RESUMEN
Spliced leader dependent trans-splicing (SLTS) has been described as an important RNA regulatory process that occurs in different organisms, including the trematode Schistosoma mansoni. We identified more than seven thousand putative SLTS sites in the parasite, comprising genes with a wide spectrum of functional classes, which underlines the SLTS as a ubiquitous mechanism in the parasite. Also, SLTS gene expression levels span several orders of magnitude, showing that SLTS frequency is not determined by the expression level of the target gene, but by the presence of particular gene features facilitating or hindering the trans-splicing mechanism. Our in-depth investigation of SLTS events demonstrates widespread alternative trans-splicing (ATS) acceptor sites occurring in different regions along the entire gene body, highlighting another important role of SLTS generating alternative RNA isoforms in the parasite, besides the polycistron resolution. Particularly for introns where SLTS directly competes for the same acceptor substrate with cis-splicing, we identified for the first time additional and important features that might determine the type of splicing. Our study substantially extends the current knowledge of RNA processing by SLTS in S. mansoni, and provide basis for future studies on the trans-splicing mechanism in other eukaryotes.
Asunto(s)
ARN Lider Empalmado/genética , Schistosoma mansoni/genética , Trans-Empalme/genética , Animales , Secuencia de Bases/genética , Eucariontes/genética , Intrones/genética , Sitios de Empalme de ARN/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Lider Empalmado/metabolismoRESUMEN
We analyzed the compositional changes and the stable base pairs in the predicted secondary structure of the 5' UTR calmodulin mRNA in T. cruzi. The three copies of calmodulin in T. cruzi genome display variable position of the trans splicing sites and give rise to several mRNA that differs slightly on 5' UTR composition in the epimastigote stage. We show that the pattern of high probability base pairs in the minimum free energy predicted secondary structures of the calmodulin 5' UTR remains unchanged despite the nucleotide composition variation. However, the 39 nt spliced leader (mini-exon, the 5' exon sequence transferred to trypanosome mRNAs by the mechanism of trans splicing) shows a variable pattern of high and low probability base pairing as consequence of the altered composition of the 5' UTR.
Asunto(s)
Regiones no Traducidas 5'/genética , Calmodulina/genética , ARN Lider Empalmado/genética , Trans-Empalme/genética , Trypanosoma cruzi/genética , Animales , Emparejamiento Base , Bovinos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL) trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i) adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779), (ii) female adult worms (LIBEST_028000: JZ139780 - JZ140379), (iii) male adult worms (LIBEST_028001: JZ140380 - JZ141002), (iv) eggs (LIBEST_028002: JZ141003 - JZ141497) and (v) schistosomula (LIBEST_028003: JZ141498 - JZ141974).
Asunto(s)
Técnicas de Silenciamiento del Gen , Precursores del ARN/aislamiento & purificación , ARN Lider Empalmado/genética , Schistosoma mansoni/genética , Trans-Empalme/fisiología , Animales , Etiquetas de Secuencia Expresada , Femenino , Regulación de la Expresión Génica/genética , Biblioteca de Genes , Larva , Estadios del Ciclo de Vida/genética , Masculino , Fenotipo , Precursores del ARN/genética , ARN Bicatenario , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Schistosoma mansoni/crecimiento & desarrollo , Trans-Empalme/genéticaRESUMEN
Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL) trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i) adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779), (ii) female adult worms (LIBEST_028000: JZ139780 - JZ140379), (iii) male adult worms (LIBEST_028001: JZ140380 - JZ141002), (iv) eggs (LIBEST_028002: JZ141003 - JZ141497) and (v) schistosomula (LIBEST_028003: JZ141498 - JZ141974).
