African trypanosomes are major pathogens of humans and livestock and represent a model for studies of unusual protozoal biology. We describe a high-throughput phenotyping approach termed RNA interference (RNAi) target sequencing, or RIT-seq that, using Illumina sequencing, maps fitness-costs associated with RNAi. We scored the abundance of >90,000 integrated RNAi targets recovered from trypanosome libraries before and after induction of RNAi. Data are presented for 7435 protein coding sequences, >99% of a non-redundant set in the Trypanosoma brucei genome. Analysis of bloodstream and insect life-cycle stages and differentiated libraries revealed genome-scale knockdown profiles of growth and development, linking thousands of previously uncharacterized and “hypothetical” genes to essential functions. Genes underlying prominent features of trypanosome biology are highlighted, including the constitutive emphasis on post-transcriptional gene expression control, the importance of flagellar motility and glycolysis in the bloodstream, and of carboxylic acid metabolism and phosphorylation during differentiation from the bloodstream to the insect stage. The current data set also provides much needed genetic validation to identify new drug targets. RIT-seq represents a versatile new tool for genome-scale functional analyses and for the exploitation of genome sequence data.

Five current human African trypanosomiasis drugs are used for genome-scale RNA interference target sequencing screens in Trypanosoma brucei, and reveal the transporters, organelles, enzymes and metabolic pathways that function to facilitate antitrypanosomal drug action. The molecular mechanisms of the drugs used to treat African trypanosomiasis are largely unknown. Use of a genome-wide screen based on RNA interference has now uncovered the pathways involved in drug action and resistance for the five most common drugs. The continued spread of resistance is a major concern, so these findings will be of value for resistance monitoring and in guiding the design of new therapies. The concept of disease-specific chemotherapy was developed a century ago. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work1,2, and the drugs that emerged remain in use for treating human African trypanosomiasis (HAT)3. The importance of understanding the mechanisms underlying selective drug action and resistance for the development of improved HAT therapies has been recognized, but these mechanisms have remained largely unknown. Here we use all five current HAT drugs for genome-scale RNA interference target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate antitrypanosomal drug action. RIT-seq profiling identifies both known drug importers4,5 and the only known pro-drug activator6, and links more than fifty additional genes to drug action. A bloodstream stage-specific invariant surface glycoprotein (ISG75) family mediates suramin uptake, and the AP1 adaptin complex, lysosomal proteases and major lysosomal transmembrane protein, as well as spermidine and N-acetylglucosamine biosynthesis, all contribute to suramin action. Further screens link ubiquinone availability to nitro-drug action, plasma membrane P-type H+-ATPases to pentamidine action, and trypanothione and several putative kinases to melarsoprol action. We also demonstrate a major role for aquaglyceroporins in pentamidine and melarsoprol cross-resistance. These advances in our understanding of mechanisms of antitrypanosomal drug efficacy and resistance will aid the rational design of new therapies and help to combat drug resistance, and provide unprecedented molecular insight into the mode of action of antitrypanosomal drugs.

Summary: Silent information regulator 2 (Sir2)‐related proteins or sirtuins function as NAD⁺‐dependent deacetylases or ADP ribosylases that target a range of substrates, thereby influencing chromatin structure and a diverse range of other biological functions. Genes encoding three Sir2‐related proteins (SIR2rp1–3) have been identified in the parasitic trypanosomatids, early branching protozoa with no previously reported transcriptional silencing machinery. Here we show that, in the mammalian‐infective bloodstream‐stage of the African trypanosome, Trypanosoma brucei, SIR2rp1 localizes to the nucleus while SIR2rp2 and SIR2rp3 are both mitochondrial proteins. The nuclear protein, SIR2rp1, controls DNA repair and repression of RNA polymerase I‐mediated expression immediately adjacent to telomeres. Antigenic variation, however, which involves the silencing and Pol I‐mediated transcriptional switching of subtelomeric variant surface glycoprotein genes, continues to operate independent of SIR2rp1.

A unifying feature of eukaryotic nuclear organization is genome segregation into transcriptionally active euchromatin and transcriptionally repressed heterochromatin. In metazoa, lamin proteins preserve nuclear integrity and higher order heterochromatin organization at the nuclear periphery, but no non-metazoan lamin orthologues have been identified, despite the likely presence of nucleoskeletal elements in many lineages. This suggests a metazoan-specific origin for lamins, and therefore that distinct protein elements must compose the nucleoskeleton in other lineages. The trypanosomatids are highly divergent organisms and possess well-documented but remarkably distinct mechanisms for control of gene expression, including polycistronic transcription and trans-splicing. NUP-1 is a large protein localizing to the nuclear periphery of Trypanosoma brucei and a candidate nucleoskeletal component. We sought to determine if NUP-1 mediates heterochromatin organization and gene regulation at the nuclear periphery by examining the influence of NUP-1 knockdown on morphology, chromatin positioning, and transcription. We demonstrate that NUP-1 is essential and part of a stable network at the inner face of the trypanosome nuclear envelope, since knockdown cells have abnormally shaped nuclei with compromised structural integrity. NUP-1 knockdown also disrupts organization of nuclear pore complexes and chromosomes. Most significantly, we find that NUP-1 is required to maintain the silenced state of developmentally regulated genes at the nuclear periphery; NUP-1 knockdown results in highly specific mis-regulation of telomere-proximal silenced variant surface glycoprotein (VSG) expression sites and procyclin loci, indicating a disruption to normal chromatin organization essential to life-cycle progression. Further, NUP-1 depletion leads to increased VSG switching and therefore appears to have a role in control of antigenic variation. Thus, analogous to vertebrate lamins, NUP-1 is a major component of the nucleoskeleton with key roles in organization of the nuclear periphery, heterochromatin, and epigenetic control of developmentally regulated loci. Eukaryotes—fungi, plants, animals, and many unicellular organisms—are defined by the presence of a cell nucleus that contains the chromosomes and is enveloped by a lipid membrane lined on the inner face with a protein network called the lamina. Among other functions, the lamina serves as an anchorage site for the ends of chromosomes. In multicellular animals (metazoa), the lamina comprises a few related proteins called lamins, which are very important for many functions related to the nucleus; abnormal lamins result in multiple nuclear defects and diseases, including inappropriate gene expression and premature aging. Until now, however, lamins had been found only in metazoa; no protein of equivalent function had been identified in plants, fungi, or unicellular organisms. Here, we describe a protein from African trypanosomes—the single-cell parasites that cause sleeping sickness—that fulfils many lamin-like roles, including maintaining nuclear structure and organizing the chromosomes of this organism. We show that this protein, which we call NUP-1 for nuclear periphery protein-1, is vital for the antigenic variation mechanisms that allow the parasite to escape the host immune response. We propose that NUP-1 is a lamin analogue that performs similar functions in trypanosomes to those of authentic lamins in metazoa. These findings, we believe, have important implications for understanding the evolution of the nucleus.