Trypanosomatids are the causative agent of three major parasitic diseases: Trypanosoma brucei causes African trypanosomiasis, Trypanosoma cruzi causes American trypanosomiasis, and a variety of Leishmania species cause leishmaniasis. These diseases are fatal if not treated, yet there are no vaccines to protect against any of them, and chemotherapy is toxic and of limited efficacy. Gene expression in these organisms is controlled mostly post-transcriptionally. We study the process of trans-splicing, which controls the production of every mRNA in the parasite cell. During the trans-splicing reaction, each of the mRNAs acquires the spliced leader (SL), a small exon from the SL RNA, which is a small RNA. We wish to reveal the mechanism and machinery underlying this essential RNA-processing event. Our aim is to understand how the SL RNA is recruited to the spliceosome, identify the SL RNA–specific protein(s) that mediates this interaction, and determine which basal splicing factor(s) specifically interacts with the SL RNA. We are investigating the structure and function of splicing factors by using RNAi silencing and by examining the interactions of these factors in large spliceosomal complexes in vivo. We identified the homologues of U2AF35, U2AF65, SF1, PTB, and SR proteins. We have also shown that, unlike in mammals, trypanosomal U2AF auxiliary factors do not interact with each other. However, as in other systems, U2AF65 interacts with SF1. Recently, we demonstrated that trypanosomes have unique Sm proteins that bind specifically to U2 and U4 snRNAs; we called them SSm proteins.
Localization of the splicing factors and the core snRNP proteins (Lsm proteins that bind only to U6 snRNA, Sm and SSm proteins) demonstrates that SL RNA transcription and assembly with Sm proteins take place in a special nuclear compartment located near the nucleolus. Based on this spatial localization of the SL RNA in the cell nucleus, we propose that the rate of trans-splicing might depend on (1) migration of the SL RNP from the “SL RNP factory” to the sites of pre-mRNA production; (2) the efficiency with which SL RNP is recruited to the spliceosome; and (3) the production of pre-mRNA, which might be a bottleneck because of a lack of conventional polymerase II promoters. Based on these considerations, we are working toward reconstituting the trans-splicing reaction in vitro, using crude extracts and synthetic pre-mRNA synthesized in vitro, as well as in a coupled transcription/splicing system. To identify the factor(s) that interacts with the SL RNA and mediate the interaction with the spliceosome, we are purifying the SL RNP complex to homogeneity, using a combination of conventional and affinity-purification techniques. We are studying the interactions between the different SL RNA-specific factors such as transcription factors, methylating enzymes, and core proteins with in vivo imaging techniques, especially fluorescence resonance energy transfer.
We recently identified a novel stress-induced mechanism that we termed SL RNA silencing (SLS). SLS was discovered by down-regulating the signal-recognition particle (SRP) receptor but was later shown to be induced under a variety of stresses including pH stress, oxidative stress, and reducing agents. Upon exposure to such stresses, trypanosomes elicit a specific shut-off of SL RNA transcription; the shut-off is mediated by the inability of the SL RNA–specific factor (tSNAP42) to bind to the SL RNA promoter and, in SL RNA transcription, leads to a shutdown of mRNA production and subsequent protein synthesis. This novel SLS pathway is reminiscent of, but distinct from, the unfolded protein response and can potentially serve as a novel target for parasite eradication. We are searching for the molecule, possibly a kinase, that transmits the signal either directly to tSNAP42 or activates a cascade leading to tSNAP42 alterations. We are developing a high-throughput screen for chemical compounds that activate the SLS pathway—chemicals that would be potential drugs against trypanosomiasis. Experiments are under way to investigate whether SLS is related to programmed cell death in higher eukaryotes.
Another line of research in our group is to characterize the trypanosome RNome. Because of the high dependence on post-transcriptional mechanisms to control gene expression, we are investigating in depth the rich repertoire of small RNAs in these parasites. So far, our studies have identified more than 100 small nucleolar RNAs (snoRNA) that guide either 2′-O-methylation or pseuoduridylation on rRNA. More recently, we investigated the genome organization and repertoire of homologous molecules in Leishmania. Our mapping analyses suggest that more snoRNAs should be found in trypanosomes. The high level of 2′-O-methyl groups on rRNA may stem from the need to preserve ribosome function during cycling between the mammalian and the insect hosts. Indeed, we find increased modification on certain rRNA positions in the bloodstream form of the parasites.
Our studies suggest that trypanosomes are also rich in small RNAs with special functions. For example, SLA1 is a guide RNA that directs pseudouridylation on the SL RNA. Our most recent results suggest that the major role of SLA1 is not to guide the modification but to serve as a chaperone during SL RNA biogenesis. Our studies further predict the need for snoRNA to guide trypanosome-specific rRNA processing events. In parallel we also identified homologues to small RNAs such as snR30 and MRP RNA that are found in other eukaryotes. We are using comparative genomics with related trypanosomatid genomes to identify potential novel conserved small RNAs. We are preparing small RNA libraries in parallel to identify such novel small RNAs extracted from RNP complexes and are developing approaches to investigate the function of these RNAs. We recently used in vivo psoralen cross-linking combined with affinity selection of the small RNA–associated targets to map the interaction of the SRP-specific tRNA-like molecule with rRNA. We are using silencing of small RNA by RNAi, which we discovered in our laboratory, to investigate the role of the trypanosome-specific snoRNA, which participates in rRNA processing, and of other small RNAs with unknown functions. In collaboration with Professor Elisabetta Ullu (Yale University), we are investigating the mechanism and machinery of snoRNAi with the aim of understanding whether and how snoRNAi is related to the cytoplasmic RNAi mechanism. Our investigations have only begun to characterize the rich RNome of these organisms and its regulatory role in the parasite's developmental cycle.
Last Updated May 2007
