In vivo analysis of trypanosome mitochondrial RNA function by artificial site-specific RNA endonuclease-mediated knockdown

Abstract

Trypanosomes possess a unique mitochondrial genome called the kinetoplast DNA (kDNA). Many kDNA genes encode premRNAs that must undergo guide RNA-directed editing. In addition, alternative mRNA editing gives rise to diverse mRNAs and several kDNA genes encode open reading frames of unknown function. To better understand the mechanism of RNA editing and the function of mitochondrial RNAs in trypanosomes, we have developed a reverse genetic approach using artificial sitespecific RNA endonucleases (ASREs) to directly silence kDNA-encoded genes. The RNA-binding domain of an ASRE can be programmed to recognize unique 8-nucleotide sequences, allowing the design of ASREs to cleave any target RNA. Utilizing an ASRE containing a mitochondrial localization signal, we targeted the extensively edited mitochondrial mRNA for the subunit A6 of the F 0 F 1 ATP synthase (A6) in the procyclic stage of Trypanosoma brucei. This developmental stage, found in the midgut of the insect vector, relies on mitochondrial oxidative phosphorylation for ATP production with A6 forming the critical proton half channel across the inner mitochondrial membrane. Expression of an A6-targeted ASRE in procyclic trypanosomes resulted in a 50% reduction in A6 mRNA levels after 24 h, a time-dependent decrease in mitochondrial membrane potential (ΔΨm), and growth arrest. Expression of the A6-ASRE, lacking the mitochondrial localization signal, showed no significant growth defect. The development of the A6-ASRE allowed the first in vivo functional analysis of an edited mitochondrial mRNA in T. brucei and provides a critical new tool to study mitochondrial RNA biology in trypanosomes.

Mitochondrial Dual-coding Genes inTrypanosomabrucei

“…If the dual-coding ARFs give BF trypanosomes a selective growth advantage similar to that observed by the COIII alternative protein AEP1, then the number of “essential” gRNA genes would increase greatly. Currently, only AEP1, A6 and RPS12 mitochondrial genes have been experimentally shown to be essential [12, 70-71]. In addition, the presence of alternative editing and dual-coding genes would complement the protection provided by gene fragmentation by also shielding the genes from deleterious point mutations within critical ETC genes.…”

Analysis of the Trypanosoma brucei EATRO 164 Bloodstream Guide RNA Transcriptome

“…The full coverage of COIII was also not surprising, as COIII was shown to be fully edited and equally abundant in both stages [ 17 ]. However, we expected to see complete coverage of ATPase 6 and RPS12 as both of these transcripts have been shown to be essential in both life cycle stages [ 3 , 44 , 45 , 50 ]. For ATPase 6, we did identify a total of 29 gRNA populations that do cover all of the editing sites.…”

Manipulating and elucidating mitochondrial gene expression with engineered proteins

“…Furthermore, northern blot analysis of total RNA showed that cytoplasmic RNAs containing perfect or nearperfect eight-nucleotide sequences were not cleaved, which strongly suggests that A6-ASRE only cleaved mtRNA targets, and is thus highly specific. Measurements of mitochondrial membrane potential (ΔΨm) also confirmed that mitochondrial activity was reduced upon A6-ASRE induction, causing a 100fold increase in sensitivity to oligomycin, an ATP synthase inhibitor [112].…”

“…The authors attributed this to inadequate protein import or low transfection efficiency, while stable cell lines producing mitoASREs enabled a reduction of 70% in MT-ND5 mRNA [110]. Further work by Choudhury and co-workers expanded the PUF-PIN ASRE system to target and knock-down specific mtRNAs of Trypanosoma brucei [112]. Trypanosomes are parasitic protists with a host life cycle that rotates between insect vectors, such as the tsetse fly, and mammals, specifically cattle, where they cause African sleeping sickness [113].…”

“…This process involves the insertion or deletion of out-of-frame uridines to produce complete open reading frames [116,117]. To study the function of mtRNAs in kinetoplastids, synthetic PUF-PIN proteins were designed to target a specific GUUAUUGG sequence in the 3 0 -UTR of the edited transcript of the A6 subunit of the mitochondrial FoF1-ATPase [112]. These PUF-PIN proteins contained an N-terminal MTS from dihydroplipoyl dehydrogenase [118].…”