Asunto(s)
Animales , Femenino , Masculino , Técnicas de Silenciamiento del Gen , Precursores del ARN/aislamiento & purificación , ARN Lider Empalmado/genética , Schistosoma mansoni/genética , Trans-Empalme/fisiología , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Regulación de la Expresión Génica/genética , Larva , Estadios del Ciclo de Vida/genética , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Precursores del ARN/genética , ARN Bicatenario , ARN Interferente Pequeño/metabolismo , Schistosoma mansoni/crecimiento & desarrollo , Trans-Empalme/genéticaRESUMEN
A regulação gênica em tripanossomatídeos é policistrônica e ocorre de maneira pós-transcricional. [...] Para verificar se UTR de tamanhos diferentes possuem impacto na transcrição e, por conseguinte, na tradução de proteínas, selecionou-se genes da família de trans-sialidases, devido à importância destas no processo de invasão celular. A metodologia envolveu a extração de RNA total de T. cruzi CL-Brener, seguido da síntese de cDNA, PCR, clonagem dos produtos amplificados e seqüenciamento por Sanger e pelo uso do 454 Junior (Next Generation Sequencing NSG). Como as trans-sialidases correspondem a uma família gênica de cópias múltiplas, usou-se a estratégia de sequenciamento de alto-desempenho na tentativa de cobrir o maior número possível de genes desta família. Para isso foram obtidos cDNAs de trans-sialidases de CL-Brener nas formas epimastigota e tripomastigota com o iniciador 5UTRTCNA, os quais apresentaram UTR com tamanhos variados entre 65 187 pb, além da obtenção de cDNAs para esta família de proteínas, em epimastigotas CL-Brener, com o iniciador 5TcTS, apresentando UTR variando de 171 a 221 pb, com similaridade de sequências entre elas. Com o intuito de avaliar a correspondência entre a transcrição dos RNAs de trans-sialidases e a sua tradução, houve a necessidade de identificação das proteínas. Desenvolvemos uma metodologia de lise celular e produção de extrato proteico (denominado TcS12) a partir de células de T. cruzi no estágio epimastigota. As cepas selecionadas para esse estudo foram CL-Brener (TcVI), Dm28c (TcI), Y (TcII) e 4167 (TcIV). O processo de lise celular foi otimizado para 107 parasitos/mL ressuspensos em 200 miL de tampão de lise hipotônica (por 30 minutos a 40C), associado ao sonicador de banho (por 30 minutos a 40C)...
Gene regulation in trypanosomatids is polycistronic and occurs in a post-transcriptionalway. [...] To verify the impact of UTRs presenting differentsizes in the transcription machinery and protein translation, genes from trans-sialidasefamily were selected due to its importance in the cell invasion process. Themethodological strategies involved the extraction of total RNA from T. cruzi CL-Brenerstrain, followed by cDNA synthesis, PCR, cloning and sequencing of amplified productsby Sanger and by using the 454 Junior (Next Generation Sequencing NGS).Considering that trans-sialidase is a multi-copy gene family, this high-throughputsequencing strategy was employed in an attempt to cover the largest number of transsialidasegenes. Trans-sialidase cDNAs from CL-Brener epimastigote andtripomastigote were obtained with 5`UTRTCNA primer showing UTR sizes between 65 -187 bp. The cDNA from this protein family were also obtained with the 5TcTS primerfrom CL-Brener epimastigotes, generating UTRs with 171 - 221 bp. Both 5UTRpresented sequence similarities between them. In order to evaluate the correspondencebetween trans-sialidase gene transcription and translation, it was necessary toaccomplish the identification of proteins. Therefore, we developed a methodology forcell disruption, which resulted in a protein extract (referred as TcS12) from epimastigoteT. cruzi cells. The strains selected for this study were CL-Brener (TcVI), Dm28c (TcI), Y(TcII) and 4167 (TcIV). The process for lysing the cells was optimized to 107parasites/mL resuspended in 200 miL hypotonic lysis buffer (30 minutes at 4oC), followedby water bath sonication (30 minutes at 4oC). The process efficacy was confirmed byFACS, showing that near 72 percent of the cells were successfully stained with propidiumiodide solution (PI)...
Asunto(s)
Animales , Biosíntesis de Proteínas , Trans-Empalme/genética , Trypanosoma cruzi/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa , Transcripción ReversaRESUMEN
Trypanosomatidae is a family of early branching eukaryotes harbouring a distinctive repertoire of gene expression strategies. Functional mature messenger RNA is generated via the trans-splicing and polyadenylation processing of constitutively transcribed polycistronic units. Recently, trans-splicing of pre-small subunit ribosomal RNA in the 5' external transcribed spacer region and of precursor tRNAsec have been described. Here, we used a previously validated semi-nested reverse transcription-polymerase chain reaction strategy to investigate internal transcribed spacer (ITS) I acceptor sites in total RNA from Leishmania (Leishmania) amazonensis. Two distinct spliced leader-containing RNAs were detected indicating that trans-splicing reactions occur at two AG acceptor sites mapped in this ITS region. These data provide further evidence of the wide spectrum of RNA molecules that act as trans-splicing acceptors in trypanosomatids.
Asunto(s)
ADN Espaciador Ribosómico/genética , Leishmania mexicana/genética , Precursores del ARN/genética , Sitios de Empalme de ARN/genética , ARN Protozoario/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trans-Empalme/genéticaRESUMEN
The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.
Asunto(s)
Leishmania mexicana/genética , Precursores del ARN/genética , ARN Lider Empalmado/genética , Trans-Empalme/genética , Exones/genética , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.
