeted proteome array. The screen also provided fine structure mapping of the hit candidates' functional domains. Our findings not only confirm the key role that RNA-binding proteins play in the regulation of gene expression in trypanosomatids, but also suggest new roles for previously uncharacterized proteins.
Assuntos
Regulação da Expressão Gênica , Genômica/métodos , Modelos Biológicos , Proteínas de Protozoários/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Trypanosoma brucei brucei/metabolismo , Regiões 3' não Traduzidas , Perfilação da Expressão Gênica , Marcadores Genéticos , Biblioteca Genômica , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Análise Serial de Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Trypanosoma brucei brucei/genética , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Pin1-type peptidyl-prolyl cis/trans isomerases (PPIases) isomerise the peptide bond of specific phosphorylated (Ser/Thr)-Pro residues, regulating various cellular events. Previously, we reported a Pin1-type PPIase in Trypanosoma cruzi, but little is known about its function and subcellular localization. Immunofluorescence analysis revealed that in contrast with Pin1-like proteins from diverse organisms, TcPin1 mainly localized in the cytoplasm and was excluded from the nuclei. In addition, RNAi-mediated downregulation of TbPin1 in Trypanosoma brucei did not abolish cell proliferation. Using yeast two-hybrid assay, we identified a MORN domain-containing protein as putative Pin1-binding partners. These data suggest that Pin1-mediated signaling mechanism plays a different role in protozoan parasites.
Assuntos
Peptidilprolil Isomerase/genética , Trypanosoma brucei brucei/enzimologia , Ciclo Celular , Regulação da Expressão Gênica , Microscopia de Fluorescência , Dados de Sequência Molecular , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Transfecção , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma cruzi , Técnicas do Sistema de Duplo-HíbridoRESUMO
The parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases) catalyzes the cis/trans isomerization of the peptide bonds preceding Pro residues. Eukaryotic parvulin-type PPIases have been shown to be involved in cell proliferation and cell cycle progression. Here we present the biochemical and molecular characterization of a novel multi-domain parvulin-type PPIase from the human pathogenic Trypanosoma cruzi, annotated as TcPar45. Like most other parvulins, Par45 has an N-terminal extension, but, in contrast to human Pin1, it contains a forkhead-associated domain (FHA) instead of a WW domain at the N-terminal end. Par45 shows a strong preference for a substrate with the basic Arg residue preceding Pro (Suc-Ala-Arg-Pro-Phe-NH-Np: k(cat)/K(M)=97.1 /M/s), like that found for human Par14. In contrast to human Pin1, but similarly to Par14, Par45 does not accelerate the cis/trans interconversion of acidic substrates containing Glu-Pro bonds. It is preferentially located in the parasite nucleus. Single RNA interference (RNAi)-mediated knock-down showed that there was a growth inhibition in procyclic Trypanosoma brucei cells. These results identify Par45 as a phosphorylation-independent parvulin required for normal cell proliferation in a unicellular eukaryotic cell.
Assuntos
Peptidilprolil Isomerase/isolamento & purificação , Trypanosomatina/genética , Sequência de Aminoácidos , Animais , Extratos Celulares/química , Células Cultivadas , Clonagem Molecular , Teste de Complementação Genética , Humanos , Dados de Sequência Molecular , Organismos Geneticamente Modificados , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Filogenia , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Transfecção , Leveduras/genética , Leveduras/metabolismoRESUMO
Trypanosomatids rely primarily on posttranscriptional mechanisms for the control of gene expression, with regulation of RNA processing, localization, degradation, and translation by RNA-binding proteins (RBPs). To determine the mechanisms by which RBPs control gene expression in trypanosomatids, transcriptome-wide identification of mRNA targets and mapping of the RNA-binding site is required. Here we present our most current RIP-Seq (RNA immunoprecipitation followed by high-throughput sequencing) protocol that we generally apply to elucidate RNA/protein interactions in Trypanosoma brucei. The technique provides valuable information about the workings of messenger ribonucleoprotein (mRNP) networks and trypanosome gene expression mechanisms.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação/métodos , Análise de Sequência de RNA/métodos , Trypanosoma brucei brucei/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , RNA de Protozoário/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/isolamento & purificação , Ribonucleoproteínas/metabolismoRESUMO
In trypanosomatids, posttranscriptional controls are very important in regulation of individual gene expression. These are achieved through combinatorial sets of RNA-binding proteins (RBPs) which recognize RNA regulatory motifs or regions of secondary structure within RNAs. To analyze the potential functional impact of an RBP on their mRNA targets, we have applied a robust technique called tethering assay. In this method, the protein under study is attached to an mRNA reporter through an artificial RNA-protein interaction. Therefore, the functional activity of a protein can be analyzed independently of its intrinsic ability to bind to RNA. By making use of a cell line expressing a chloramphenicol acetyltransferase (CAT) reporter mRNA, we have characterized dozens of novel mRNA-fate regulators in cultured Trypanosoma brucei. After induction of the candidate fusion protein, the effect on the reporter expression is determined by a rapid CAT assay. The protocol is simple and typically takes one working day for analysis of a single protein and controls. In this chapter, we provide a description of materials and methods for the tethering method and should allow the assay to be successfully deployed in any laboratory with minimal user training.
