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Infections with parasitic nematodes are among the most significant of the neglected tropical diseases affecting about a billion people living mainly in tropical regions with low economic activity. The most effective current strategy to control nematode infections involves large scale treatment programs with anthelmintic drugs. This strategy is at risk from the emergence of drug resistant parasites. Parasitic nematodes also affect livestock, which are treated with the same limited group of anthelmintic drugs. Livestock parasites resistant to single drugs, and even multi-drug resistant parasites, are appearing in many areas. There is therefore a pressing need for new anthelmintic drugs. Here we use the nematode Caenorhabditis elegans as a model for parasitic nematodes and demonstrate that sinefungin, a competitive inhibitor of methyltransferases, causes a delay in development and reduced fecundity, and inhibits spliced leader trans-splicing. Spliced leader trans-splicing is an essential step in gene expression that does not occur in the hosts of parasitic nematodes, and is therefore a potential target for new anthelmintic drugs. We have exploited the ability of sinefungin to inhibit spliced leader trans-splicing to adapt a green fluorescent protein based reporter gene assay that monitors spliced leader trans-splicing for high-throughput screening for new anthelmintic compounds. We have established a protocol for robust high-throughput screening, combining mechanical dispensing of living C. elegans into 384- or 1536- well plates with addition of compounds using an acoustic liquid dispenser, and the detection of the inhibition of SL trans-splicing using a microplate reader. We have tested this protocol in a first pilot screen and envisage that this assay will be a valuable tool in the search for new anthelmintic drugs., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Trypanosomatids transcribe their genes in large polycistronic clusters that are further processed into mature mRNA molecules by trans-splicing. During this maturation process, a conserved spliced leader RNA (SL-RNA) sequence of 39 bp is physically linked to the 5' end of the pre-mRNA molecules. Trypanosomatid infections cause a series of devastating diseases in man (sleeping sickness, leishmaniasis, Chagas disease) and animals (nagana, surra, dourine). Here, we investigated the SL-RNA molecule for its diagnostic potential using reverse transcription followed by real-time PCR. As a model, we used Trypanosoma brucei gambiense, which causes sleeping sickness in west and central Africa. We showed that the copy number of the SL-RNA molecule in one single parasitic cell is at least 8600. We observed a lower detection limit of the SL-RNA assay in spiked blood samples of 100 trypanosomes per milliliter of blood. We also proved that we can detect the trypanosome's SL-RNA in the blood of sleeping sickness patients with a sensitivity of 92% (95% CI, 78%-97%) and a specificity of 96% (95% CI, 86%-99%). The SL-RNA is thus an attractive new molecular target for next-generation diagnostics in diseases caused by trypanosomatids., (Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

The HIV-1 5' untranslated region (UTR) contains conserved sequences and unique structural motifs associated with many steps in virus replication. Because unspliced HIV mRNA containing the full-length UTR serves as a template for replication and transcription as well as packaging genomic RNA into virion, it has been postulated that the UTR may play a role in translational regulation. However, the effect and the region(s) responsible for translation control remain controversial. We used deletion mutations of the 5' UTR region in both cell-based and in vitro assays to determine if secondary structural elements within the 5' UTR confer translation inhibition, and to identify which of these elements are involved. The results indicate clearly that the entire HIV-1 5' UTR confers translation inhibition in vitro and in cells; the Psi (Ψ) region specifically has the most translation inhibitory activity among the highly-structured elements in the HIV-1 5' UTR. Moreover, it was found that the SL4 structure in the Psi (Ψ) region is the major determinant of translation inhibition, and that elimination of the SL4 RNA sequence led to increased translation. The results suggest a functional role for the Psi element and the SL4 structure in the translational control of HIV-1 full-length mRNA., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Spliced leader intergenic region (SL-IR) sequences from 23 Trypanosoma rangeli strains isolated from the salivary glands of Rhodnius colombiensis, R. ecuadoriensis, R. pallescens and R. prolixus and two human strains revealed the existence of 4 genotypes with CA, GT, TA, ATT and GTAT microsatellite repeats and the presence of insertions/deletions (INDEL) and single nucleotide polymorphism (SNP) characterizing each genotype. The strains isolated from the same vector species or the same Rhodnius evolutionary line presented the same genotypes, even in cases where strains had been isolated from vectors captured in geographically distant regions. The dendrogram constructed from the SL-IR sequences separated all of them into two main groups, one with the genotypes isolated from R. prolixus and the other group containing three well defined sub-groups with the genotypes isolated from R. pallescens, R. colombiensis and R. ecuadoriensis. Random amplified polymorphic DNA (RAPD) analysis showed the same two main groups and sub-groups supporting strict T. rangeli genotypes' association with Rhodnius species. Combined with other studies, these results suggest a possible co-evolutionary association between T. rangeli genotypes and their vectors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Trypanosoma rangeli infects several mammalian orders but has never confidently been described in Chiroptera, which are commonly parasitized by many trypanosome species. Here, we described trypanosomes from bats captured in Central Brazil identified as T. rangeli, T. dionisii, T. cruzimarinkellei and T. cruzi. Two isolates, Tra643 from Platyrrhinus lineatus and Tra1719 from Artibeus planirostris were identified as T. rangeli by morphological, biological and molecular methods, and confirmed by phylogenetic analyses. Analysis using SSU rDNA sequences clustered these bat trypanosomes together with T. rangeli from other hosts, and separated them from other trypanosomes from bats. Genotyping based on length and sequence polymorphism of PCR-amplified intergenic spliced-leader gene sequences assigned Tra1719 to the lineage A whereas Tra643 was shown to be a new genotype and was assigned to the new lineage E. To our knowledge, these two isolates are the earliest T. rangeli from bats and the first isolates from Central Brazil molecularly characterized. Rhodnius stali captured for this study was found infected by T. rangeli and T. cruzi.

