Functions of nuclear non-canonical poly(A) polymerases in Trypanosomes

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DESCRIPTION (provided by applicant): Trypanosomes are hemoflagellate parasitic protozoans that diverged early in evolution from other eukaryotes and possess many unique features with respect to regulation of gene expression, energy metabolism, and other cellular processes. The gene expression patterns that ensure adaptation to environmental conditions in mammalian and insect hosts do not include transcription initiation control of RNA polymerase II (Pol II) activity on protein-encoding genes. The promiscuous transcription, which presumably occurs on both DNA strands, should be toxic to cells, especially for species with active RNA interference machinery. To resolve this paradox, we put forward a hypothesis that a nuclear polyadenylation-directed/exosome-mediated RNA surveillance system exists in Trypanosomatids. Our goal is to combine transcriptome and protein interaction network analyses to determine whether the distinct non-canonical poly(A) polymerase 1 (ncPAP1) complexes mark specific RNA classes for 32-52 degradation by the nuclear exosome. We propose to: 1). Identify RNA targets of ncPAP1 in vivo. The genome of T. brucei encodes two canonical nuclear poly(A) polymerases, which presumably catalyze mRNA polyadenylation. In preliminary studies, we have identified ncPAP1, the main subject of this application, and related ncPAP2, which is a processive PAP that is nonessential for parasites. The working model is that antisense, and probably other transcripts, are adenylated by ncPAP1 and thus targeted for degradation. Because we cannot predict specific targets of ncPAP1, two global approaches will be undertaken to identify RNAs bound to ncPAP1 in vivo and assess changes in the transcriptome. 2). Define protein interaction network for ncPAP1. The individual ncPAP1 polypeptide is enzymatically inactive and most likely requires an RNA binding subunit to exert its PAP activity. We have identified four RNA binding proteins, Mtr4 helicase, and a second putative DEAD box helicase in affinity-purified ncPAP1 complex, but no exosome components. We intend to assess hypotheses that 1) each of these RNA binding proteins forms a specific complex with ncPAP1; and 2) transcripts polyadenylated by ncPAP1 are targeted to the exosome via RNA helicases. PUBLIC HEALTH RELEVANCE: Trypanosomatids are the causative agents of major parasitic diseases in developing countries around the world. Considering the occurrence of visceral leishmaniasis among U.S. troops stationed in the Persian Gulf and Afghanistan, a demand for effective anti-trypanosomal therapies may be anticipated in the United States. Available treatments are often toxic and ineffective, creating demand for the discovery of new drugs. Targeting essential parasite-specific enzymes, such as nuclear non-canonical poly(A) polymerase introduced in our application, is a promising approach toward creating a new generation of trypanocides.