The nematode Caenorhabditis elegans is a bacterivore filter feeder. Through the contraction of the worm’s pharynx, a bacterial suspension is sucked into the pharynx’s lumen. Excess liquid is then shunted out of the buccal cavity through ancillary channels made by surrounding marginal cells. We find that many worm-bioactive small molecules ( a.k.a . wactives) accumulate inside of the marginal cells as crystals or globular spheres. Through screens for mutants that resist the lethality associated with one crystallizing wactive we identify a presumptive sphingomyelin-synthesis pathway that is necessary for crystal and sphere accumulation. We find that expression of sphingomyelin synthase 5 (SMS-5) in the marginal cells is not only sufficient for wactive accumulation but is also important for absorbing exogenous cholesterol, without which C. elegans cannot develop. We conclude that sphingomyelin-rich marginal cells act as a sink to scavenge important nutrients from filtered liquid that might otherwise be shunted back into the environment.

The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org.

RNA editing, catalyzed by the multiprotein editosome complex, is an essential step for the expression of most mitochondrial genes in trypanosomatid pathogens. It has been shown previously that Trypanosoma brucei RNA editing ligase 1 (TbREL1), a core catalytic component of the editosome, is essential in the mammalian life stage of these parasitic pathogens. Because of the availability of its crystal structure and absence from human, the adenylylation domain of TbREL1 has recently become the focus of several studies for designing inhibitors that target its adenylylation pocket. Here, we have studied new and existing inhibitors of TbREL1 to better understand their mechanism of action. We found that these compounds are moderate to weak inhibitors of adenylylation of TbREL1 and in fact enhance adenylylation at higher concentrations of protein. Nevertheless, they can efficiently block deadenylylation of TbREL1 in the editosome and, consequently, result in inhibition of the ligation step of RNA editing. Further experiments directly showed that the studied compounds inhibit the interaction of the editosome with substrate RNA. This was supported by the observation that not only the ligation activity of TbREL1 but also the activities of other editosome proteins such as endoribonuclease, terminal RNA uridylyltransferase, and uridylate-specific exoribonuclease, all of which require the interaction of the editosome with the substrate RNA, are efficiently inhibited by these compounds. In addition, we found that these compounds can interfere with the integrity and/or assembly of the editosome complex, opening the exciting possibility of using them to study the mechanism of assembly of the editosome components.Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major are three major trypanosomatid pathogens that cause hundreds of thousands of deaths and infect millions of people in tropical and subtropical areas of the world (1). Current trypanocidal drugs have a number of limitations such as high rate of toxicity, low rate of efficacy, and drug resistance (2, 3). Therefore, it is important to look for a drug that is effective and does not produce harmful side effects. RNA editing is a unique posttranscriptional modification of mitochondrial mRNAs that is shared in all trypanosomatid pathogens (4, 5). Modification of specific editing sites, dictated by complementary guide RNAs (gRNAs), 5 constitutes essential steps to ensure the production of translatable mRNAs that encode essential components of the mitochondrial respiratory system. Although gRNAs specify the number of uridylates (Us) to be added or deleted by base pairing at each editing block (6, 7), a 1.6-MDa multiprotein complex, the editosome, is responsible for catalysis of different steps of RNA editing. Although the complete composition of the editosome is being elucidated, most purified functional editosomes contain over 20 proteins (8,9). The editosomes differ in their compositions, having at least three different complexes that sediment at ϳ20 S on glycerol gradie...

RNA editing regulates mitochondrial gene expression in trypanosomatid pathogens by creating functional mRNAs. It is catalyzed by a multi-protein complex (the editosome), and is found to be essential in both insect stage and mammalian blood stream form of Trypanosoma brucei. This particular form of RNA editing is unique to trypanosomatids, and thus provides a suitable drug target in trypanosomatid pathogens. Here, we demonstrate the feasibility of a rapid and sensitive fluorescence-based reporter assay to monitor RNA editing based on ribozyme activity. We could validate our new assay using previously identified inhibitors against the essential RNA editing ligase. The principle advantages of this assay are: (i) the use of non-radioactively labeled materials, (ii) sensitivity afforded by fluorescence instrumentation applicable to high-throughput screening of chemical inhibitors against the essential editosome and (iii) a rapid and convenient ‘mix and measure’ type of assay in low volume with a high signal to noise ratio. This assay should enhance rapid identification and characterization of the editosome inhibitors primarily based on the overall composition of the editosomes from T. brucei. These inhibitors could also be tested against the editosomes from the closely related pathogens including T. cruzi and Leishmania species.

The related trypanosomatid pathogens, Trypanosoma brucei spp., Trypanosoma cruzi and Leishmania spp. cause devastating diseases in humans and animals and continue to pose a major challenge in drug development. Mitochondrial RNA editing, catalyzed by multi-protein complexes known as editosomes, has provided an opportunity for development of efficient and specific chemotherapeutic targets against trypanosomatid pathogens. This review will discuss both methods for discovery of RNA editing inhibitors, as well as inhibitors against the T. brucei editosome that were recently discovered through creative virtual and high throughput screening methods. In addition, the use of these inhibitors as agents that can block or perturb one or more steps of the RNA editing process will be discussed. These inhibitors can potentially be used to study the dynamic processing and assembly of the editosome proteins. A thorough understanding of the mechanisms and specificities of these new inhibitors is needed in order to contribute to both the functional studies of an essential gene expression mechanism and to the possibility of future drug development against the trypanosomatid pathogens.