Cis- and trans-acting factors in Trypanosoma brucei mitochondrial RNA metabolism
MetadataShow full item record
In Trypanosoma brucei , mitochondrial gene expression is regulated primarily at the posttranscriptional level, due to polycistronic transcription. Our work in this dissertation focused on elucidating the role of posttranscriptional processing events such as polyadenylation and RNA editing in regulating RNA turnover in this system. Previously, it was shown that decay of unedited RNAs is stimulated by polyadenylation in both in organello and in vitro systems. However, the turnover mechanism of edited RNAs remained largely unknown. Utilizing an in vitro RNA degradation system, we demonstrated that polyadenylation stabilizes both fully and partially edited RNAs, opposite to its role in unedited RNA turnover. The presence of a very small edited cis -acting element switches the poly(A) tail from a destabilizing to a stabilizing element. We proposed that this polyadenylation-mediated stabilization is conferred by a protective protein(s) that simultaneously recognizes the poly(A) tail and small edited element, and thereby blocks the action a 3'-5' exonuclease. To expand our knowledge on the in vivo role of polyadenylation, we identified and characterized a trypanosome homolog of the human mitochondrial poly(A) polymerase, which was previously termed TbTUT6. Targeted gene depletion using RNAi showed that TbTUT6 is dispensable for growth of both bloodstream- and procyclic-form trypanosomes, and for differentiation from bloodstream- to procyclic-form. Upon TbTUT6 down-regulation, 3' tail length and steady state abundance of several mitochondrial RNAs were largely unaffected. However, the percentage of U residues in 3' tails was increased. These data suggest that, in this system, formation of mRNA 3' tails involves multiple nucleotidyltransferases whose functions are somewhat redundant. We also characterized the decay of mitochondrial guide RNAs (gRNAs), which contain the information for RNA editing. In organello and in vitro studies reveal that gRNAs are degraded by a biphasic mechanism. The first phase involves rapid removal of 3' gRNA sequences encompassing primarily the posttranscriptionally added oligo(U) tail. This is followed by a second phase, in which the encoded gRNA body is degraded at a comparatively slower rate. Taken together, these studies demonstrate that regulation of RNA stability in T. brucei mitochondria involves collaborative efforts of multiple cis- and trans-acting factors.