Protozoan parasites of the genus Leishmania are found as promastigotes in the sandfly vector and as amastigotes in mammalian macrophages. Mechanisms controlling stage-regulated gene expression in these organisms are poorly understood. Here, we applied a comprehensive approach consisting of protein prefractionation, global proteomics and targeted DNA microarray analysis to the study of stage differentiation in Leishmania. By excluding some abundant structural proteins and reducing complexity, we detected and identified numerous novel differentially expressed protein isoforms in L. infantum. Using 2-D gels, over 2200 protein isoforms were visualized in each developmental stage. Of these, 6.1% were strongly increased or appeared unique in the promastigote stage, while the relative amounts of 12.4% were increased in amastigotes. Amastigote-specific protein isoform and mRNA expression trends correlated modestly (53%), while no correlation was found for promastigote-specific spots. Even where direction of regulation was similar, fold-changes were more modest at the RNA than protein level. Many proteins were present in multiple spots, suggesting that PTM is extensive in this organism. In several cases, different isoforms appeared to be specific to different life stages. Our results suggest that post-transcriptional controls at translational and post-translational levels could play major roles in differentiation in Leishmania parasites.

Trypanosomatids are unicellular protists that include the human pathogens Leishmania spp. (leishmaniasis), Trypanosoma brucei (sleeping sickness), and Trypanosoma cruzi (Chagas disease). Analysis of their recently completed genomes confirmed the presence of non–long-terminal repeat retrotransposons, also called retroposons. Using the 79-bp signature sequence common to all trypanosomatid retroposons as bait, we identified in the Leishmania major genome two new large families of small elements—LmSIDER1 (785 copies) and LmSIDER2 (1,073 copies)—that fulfill all the characteristics of extinct trypanosomatid retroposons. LmSIDERs are ∼70 times more abundant in L. major compared to T. brucei and are found almost exclusively within the 3′-untranslated regions (3′UTRs) of L. major mRNAs. We provide experimental evidence that LmSIDER2 act as mRNA instability elements and that LmSIDER2-containing mRNAs are generally expressed at lower levels compared to the non-LmSIDER2 mRNAs. The considerable expansion of LmSIDERs within 3′UTRs in an organism lacking transcriptional control and their role in regulating mRNA stability indicate that Leishmania have probably recycled these short retroposons to globally modulate the expression of a number of genes. To our knowledge, this is the first example in eukaryotes of the domestication and expansion of a family of mobile elements that have evolved to fulfill a critical cellular function.

BACKGROUND. Transcriptome sequencing (RNA-seq) improves diagnostic rates in individuals with suspected Mendelian conditions to varying degrees, primarily by directing the prioritization of candidate DNA variants identified on exome or genome sequencing (ES/GS). Here we implemented an RNA-seq guided method to diagnose individuals across a wide range of ages and clinical phenotypes. METHODS. One hundred fifteen undiagnosed adult and pediatric patients with diverse phenotypes and 67 family members (182 total individuals) underwent RNA-seq from whole blood and fibroblasts at the Baylor College of Medicine (BCM) Undiagnosed Diseases Network (UDN) clinical site from 2014-2020. We implemented a workflow to detect outliers in gene expression and splicing for cases that remained undiagnosed despite standard genomic and transcriptomic analysis. RESULTS. The transcriptome-directed approach resulted in a diagnostic rate of 12% across the entire cohort, or 17% after excluding cases solved on ES/GS alone. Newly diagnosed conditions included Koolen-de Vries syndrome (KANSL1), Renpenning syndrome (PQBP1), TBCKassociated encephalopathy, NSD2and CLTC-related intellectual disability, and others, all with negative conventional genomic testing, including ES and chromosomal microarray (CMA). Fibroblasts exhibited higher and more consistent expression of clinically relevant genes than whole blood. In solved cases with RNA-seq from both tissues, the causative defect was missed in blood in half the cases but none from fibroblasts. CONCLUSION. For our cohort of undiagnosed individuals with suspected Mendelian conditions, transcriptome-directed genomic analysis facilitated diagnoses, primarily through the identification of variants missed on ES and CMA. 4 TRIAL REGISTRATION. Not applicable.

Background Lack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics. Methods We implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage. Results We detected on average 12,500 genes per sample including around 60% of all disease genes—a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions. Conclusion Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.

