Targeting of the cytosolic poly(A) binding protein PABPC1 to mitochondria causes mitochondrial translation inhibition

Mammalian mitochondria contain their own genome that is almost fully transcribed from both strands, generating polycistronic RNA units that are processed and matured. The mitochondrial mRNA is modified by oligo- or polyadenylation at the 3′ termini, but the exact function of this post-transcriptional addition is unclear. Current debate focuses on the role of polyadenylation in transcript stability. An equally likely function that has received little attention is that, as in the cytosol of eukaryotes, polyadenylation facilitates translation in the mitochondrion. To address this issue, we have targeted cytosolic proteins to the mitochondrion, a poly(A) specific 3′ exoribonuclease, mtPARN, and a poly(A)binding protein, mtPABP1. Removal of the 3′ adenylyl extensions had a variable effect on mt-mRNA steady-state levels, increasing (MTND1, 2, 5) or decreasing (MTCO1, 2, RNA14) certain species with minimal effect on others (RNA7, MTND3). Translation was markedly affected, but interpretation of this was complicated by the concomitant 3′ truncation of the open reading frame in most cases. Coating of the poly(A) tail by mtPABP1, however, did not lead to transcript decay but caused a marked inhibition of mitochondrial translation. These data are consistent with endogenous RNA-binding factor(s) interacting with the poly(A) to optimize mitochondrial protein synthesis

Human ERAL1 is a mitochondrial RNA chaperone involved in the assembly of the 28S small mitochondrial ribosomal subunit

The bacterial Ras-like protein Era has been reported previously to bind 16S rRNA within the 30S ribosomal subunit and to play a crucial role in ribosome assembly. An orthologue of this essential GTPase ERAL1 (Era G-protein-like 1) exists in higher eukaryotes and although its exact molecular function and cellular localization is unknown, its absence has been linked to apoptosis. In the present study we show that human ERAL1 is a mitochondrial protein important for the formation of the 28S small mitoribosomal subunit. We also show that ERAL1 binds in vivo to the rRNA component of the small subunit [12S mt (mitochondrial)-rRNA]. Bacterial Era associates with a 3′ unstructured nonanucleotide immediately downstream of the terminal stem–loop (helix 45) of 16S rRNA. This site contains an AUCA sequence highly conserved across all domains of life, immediately upstream of the anti-Shine–Dalgarno sequence, which is conserved in bacteria. Strikingly, this entire region is absent from 12S mt-rRNA. We have mapped the ERAL1-binding site to a 33 nucleotide section delineating the 3′ terminal stem–loop region of 12S mt-rRNA. This loop contains two adenine residues that are reported to be dimethylated on mitoribosome maturation. Furthermore, and also in contrast with the bacterial orthologue, loss of ERAL1 leads to rapid decay of nascent 12S mt-rRNA, consistent with a role as a mitochondrial RNA chaperone. Finally, whereas depletion of ERAL1 leads to apoptosis, cell death occurs prior to any appreciable loss of mitochondrial protein synthesis or reduction in the stability of mitochondrial mRNA

