UTP-dependent turnover of <i>Trypanosoma brucei</i> mitochondrial mRNA requires UTP polymerization and involves the RET1 TUTase

Ryan, Christopher M.; Read, Laurie K.

doi:10.1261/rna.7248605

Cited by 36 publications

(35 citation statements)

References 63 publications

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“…Interestingly, these extensions, comprised mostly of uridines, were detected in eight out of the 17 PNPase-RNAi COX1 clones. The addition of post-transcriptional added poly(U) tails in trypanosome mitochondria and a recently discovered family of poly(U) polymerases, present in many organisms, were previously reported (Ryan and Read 2005;Kwak and Wickens 2007). However, the type of extension reported here has not been described before in human mitochondria.…”

Section: Stable Shrna-mediated Silencing Of Pnpase In Hela Cellsmentioning

confidence: 44%

Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA

Slomovic

Schuster²

2007

RNA

104

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Polynucleotide phosphorylase (PNPase) is a diverse enzyme, involved in RNA polyadenylation, degradation, and processing in prokaryotes and organelles. However, in human mitochondria, PNPase is located in the intermembrane space (IMS), where no mitochondrial RNA (mtRNA) is known to be present. In order to determine the nature and degree of its involvement in mtRNA metabolism, we stably silenced PNPase by establishing HeLa cell lines expressing PNPase short-hairpin RNA (shRNA). Processing and polyadenylation of mt-mRNAs were significantly affected, but, to different degrees in different genes. For instance, the stable poly(A) tails at the 39 ends of COX1 transcripts were abolished, while COX3 poly(A) tails remained unaffected and ND5 and ND3 poly(A) extensions increased in length. Despite the lack of polyadenylation at the 39 end, COX1 mRNA and protein accumulated to normal levels, as was the case for all 13 mt-encoded proteins. Interestingly, ATP depletion also altered poly(A) tail length, demonstrating that adenylation of mtRNA can be manipulated by indirect, environmental means and not solely by direct enzymatic activity. When both PNPase and the mitochondrial poly(A)-polymerase (mtPAP) were concurrently silenced, the mature 39 end of ND3 mRNA lacked poly(A) tails but retained oligo(A) extensions. Furthermore, in mtPAP-silenced cells, truncated adenylated COX1 molecules, considered to be degradation intermediates, were present but harbored significantly shorter tails. Together, these results suggest that an additional mitochondrial polymerase, yet to be identified, is responsible for the oligoadenylation of mtRNA and that PNPase, although located in the IMS, is involved, most likely by indirect means, in the processing and polyadenylation of mtRNA.

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Section: Stable Shrna-mediated Silencing Of Pnpase In Hela Cellsmentioning

confidence: 44%

Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA

Slomovic

Schuster²

2007

RNA

104

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“…Of these, KRET1 and KRET2 are mitochondrial enzymes that have a function in RNA editing (51). Interestingly, KRET1 has also been shown to be required for UTP-dependent decay of polyadenylated RNAs in isolated T. brucei mitochondria by a mechanism involving RNA polymerization, suggesting it may participate in formation of mRNA 3′ ends (52). TUT3 and TUT4 are cytoplasmic, and thus unlikely to participate in mitochondrial mRNA maturation.…”

Section: Discussionmentioning

confidence: 99%

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

Kao

Read

2007

Molecular and Biochemical Parasitology

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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.

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“…RNA editing TUTase 1 (RET1) is a processive enzyme and is required for the uridylylation of guide RNAs [23] and is also involved in mitochondrial RNA turnover [24]. RNA editing TUTase 2 (RET 2) is a subunit of a multi-protein editing complex (or 20 S editosome) and participates in guide-RNA-dependent U-insertion into mitochondrial pre-mRNAs [25].…”

Section: Structure Of Active Sites In U-adding Enzymesmentioning

confidence: 99%

Determinants of substrate specificity in RNA-dependent nucleotidyl transferases

Martín

Doublié

Keller

2008

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Poly(A) polymerases were identified almost fifty years ago as enzymes that add multiple AMP residues to the 3′ ends of primer RNAs without use of a template from ATP as cosubstrate and with release of pyrophosphate. Based on sequence homology of a signature motif in the catalytic domain, poly(A) polymerases were later found to belong to a superfamily of nucleotidyl transferases acting on a very diverse array of substrates. Enzymes belonging to the superfamily can add from single nucleotides of AMP, CMP or UMP to RNA, antibiotics and proteins but also homopolymers of many hundred residues to the 3′ ends of RNA molecules.The recently reported structures of several nucleotidyl transferases facilitate the study of the catalytic mechanisms of these very diverse enzymes. Numerous structures of CCA-adding enzymes have now revealed all steps in the formation of a CCA tail at the 3′ end of tRNAs. In addition, structures of poly(A) polymerases and uridylyl transferases are now available as binary and ternary complexes with incoming nucleotide and RNA primer. Some of these proteins undergo significant conformational changes after substrate binding. This is proposed to be an indication for an induced fit mechanism that drives substrate selection and leads to catalysis. Insights from recent structures of ternary complexes indicate an important role for the primer molecule in selecting the incoming nucleotide.

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UTP-dependent turnover of Trypanosoma brucei mitochondrial mRNA requires UTP polymerization and involves the RET1 TUTase

Cited by 36 publications

References 63 publications

Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA

Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA

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

Determinants of substrate specificity in RNA-dependent nucleotidyl transferases

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