We present a general computational approach to simulate RNA folding kinetics that can be used to extract population kinetics, folding rates and the formation of particular substructures that might be intermediates in the folding process. Simulating RNA folding kinetics can provide unique insight into RNA whose functions are dictated by folding kinetics and not always by nucleotide sequence or the structure of the lowest free-energy state. The method first builds an approximate map (or model) of the folding energy landscape from which the population kinetics are analyzed by solving the master equation on the map. We present results obtained using an analysis technique, map-based Monte Carlo simulation, which stochastically extracts folding pathways from the map. Our method compares favorably with other computational methods that begin with a comprehensive free-energy landscape, illustrating that the smaller, approximate map captures the major features of the complete energy landscape. As a result, our method scales to larger RNAs. For example, here we validate kinetics of RNA of more than 200 nucleotides. Our method accurately computes the kinetics-based functional rates of wild-type and mutant ColE1 RNAII and MS2 phage RNAs showing excellent agreement with experiment.

The development of multicellular organisms requires precise spatiotemporal gene expression and the expression of cell/tissue specific isoforms of some genes. This task may require more efficient genome organization in Caenorhabditis elegans and other organisms with relatively small genome size. The SL1 leader sequence is trans-spliced to many mRNAs in C. elegans. We hypothesize that introns coupled to internal SL1 acceptors contain independent promoters. We identify 238 genes that have introns coupled to internal SL1 acceptors. We find that the mean length of the internal SL1-coupled introns is significantly longer than the genome mean. For twelve of the genes, evidence exists that the intronic promoter provides tissue specificity different from that of the primary promoter. We estimate that 2.7% of the genome is regulated through this two-promoter system. We propose that internal SL1-coupled introns function as independent promoters and that this two-promoter system represents a major mechanism in C. elegans, in addition to alternative splicing, that serves to promote tissue-specific expression of protein isoforms. Our finding of the frequent coupling between an internal SL1 and a large immediately upstream intron will make promoters and transcription start sites predictable.

Both HIV-1 reverse transcriptase (RT) and topoisomerase I bind to structural RNAs and they cooperate to synthesize cDNA during the replication of HIV-1. In this study, we find that human topoisomerase I exclusively dissociated HIV-1 reverse transcriptase, which strongly binds to structural RNAs. Meanwhile, topoisomerase I did not dissociate either HIV-1 nucleocapsid proteins or murine leukemia virus RT which was bound to RNA. We propose that human topoisomerase I may regulate the binding of RT to RNAs and play a pivotal role in HIV-1 replication.

Transcription in the kinetoplastid protozoa shows substantial variation from the paradigms of eukaryotic gene expression, including polycistronic transcription, a paucity of RNA polymerase (RNAP) II promoters, no qualitative regulated transcription initiation for most protein-coding genes, transcription of some protein-coding genes by RNAP I, an exclusive subnuclear location for VSG transcription, the dependence of small nuclear RNA gene transcription on an upstream tRNA gene, and the synthesis of mitochondrial tRNAs in the nucleus. Here, we present a broad overview of what is known about transcription in the kinetoplastids and what has yet to be determined.