Regulation of Trypanosoma brucei mitochondrial RNA turnover by cis-acting factors and UTP
Ryan, Christopher M
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Trypanosoma brucei is a parasitic protozoan that causes African sleeping sickness in humans and nagana in wildlife and livestock. Study of this medically and economically important parasite has focused on finding differences between the parasite and host, with the ultimate goal of exploiting these differences for drug treatment. T. brucei , like all species of the order Kinetoplastida, contain a single large mitochondria. Recent studies have revealed a divergence across species in the mechanisms which regulate RNA stability in mitochondria. In this work, we focused on examining the mechanisms of RNA turnover in T. brucei mitochondria. The mechanisms that regulate RNA stability in T. brucei mitochondria are poorly understood. In organello pulse chase experiments performed in this laboratory have shown that two distinct mRNA turnover pathways exist in T. brucei mitochondria. The first pathway involves specific and rapid degradation of polyadenylated RNAs and requires the addition of UTP. The second pathway involves a slower degradation of RNAs in both a UTP independent and poly(A) tail independent manner. To test if 3 ' poly(A) tails can act as cis -acting elements to facilitate RNA decay in T. brucei mitochondria, we developed an in vitro assay that allows us to directly examine the roles of specific 3 ' sequences in RNA decay. Using these assays, a hydrolytic exoribonuclease activity was identified that preferentially degrades polyadenylated mitochondrial encoded and reporter RNAs as compared to their non-adenylated counterparts. This activity was localized to the mitochondrial membranes of T. brucei . In contrast to in organello assays, addition of UTP had no effect on degradation of polyadenylated RNAs in this in vitro assay. To determine how UTP functioned to stimulate decay of polyadenylated RNAs in isolated mitochondria, in organello pulse-chase assays were performed under different conditions, in the presence of UTP analogs, or in mitochondria down-regulated for specific mitochondrial proteins. The results from these experiments demonstrated that UTP-dependent degradation of polyadenylated RNAs requires the polymerization of UTP into RNAs, presumably by the RET1 terminal uridylyl transferase. To determine if decay of polyadenylated RNAs in organello and in vitro are mechanistically connected, future experiments will focus on isolating protein factors involved in these pathways. T. brucei mitochondria encode a unique class of RNAs known as guide RNAs (gRNAs) that contain the information for RNA editing. In organello pulse chase assays and in vitro decay assays were utilized to study the mechanisms of gRNA decay. These assays revealed that gRNAs are degraded by a biphasic mechanism. In the first step of decay, 3 ' gRNA sequences encompassing primarily the posttranscriptionally added oligo(U) tail are rapidly removed. This is followed by a second step, which entails a comparatively slower degradation of the encoded gRNA body. These experiments are the first to address the mechanism of gRNA decay. Future experiments will utilize this in vitro system to study the cis - and trans -acting factors involved in this process.