Poly(A) binding proteins in human mitochondria

Mitochondria are organelles present in all nucleated eukaryotic cells. They play a central role in the conversion of metabolic fuels to a readily ·utilisable source of energy in the form of ATP. One of the most distinct features of mitochondria is the possession of their own genome (mtDNA). The mammalian mitochondrial genome encodes only 13 peptides, all of which all are involved in oxidative phosphorylation, in addition to all the RNA components necessary for the intra-mitochondrial translation machinery. Since mitochondria comprise greater than 1000 proteins, the majority of mitochondrial proteins are encoded by the nucleus, synthesized in the cytosol and then imported into mitochondria. In mammals, all mRNA species encoded by mtDNA exhibit constitutive oligo- or poly- adenylation. The poly(A) tails are generally 50-60 nt in length and stable, suggesting that they are protected from nucleases, potentially by the existence of a mitochondrial poly(A) binding protein. In the eukaryotic cytosol, a number of forms of poly(A)-binding proteins (PABPs) exist and are central to the regulation of RNA stability and translation. To date, no such protein has been isolated from mammalian mitochondria. Extensive in silico studies have not yielded any candidates, and so a biochemical approach with an extended FPLC purification scheme has been used to isolate the putative candidates for this activity from rat liver mitochondria. A subset of fractions .derived from this purification have been analysed by electrophoretic mobility shift assay and a number have demonstrated specific poly(A) binding activity using an in vitro derived radiolabelled substrate. The Mass spectro~copy analysis of these 'poly(A) binding' fractions indicated that the highest abundance proteins are ACATI and ACAA2. These are mitochondrial proteins with well characterised metabolic activities but no recognised RNA binding motifs. Over- Poly(A) binding proteins in human mitochondria Mateusz Wydro expression of recombinant ACATI and ACAA2 followed by in vitro studies revealed that both candidates exhibit specific poly(A) binding with weak affinity. The physiological role of this interaction, if any, remains unclear. A second approach that I have taken has been to target a known cytosolic poly(A) binding protein, PABPC1, to the human mitochondria in an inducible manner and observe any consequent changes in mt-mRNA metabolism. Here, I show that mitochondrially targeted (mt-PABPCI) is able to interact with the mitochondrial mRNA poly(A) region. Binding of mt-PABPCI resulted in shortening of all investigated mitochondrial mRNAs, but does not induce a concomitant loss of RNA stability. In response to mt-PABPC1 binding, however, mitochondrial translation is compromised, leading to a severe respiratory phenotype, consistent with the loss of mitochondrially-encoded polypeptides. I conclude that the mitochondrial translation inhibition is due to mt-PABPCI competition with endogenous protein(s) that interact with the poly(A) that are necessary to promote mitochondrial protein synthesis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Optimization of transient Agrobacterium-mediated gene expression system in leaves of Nicotioana benthamiana

system in leaves of Nicotiana benthamian

Probing the orientation of yeast VDAC1 in vivo

AbstractVoltage dependent anion channel (VDAC) is a vital ion channel in mitochondrial outer membranes and its structure was recently shown to be a 19 stranded β-barrel. However the orientation of VDAC in the membrane remains unclear. We probe here the topology and membrane orientation of yeast Saccharomyces cerevisiae in vivo. Five FLAG-epitopes were independently inserted into scVDAC1 and their surface exposure in intact and disrupted mitochondria detected by immunoprecipitation. Functionality was confirmed by measurements of respiration. Two epitopes suggest that VDAC (scVDAC) has its C-terminus exposed to the cytoplasm whilst two others are more equivocal and, when combined with published data, suggest a dynamic behavior

Human mitochondrial mRNAs—like members of all families, similar but different

The messenger RNAs containing the thirteen protein coding sequences of the human mitochondrial genome have frequently been regarded as a single functional category, alike in arrangement and hence in mode of expression. The “generic” mitochondrial mRNA is perceived as having (i) an arrangement within the polycistronic unit that permits its liberation following mt-tRNA processing, (ii) no 5′ cap structure or introns, (iii) essentially no untranslated regions, and (iv) a poly(A) tail of approximately fifty nucleotides that is required in part to complete the termination codon. Closer inspection reveals that only two molecules fit this pattern. This article examines the extent to which human mitochondrial mRNA species differ from one another

The evolutionarily conserved iron-sulfur protein INDH is required for complex I assembly and mitochondrial translation in Arabidopsis.

The assembly of respiratory complexes is a multistep process, requiring coordinate expression of mitochondrial and nuclear genes and cofactor biosynthesis. We functionally characterized the iron-sulfur protein required for NADH dehydrogenase (INDH) in the model plant Arabidopsis thaliana. An indh knockout mutant lacked complex I but had low levels of a 650-kD assembly intermediate, similar to mutations in the homologous NUBPL (nucleotide binding protein-like) in Homo sapiens. However, heterozygous indh/+ mutants displayed unusual phenotypes during gametogenesis and resembled mutants in mitochondrial translation more than mutants in complex I. Gradually increased expression of INDH in indh knockout plants revealed a significant delay in reassembly of complex I, suggesting an indirect role for INDH in the assembly process. Depletion of INDH protein was associated with decreased (35)S-Met labeling of translation products in isolated mitochondria, whereas the steady state levels of several mitochondrial transcripts were increased. Mitochondrially encoded proteins were differentially affected, with near normal levels of cytochrome c oxidase subunit2 and Nad7 but little Nad6 protein in the indh mutant. These data suggest that INDH has a primary role in mitochondrial translation that underlies its role in complex I assembly