Double-stranded RNA (dsRNA) recently has been shown to give rise to genetic interference in Caenorhabditis elegans and also is likely to be the basis for phenotypic cosuppression in plants in certain instances. While constructing a plasmid vector for transfection of trypanosome cells, we serendipitously discovered that in vivo expression of dsRNA of the ␣-tubulin mRNA 5 untranslated region (5 UTR) led to multinucleated cells with striking morphological alterations and a specific block of cytokinesis. Transfection of synthetic ␣-tubulin 5 UTR dsRNA, but not of either strand individually, caused the same phenotype. On dsRNA transfection, tubulin mRNA, but not the corresponding pre-mRNA, was rapidly and specifically degraded, leading to a deficit of ␣-tubulin synthesis. The transfected cells were no longer capable of carrying out cytokinesis and eventually died. Analysis of cytoskeletal structures from these trypanosomes revealed defects in the microtubules of the f lagellar axoneme and of the f lagellar attachment zone, a complex cortical structure that we propose is essential for establishing the path of the cleavage furrow at cytokinesis. Last, dsRNA-mediated mRNA degradation is not restricted to ␣-tubulin mRNA but can be applied to other cellular mRNAs, thus establishing a powerful tool to genetically manipulate these important protozoan parasites.Over the last 10 years, the study of RNA metabolism in trypanosomatid protozoa has unraveled novel mechanisms of eukaryotic gene expression such as polycistronic transcription (1), trans-splicing (2, 3), mitochondrial RNA editing (4-6), and coupling of trans-splicing and polyadenylation (7). Furthermore, in these organisms, RNA polymerase II promoters have not been identified. So far, regulation of expression of polymerase II genes has been documented only at the posttranscriptional level, with clear evidence that cis-acting mRNA sequences play a role in mRNA stability. However, regulation of pre-mRNA turnover, trans-splicing, polyadenylation, and mRNA export from the nucleus to the cytoplasm likely are additional steps for modulating the output of mature mRNA from a given gene. Notwithstanding our present limited knowledge of these subjects, it appears that trypanosome mRNA abundance is primarily, if not solely, regulated at the level of RNA metabolism rather that at the level of transcription initiation, as is the case in most eukaryotic organisms. How the various steps involved in mRNA metabolism are integrated to generate diversity at the level of mRNA abundance is not known.We have observed that challenging Trypanosoma brucei cells with gene-specific double-stranded RNAs (dsRNAs) leads to specific degradation of the homologous mRNA. We serendipitously discovered this pathway because trypanosomes expressing dsRNA representing a portion of ␣-tubulin mRNA acquired an abnormal phenotype, namely, they became multinucleated and lost their typical slender morphology. In addition to providing evidence for dsRNA-mediated mRNA degradation in trypanosomes, our analy...

The genome of Trypanosoma brucei, the causative agent of African trypanosomiasis, was published five years ago, yet identification of all genes and their transcripts remains to be accomplished. Annotation is challenged by the organization of genes transcribed by RNA polymerase II (Pol II) into long unidirectional gene clusters with no knowledge of how transcription is initiated. Here we report a single-nucleotide resolution genomic map of the T. brucei transcriptome, adding 1,114 new transcripts, including 103 non-coding RNAs, confirming and correcting many of the annotated features and revealing an extensive heterogeneity of 5′ and 3′ ends. Some of the new transcripts encode polypeptides that are either conserved in T. cruzi and Leishmania major or were previously detected in mass spectrometry analyses. High-throughput RNA sequencing (RNA-Seq) was sensitive enough to detect transcripts at putative Pol II transcription initiation sites. Our results, as well as recent data from the literature, indicate that transcription initiation is not solely restricted to regions at the beginning of gene clusters, but may occur at internal sites. We also provide evidence that transcription at all putative initiation sites in T. brucei is bidirectional, a recently recognized fundamental property of eukaryotic promoters. Our results have implications for gene expression patterns in other important human pathogens with similar genome organization (Trypanosoma cruzi, Leishmania sp.) and revealed heterogeneity in pre-mRNA processing that could potentially contribute to the survival and success of the parasite population in the insect vector and the mammalian host.

7SL RNA is an abundant cytoplasmic RNA which functions in protein secretion as a component of the signal recognition particle. Alu sequences are the most abundant family of human and rodent middle repetitive DNA sequences (reviewed in ref. 2). The primary structure of human 7SL RNA consists of an Alu sequence interrupted by a 155-base pair (bp) sequence that is unique to 7SL RNA. In order to obtain information about the evolution of the Alu domain of 7SL RNA, we have determined the nucleotide sequence of a cDNA copy of Xenopus laevis 7SL RNA and of the 7SL RNA gene of Drosophila melanogaster. We find that the Xenopus sequence is 87% homologous with its human counterpart and the Drosophila 7SL RNA is 64% homologous to both the human and amphibian molecules. Despite the evolutionary distance between the species, significant blocks of homology to both the Alu and 7SL-specific portions of mammalian 7SL RNA can be found in the insect sequence. These results clearly demonstrate that the Alu sequence in 7SL RNA appeared in evolution before the mammalian radiation. We suggest that mammalian Alu sequences were derived from 7SL RNA (or DNA) by a deletion of the central 7SL-specific sequence, and are therefore processed 7SL RNA genes.

RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite virulence.