Targeted depletion of a mitochondrial nucleotidyltransferase suggests the presence of multiple enzymes that polymerize mRNA 3′ tails in Trypanosoma brucei mitochondria

Abstract

Polyadenylation plays an important role in regulating RNA stability in Trypanosoma brucei mitochondria. To date, little is known about the enzymes responsible for the addition of mRNA 3′ tails in this system. In this study, we characterize a trypanosome homolog of the human mitochondrial poly(A) polymerase, which we term kPAP2. kPAP2 is mitochondrially localized and expressed in both bloodstream and procyclic form trypanosomes. Targeted gene depletion using RNAi showed that kPAP2 is not required for T. brucei growth in either bloodstream or procyclic life stages, nor is it essential for differentiation from bloodstream to procyclic form. We also demonstrate that steady state abundance of several mitochondrial RNAs was largely unaffected upon kPAP2 downregulation. Interestingly, mRNA 3′ tail analysis of several mRNAs from both life cycle stages in uninduced kPAP2 RNAi cells demonstrated that tail length and uridine content are both regulated in a transcript-specific manner. mRNA-specific tail lengths were maintained upon kPAP2 depletion. However, the percentage of uridine residues in 3′ tails was increased, and conversely the percentage of adenosine residues was decreased, in a distinct subset of mRNAs when kPAP2 levels were downregulated. Thus, kPAP2 apparently contributes to the incorporation of adenosine residues in 3′ tails of some, but not all, mitochondrial mRNAs. Together, these data suggest that multiple nucleotidyltransferases act on mitochondrial mRNA 3′ ends, and that these enzymes are somewhat redundant and subject to complex regulation.

circTAIL-seq, a targeted method for deep analysis of RNA 3′ tails, reveals transcript-specific differences by multiple metrics

“…Thus it should not be associated with the ribosome and we would not expect it to be ex-tailed . Additionally, limited published tail sequences suggest that the typical length and composition of CO1 and CO3 in-tails would likely be different (Decker and Sollner-Webb 1990;Kao and Read 2007). Because of these differences, tail populations of CO1 and CO3p transcripts were used to illustrate the functionality of circTAIL-seq results.…”

Mitochondrial poly(A) polymerase and polyadenylation

“…Polyadenylation is related to the editing status and has diverse effects on these mRNAs at various stages of processing. Pre-edited, partially edited and fully edited transcripts are stabilized by the addition of a short (20-25 nt) poly(A) tail, catalyzed by KPAP1 (kinetoplast PAP1) [41-43]. Fully edited transcripts that lack an adenylate tail are degraded much faster than its precursor in an in vitro turnover assay [42].…”

Novel TUTase associates with an editosome-like complex in mitochondria of Trypanosoma brucei

“…Indeed, the cytosolic TUT3 ) and TUT4 (Stagno et al 2007b) have been characterized as RNA uridylyl transferases, while TUT5 (KPAP1) (Etheridge et al 2008a) and possibly TUT6 (KPAP2) (Kao and Read 2007), were shown to be mitochondrial poly(A) polymerases. Beyond Kinetoplastida, data mining of eukaryotic genomes identified ''noncanonical'' PAPs, such as animal cytoplasmic Gld-2-type and yeast nuclear ''quality control'' Trf4/5 poly(A) polymerases, as the proteins most closely related to TUTases.…”