We recently characterized a large developmentally regulated gene family in Leishmania encoding the amastin surface proteins. While studying the regulation of these genes, we identified a region of 770 nucleotides (nt) within the 2055-nt 3-untranslated region (3-UTR) that regulates stage-specific gene expression at the level of translation. An intriguing feature of this 3-UTR regulatory region is the presence of a ϳ450-nt element that is highly conserved among several Leishmania mRNAs. Here we show, using a luciferase reporter system and polysome profiling experiments, that the 450-nt element stimulates translation initiation of the amastin mRNA in response to heat shock, which is the main environmental change that the parasite encounters upon its entry into the mammalian host. Deletional analyses depicted a second region of ϳ100 nucleotides located at the 3-end of several amastin transcripts, which also activates translation in response to elevated temperature. Both 3-UTR regulatory elements act in an additive manner to stimulate amastin mRNA translation. In addition, we show that acidic pH encountered in the phagolysosomes of macrophages, the location of parasitic differentiation, triggers the accumulation of amastin transcripts by a distinct mechanism that is independent of the 450-nt and 100-nt elements. Overall, these important findings support the notion that stage-specific post-transcriptional regulation of the amastin mRNAs in Leishmania is complex and involves the coordination of distinct mechanisms controlling mRNA stability and translation that are independently triggered by key environmental signals inducing differentiation of the parasite within macrophages.The protozoan parasite Leishmania constitutes a major health problem in several endemic tropical and sub-tropical regions around the world, threatening over 350 million people of which more than 15 million are infected (1, 2). At least 20 different Leishmania species are responsible for the various clinical manifestations of leishmaniasis, ranging from chronic skin ulcers (L. major, L. tropica, and L. mexicana) to more severe naso-pharynx mucosal destruction (L. braziliensis) or life-threatening visceral diseases (L. donovani, L. infantum, and L. chagasi) (3). No effective vaccine is currently available against Leishmania infections, and resistance to the main anti-leishmanial agents is dramatically increasing in several endemic areas (4). These facts have underscored the urgency of identification of new drug targets for chemotherapy and/or vaccine development.The life cycle of Leishmania includes two developmental stages: the extracellular promastigote form, transmitted to the mammalian host by the sand fly vector, and the amastigote form, adapted to resist and replicate within the threatening environment of the phagolysosomes. This adaptation requires a dynamic process implicating morphological and physiological changes within the parasite (5-9) that are mainly orchestrated by the differential expression of a variety of genes. To date, sever...

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RNA sequencing (RNA-seq) has emerged as a powerful approach to discover disease-causing gene regulatory defects for individuals affected with a genetically undiagnosed rare disorder. Pioneer studies have shown that RNA-seq could increase diagnostic rates over DNA sequencing alone by 8% to 36 % depending on disease entities and probed tissues. To accelerate the adoption of RNA-seq among human genetic centers, detailed analysis protocols are now needed. Here, we present a step-by-step protocol that instructs how to robustly detect aberrant expression, aberrant splicing, and mono-allelic expression of a rare allele in RNA-seq data using dedicated statistical methods. We describe how to generate and assess quality control plots and interpret the analysis results. The protocol is based on DROP (Detection of RNA Outliers Pipeline), a modular computational work ow that integrates all analysis steps, can leverage parallel computing infrastructures, and generates browsable web page reports.

BackgroundLeishmania (Leishmania) major, one of the agents causing cutaneous leishmaniasis (CL) in humans, is widely distributed in the Old World where different species of wild rodent and phlebotomine sand fly serve as animal reservoir hosts and vectors, respectively. Despite this, strains of L. (L.) major isolated from many different sources over many years have proved to be relatively uniform. To investigate the population structure of the species highly polymorphic microsatellite markers were employed for greater discrimination among it's otherwise closely related strains, an approach applied successfully to other species of Leishmania.ResultsMultilocus Microsatellite Typing (MLMT) based on 10 different microsatellite markers was applied to 106 strains of L. (L.) major from different regions where it is endemic. On applying a Bayesian model-based approach, three main populations were identified, corresponding to three separate geographical regions: Central Asia (CA); the Middle East (ME); and Africa (AF). This was congruent with phylogenetic reconstructions based on genetic distances. Re-analysis separated each of the populations into two sub-populations. The two African sub-populations did not correlate well with strains' geographical origin. Strains falling into the sub-populations CA and ME did mostly group according to their place of isolation although some anomalies were seen, probably, owing to human migration.ConclusionThe model- and distance-based analyses of the microsatellite data exposed three main populations of L. (L.) major, Central Asia, the Middle East and Africa, each of which separated into two sub-populations. This probably correlates with the different species of rodent host.