Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples.

Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans.

Evaluation of a Pan-Leishmania Spliced-Leader RNA Detection Method in Human Blood and Experimentally Infected Syrian Golden Hamsters.

Several methods have been developed for the detection of Leishmania, mostly targeting the minicircle kinetoplast DNA (kDNA). A new RNA real-time quantitative PCR (qPCR) assay was developed targeting the conserved and highly expressed spliced-leader (SL) mini-exon sequence. This study compared the limits of detection of various real-time PCR assays in hamsters infected with Leishmania infantum, in spiked human blood, and in clinical blood samples from visceral leishmaniasis patients. The SL-RNA assay showed an excellent analytical sensitivity in tissues (0.005 and 0.002 parasites per mg liver and spleen, respectively) and was not prone to false-positive reactions. Evaluation of the SL-RNA assay on clinical samples demonstrated lower threshold cycle values than the kDNA qPCR, an excellent interrun stability of 97%, a 93% agreement with the kDNA assay, and an estimated sensitivity, specificity, and accuracy of 93.2%, 94.3%, and 93.8%, respectively. The SL-RNA qPCR assay was equally efficient for detecting Leishmania major, Leishmania tropica, Leishmania mexicana, Leishmania guayensis, Leishmania panamensis, Leishmania braziliensis, L. infantum, and Leishmania donovani and revealed similar SL-RNA levels in the different species and the occurrence of polycistronic SL-containing transcripts in Viannia species. Collectively, this single SL-RNA qPCR assay enables universal Leishmania detection and represents a particularly useful addition to the widely used kDNA assay in clinical studies in which the detection of viable parasites is pivotal to assess parasitological cure.

Trypanosoma brucei gambiense Spliced Leader RNA Is a More Specific Marker for Cure of Human African Trypanosomiasis Than T. b. gambiense DNA.

To assess the efficacy of treatment for human African trypanosomiasis, accurate tests that can discriminate relapse from cure are needed. We report the first data that the spliced leader (SL) RNA is a more specific marker for cure of human African trypanosomiasis than parasite DNA. In blood samples obtained from 61 patients in whom human African trypanosomiasis was cured, SL RNA detection had specificities of 98.4%-100%, while DNA detection had a specificity of only 77%. Data from our proof-of-concept study show that SL RNA detection has high potential as a test of cure.

RNAi interference of XPO1 and Sm genes and their effect on the spliced leader RNA in Trypanosoma brucei.

In trypanosomes, trans-splicing is a major essential RNA-processing mechanism that involves the addition of a spliced leader sequence to all mRNAs from a small RNA species, known as the spliced leader RNA (SL RNA). SL RNA maturation is poorly understood and it is not clear where assembly with Sm proteins takes place. In this study, we followed the localization of the SL RNA during knockdown of Sm proteins and XPO1, which in metazoa functions in transport of mRNA and U snRNAs from the nucleus to the cytoplasm. We found that XPO1 has no role in SL RNA biogenesis in wild-type cells, or when the cells are depleted of Sm proteins. During Sm depletion, 'defective' SL RNA lacking cap modification at position +4 first accumulates in the nucleus, suggesting that Sm assembly on SL RNA most probably takes place in this compartment. Only after massive nuclear accumulation is the 'defective' SL RNA exported to the cytoplasm to form SL RNP-C, which may be a route to dispose of SL RNA when its normal biogenesis is blocked.

Diversity of insect trypanosomatids assessed from the spliced leader RNA and 5S rRNA genes and intergenic regions.

We have determined the sequences of 5S rRNA and spliced leader (SL) RNA genes, and adjacent intergenic regions for representatives of all known trypanosomatid genera parasitizing insects. The genetic loci have been analyzed separately as well as by a combined approach. Several isolates, assigned by morphology to different genera (Leptomonas spp., Blastocrithidia spp.), seem to belong to a single species with an unexpectedly wide host and geographical range. An unnamed trypanosomatid isolated from rats in Egypt was found to belong to the genus Herpetomonas, so far associated with insect hosts only. It is closely related to Herpetomonas ztiplika, a parasite of a blood-sucking biting midge. Apparently several different trypanosomatid species can infect one insect species, as exemplified by Leptomonas sp. PL and Wallaceina sp. Wsd, which were isolated from different specimens of Salda littoralis on the same locality and day. However, since the same species of Leptomonas was obtained from insect hosts belonging to different genera, some insect trypanosomatids may have low host specificity. Our data revealed additional discrepancies between molecular phylogenetic data and cell morphology, rendering current trypanosomatid taxonomy unreliable.