Assuntos
Ensaios Enzimáticos/métodos , Proteínas de Protozoários/análise , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/análise , Trypanosoma brucei brucei/genética , Cloranfenicol O-Acetiltransferase/genética , Regulação da Expressão Gênica , Genes Reporter , Parasitologia/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Translation and RNA decay, two processes in which all mRNAs are engaged, are intimately related processes. Two new studies demonstrate that, in trypanosomatids, codon usage largely shapes mRNA abundance in a translation-dependent manner. The findings indicate that mRNA decay control by codon choice is an ancient and conserved mechanism.
Assuntos
Códon , Estabilidade de RNA , Biossíntese de Proteínas , RNA MensageiroRESUMO
Parvulins are a conserved group of peptidyl-prolyl cis/trans isomerases (PPIases) that catalyze the cis/trans isomerization of proline-preceding peptide bonds. Parvulin-class PPIases are structurally unrelated to cyclophilins and FK506-binding proteins that are defined as receptors for immunosuppressive drugs. In Trypanosoma cruzi we identified parvulin TcPIN1 as a homolog of the human hPin1 PPIase. The 117 amino acids of the TcPIN1 display 40% identity with the catalytic core of hPin1 and exhibit prolyl cis/trans isomerase activity. TcPIN1 lacks the WW domain at the N-terminus, and is able to rescue the temperature-sensitive phenotype on a mutation in the Saccharomyces cerevisiae hPin1 homolog, ESS1/PTF1. Western blot analysis revealed that the enzyme was present both in dividing and non-dividing forms of T. cruzi. In epimastigote cells neither cell growth kinetics nor cell morphology was affected by the overexpression of the small parvulin TcPIN1. These results suggest the occurrence of a supplementary conserved level of post-translational control in trypanosomatids.
Assuntos
Peptidilprolil Isomerase/genética , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Ciclo Celular , Regulação da Expressão Gênica , Teste de Complementação Genética , Humanos , Dados de Sequência Molecular , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae , Alinhamento de Sequência , Análise de Sequência de DNA , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Methionine is an amino acid susceptible to being oxidized to methionine sulfoxide (MetSO). The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductase (MSR), an enzyme present in almost all organisms. In trypanosomatids, the study of antioxidant systems has been mainly focused on the involvement of trypanothione, a specific redox component in these organisms. However, no information is available concerning their mechanisms for repairing oxidized proteins, which would be relevant for the survival of these pathogens in the various stages of their life cycle. We report the molecular cloning of three genes encoding a putative A-type MSR in trypanosomatids. The genes were expressed in Escherichia coli, and the corresponding recombinant proteins were purified and functionally characterized. The enzymes were specific for L-Met(S)SO reduction, using Trypanosoma cruzi tryparedoxin I as the reducing substrate. Each enzyme migrated in electrophoresis with a particular profile reflecting the differences they exhibit in superficial charge. The in vivo presence of the enzymes was evidenced by immunological detection in replicative stages of T. cruzi and Trypanosoma brucei. The results support the occurrence of a metabolic pathway in Trypanosoma spp. involved in the critical function of repairing oxidized macromolecules.
Assuntos
Metionina Sulfóxido Redutases/genética , Metionina Sulfóxido Redutases/metabolismo , Trypanosoma/enzimologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Chlorocebus aethiops , Clonagem Molecular , Desintoxicação Metabólica Fase I/genética , Redes e Vias Metabólicas/genética , Metionina Sulfóxido Redutases/química , Metionina Sulfóxido Redutases/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Estresse Oxidativo/genética , Homologia de Sequência , Trypanosoma/genética , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Células VeroRESUMO
ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for the E. coli cells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between Leu(102) and Pro(103) was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains.