A directed approach for the identification of transcripts harbouring the spliced leader sequence and the effect of trans-splicing knockdown in Schistosoma mansoni

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).

Os sítios adicionais de trans-splicing na 5' UTR dos genes trans-sialidase de Trypanosoma cruzi e avaliação de seus efeitos sobre a tradução / Additional trans-splicing sites in the 5 'UTR of the trans-sialidase gene Trypanosoma cruzi and evaluation of their effects on translation

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 5’UTRTCNA, 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 5’TcTS, 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 5’TcTS primerfrom CL-Brener epimastigotes, generating UTRs with 171 - 221 bp. Both 5’UTRpresented 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)...

Identification of SL addition trans-splicing acceptor sites in the internal transcribed spacer I region of pre-rRNA in Leishmania (Leishmania) amazonensis

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.

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

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.

Gene expression responses in vivo by human telomerase reverse transcriptase (hTERT)-targeting trans-splicing ribozyme

A trans-splicing ribozyme which can specifically reprogram human telomerase reverse transcriptase (hTERT) RNA was previously suggested as a useful agent for tumor-targeted gene therapy. In this study, we evaluated in vivo function of the hTERT-targeting trans-splicing ribozymes by employing the molecular analysis of expression level of genes affected by the ribozyme delivery into peritoneal carcinomatosis mice model. To this effect, we constructed adenoviral vector encoding the specific ribozyme. Noticeably, more than four-fold reduction in the level of hTERT RNA was observed in tumor nodules by the systemic infection of the ribozyme-encoding virus. Such hTERT RNA knockdown in vivo induced changes in the global gene expression profile, including the suppression of specific genes associated with anti-apoptosis including bcl2, and genes for angiogenesis and metastasis. In addition, specific trans-splicing reaction with the targeted hTERT RNA took place in the tumors established as peritoneal carcinomatosis in mice by systemic delivery of the ribozyme. In conclusion, this study demonstrates that an hTERT-specific RNA replacement approach using trans-splicing ribozyme represents a potential modality to treat cancer.

Pre-mRNA trans-splicing: from kinetoplastids to mammals, an easy language for life diversity

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.