Diagnóstico molecular de Enterobius vermicularis (Lannaeus, 1758) em populações pré-históricas: análise da região intergênica do gene ribossomal 5S RNA e do gene SL 1 RNA / Molecular diagnosis of Enterobius vermicularis (Lannaeus, 1758) in prehistoric populations: analysis of the intergenic region of the ribossomal gene 5S RNA and gene SL 1 RNA

As análises de DNA antigo (aDNA) recuperado de espécimes arqueológicos têm revolucionado áreas das Ciências especialmente na evolução humana e na origem das doenças humanas. Estudos paleoparasitológicos têm mostrado a presença do parasito Enterobius vermicularis em coprólitos de populações antigas das Américas. Até agora o diagnóstico de E. vermiculares em restos arqueológicos depende do exame microscópico. O propósito deste trabalho foi desenvolver uma abordagem metodológica para a recuperação de aDNA e o diagnóstico molecular de E. vermicularis a partir de coprólitos humanos. Propomos o diagnóstico molecular usando como alvo a região intergênica do gene ribossomal 5S RNA de E. vermicularis. Esta estratégia foi analisada quanto a sua especificidade e sensibilidade em parasitos recuperados de amostras fecais e em coprólitos experimentais...Cinqüenta e uma amostras fecais coletadas de pacientes de distintas regiões geográficas do Brasil, e positivas microscopicamente para diversos enteroparasitos incluindo 24 para E. vermicularis fizeram parte deste estudo. O diagnóstico molecular, considerando 420pb da região intergênica do gene 5S rRNA de E. vermicularis identificou 20 das 24 amostras positivas microscopicamente para o parasito, e quando a região de 198pb foi considerada, 100 por cento das amostras analisadas positivas pela análise microscópica, inclusive amostras co-infetadas, foram positivas. A PCR da de 198pb foi sensível para diagnosticar E. vermicularis em amostras fecais com apenas um único ovo do parasito. Os métodos de polimerização reconstrutora e de Nested PCR previamente aplicados em coprólitos experimentais mostraram se úteis na recuperação de aDNA de E. vermicularis em 9 amostras de coprólitos do total de 27 amos5S rRNA de 9 amostras fecais de distintas regiões do Brasil e 7 de coprólitos da América do Norte e da América do Sul, revelou um alto grau de conservação nesta região e a presença do gene SL1 RNA. Nós sugerimos a estrutura secundária do gene SL1 de E. vermicularis, a qual é similar à estrutura modelo de três hastes-alças, e com as mesmas peculiaridades que, em outros organismos são essenciais para a reação de trans-splincing. Pela primeira vez é demonstrada a recuperação de seqüências de aDNA de E. vermicularis provenientes de restos arqueológicos. O diagnóstico molecular específico e sensível para o parasito e a identificação da seqüência e estrutura secundária do gene SL1 RNA de E. vermicularis, também são resultados inéditos.

Characterization of a candidate Trypanosoma brucei U1 small nuclear RNA gene.

We have previously shown that the poly(A) polymerase (PAP) gene of Trypanosoma brucei is interrupted by an intervening sequence. It was postulated that removing this intron by cis-splicing requires a yet unidentified U1 small nuclear RNA (snRNA), which in other organisms engages in base-pair interactions across the 5' splice site during early spliceosome assembly. Here we present a characterization of a 75 nucleotide long candidate T. brucei U1 snRNA. Immunoprecipitation studies indicate that a trimethylguanosine cap structure is present at the 5' end and that the RNA is bound to core proteins common to spliceosomal ribonucleoprotein particles. The U1 snRNA has the potential for extensive intermolecular base pairing with the PAP 5' splice site. We used block replacement mutagenesis to identify sequences necessary for in vivo expression of U1 snRNA. We found that at least two cis-acting elements, tRNA-like A and B boxes, located in the 5'-flanking region are necessary for U1 snRNA synthesis; no internal sequences close to the transcription start site are essential, suggesting a promoter architecture distinct from other trypanosome U-snRNA genes.

Identification of a novel RNA splicing pattern as a basis of restricted cell tropism of erythrovirus B19.

Prior studies on the transcription of erythrovirus B19 have identified a short leader sequence associated with all spliced viral transcripts. While some variability has been observed in the acceptor for this first intron, studies to date in both permissive and nonpermissive cell types have reported a unique splice donor site. In the semipermissive MB-02 cell line, we have found that splicing of this first intron proceeds almost exclusively via a cryptic CT donor downstream of the previously reported GT donor at nucleotide 406. The resulting messages for the viral structural proteins and 11-kDa protein are thereby made bicistronic, with the first expressible polypeptide being a 34 amino acid fusion of the NS-1 and 7.5-kDa proteins. The presence of an upstream open-reading frame on these messages is likely to block effective translation of the downstream structural protein products. We propose this as a significant mechanism in determining B19's tropism on the basis of host cell splicing machinery, and present evidence in support of this model. Additionally, this is the first report of usage of a noncanonical splice donor in B19, and to our knowledge the first report of a CT-AG splice in any system.