Asunto(s)
Leishmania mexicana/genética , Precursores del ARN/genética , ARN Lider Empalmado/genética , Trans-Empalme/genética , Exones/genética , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Trypanosomatidae is a family of early branching eukaryotes harbouring a distinctive repertoire of gene expression strategies. Functional mature messenger RNA is generated via the trans-splicing and polyadenylation processing of constitutively transcribed polycistronic units. Recently, trans-splicing of pre-small subunit ribosomal RNA in the 5' external transcribed spacer region and of precursor tRNAsec have been described. Here, we used a previously validated semi-nested reverse transcription-polymerase chain reaction strategy to investigate internal transcribed spacer (ITS) I acceptor sites in total RNA from Leishmania (Leishmania) amazonensis. Two distinct spliced leader-containing RNAs were detected indicating that trans-splicing reactions occur at two AG acceptor sites mapped in this ITS region. These data provide further evidence of the wide spectrum of RNA molecules that act as trans-splicing acceptors in trypanosomatids.
Asunto(s)
ADN Espaciador Ribosómico/genética , Leishmania mexicana/genética , Precursores del ARN/genética , Sitios de Empalme de ARN/genética , ARN Protozoario/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trans-Empalme/genéticaRESUMEN
Trypanosoma cruzi, a protozoan parasite that causes Chagas disease, exhibits unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes, RNA editing and trans-splicing. In the absence of mechanism controlling transcription initiation, organized subsets of T. cruzi genes must be post-transcriptionally co-regulated in response to extracellular signals. The mechanisms that regulate stage-specific gene expression in this parasite have become much clearer through sequencing its whole genome as well as performing various proteomic and microarray analyses, which have demonstrated that at least half of the T. cruzi genes are differentially regulated during its life cycle. In this review, we attempt to highlight the recent advances in characterising cis and trans-acting elements in the T. cruzi genome that are involved in its post-transcriptional regulatory machinery.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/genética , Transcripción Genética/genética , Trypanosoma cruzi/genética , Genoma de Protozoos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Protozoario/genética , Trans-Empalme/genética , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Since the discovery that genes are split into intron and exons, the studies of the mechanisms involved in splicing pointed to presence of consensus signals in an attempt to generalize the process for all living cells. However, as discussed in the present review, splicing is a theme full of variations. The trans-splicing of pre-mRNAs, the joining of exons from distinct transcripts, is one of these variations with broad distribution in the phylogenetic tree. The biological meaning of this phenomenon is discussed encompassing reactions resembling a possible noise to mechanisms of gene expression regulation. All of them however, can contribute to the generation of life diversity.
Asunto(s)
Variación Genética/genética , Kinetoplastida/genética , Mamíferos/genética , Nematodos/genética , Precursores del ARN/genética , Trans-Empalme/genética , Animales , Regulación de la Expresión Génica , FilogeniaRESUMEN
Since the discovery that genes are split into intron and exons, the studies of the mechanisms involved in splicing pointed to presence of consensus signals in an attempt to generalize the process for all living cells. However, as discussed in the present review, splicing is a theme full of variations. The trans-splicing of pre-mRNAs, the joining of exons from distinct transcripts, is one of these variations with broad distribution in the phylogenetic tree. The biological meaning of this phenomenon is discussed encompassing reactions resembling a possible noise to mechanisms of gene expression regulation. All of them however, can contribute to the generation of life diversity.
Asunto(s)
Animales , Variación Genética , Kinetoplastida/genética , Mamíferos/genética , Nematodos/genética , Precursores del ARN/genética , Trans-Empalme/genética , Regulación de la Expresión Génica , FilogeniaRESUMEN
BACKGROUND: Parasitism is a highly successful mode of life and one that requires suites of gene adaptations to permit survival within a potentially hostile host. Among such adaptations is the secretion of proteins capable of modifying or manipulating the host environment. Nippostrongylus brasiliensis is a well-studied model nematode parasite of rodents, which secretes products known to modulate host immunity. RESULTS: Taking a genomic approach to characterize potential secreted products, we analyzed expressed sequence tag (EST) sequences for putative amino-terminal secretory signals. We sequenced ESTs from a cDNA library constructed by oligo-capping to select full-length cDNAs, as well as from conventional cDNA libraries. SignalP analysis was applied to predicted open reading frames, to identify potential signal peptides and anchors. Among 1,234 ESTs, 197 (~16%) contain predicted 5' signal sequences, with 176 classified as conventional signal peptides and 21 as signal anchors. ESTs cluster into 742 distinct genes, of which 135 (18%) bear predicted signal-sequence coding regions. Comparisons of clusters with homologs from Caenorhabditis elegans and more distantly related organisms reveal that the majority (65% at P < e-10) of signal peptide-bearing sequences from N. brasiliensis show no similarity to previously reported genes, and less than 10% align to conserved genes recorded outside the phylum Nematoda. Of all novel sequences identified, 32% contained predicted signal peptides, whereas this was the case for only 3.4% of conserved genes with sequence homologies beyond the Nematoda. CONCLUSIONS: These results indicate that secreted proteins may be undergoing accelerated evolution, either because of relaxed functional constraints, or in response to stronger selective pressure from host immunity.