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

A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome

Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes

Identification of the Main Translation Release Factor in Human Mitochondria

Mitochondria, the powerhouses of the cell, possess their own translational system seemingly inherited from their proteo-bacterial ancestor. Termination of translation in bacteria is mediated by Release Factors 1, 2 and 3 (RF1, 2 & 3), the first two of which recognise stop codons UAA, UAG and UGA, while RF3 enhances the efficiency of termination. Codon discrimination in RF1 is ascribed to a highly conserved three-peptide motif, namely 'PXT', in its anticodon-like domain. In human mitochondria where translation termination is triggered by four stop codons (UAA, UAG, AGG and AGA), only one candidate gene (MTRFl) was purported in silico to encode a functional mitochondrial release factor. Having searched the human genome database, we have found another candidate (UniprotKB/TrEMBL Q96EX4), potentially capable of acting as mitochondrial release factor based upon its high similarity to bacterial RFI and MTRFI. An alignment analysis of the highly conserved PXT motif revealed that the new candidate harbours the same three-peptide motif in the form of 'PKT', whereas the previously identified mtRF1 carried a hexa-peptide motif of'PEVGLS'. GFP fusion studies demonstrated co-localization of this new candidate with mitochondria. But to confirm whether this new candidate is actually imported into mitochondria, a radiolabelled product was generated in vitro and incubated with isolated rat liver mitochondria. N-terminal cleavage was demonstrated and the matured protein became insensitive to added proteinase, consistent with this protein accessing the mitochondrial matrix. In vitro translation termination assays indicated that this new candidate is capable of codon discrimination, recognising UAA and UAG as stop codons, thus implicating the new protein as the main translational release factor in human mitochondria. Further, this protein was shown to be capable of rescuing the non-respiratory phenotype of the I1mr/] yeast strains (8. cerevisiae and S. pombe) in non-fermentable carbon sources owing to restoration of mitochondrial respiration. RNAi knock-down of mtRF1a in human HeLa cells increased mitochondrial mass and ROS generation, in addition to retarding their growth in galactose medium consistent with the new candidate having a vital role in human mitochondrial respiration. This protein has now been re-designated as 'mtRF1a', mutations of which may prove responsible for mitochondrial disorders in some patients.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Effects of left prefrontal transcranial direct current stimulation on the acquisition of contextual and cued fear memory

Objective(s): Behavioral and neuroimaging studies have shown that transcranial direct current stimulation, as a non-invasive neuromodulatory technique, beyond regional effects can modify functionally interconnected remote cortical and subcortical areas. In this study, we hypothesized that the induced changes in cortical excitability following the application of cathodal or anodal tDCS over the left frontal cortex as pre-training would affect functional connectivity in resting-state circuits of fear memory and consequently could improve or disturb the acquisition of fear memory. Materials and Methods: In order to evaluate the polarity-dependent effects of tDCS on the acquisition of fear memory and the functional connectivity, we applied left prefrontal anodal or cathodal stimulation at 200 μA for one session to healthy mice for the durations of 20 and 30 min prior to fear conditioning. Results: Our results revealed that the administration of left prefrontal anodal (for both 20 and 30 min durations) and cathodal (at 30 min duration) tDCS impaired the acquisition of both contextual and cued fear memory. In addition, we did not observe a direct correlation between stimulation duration and the efficacy of tDCS on the acquisition of contextual and cued fear memory. Conclusion: In this study, the impairments of both contextual and cued memory further confirmed the previous studies reporting that the administration of transcranial stimulation would affect the activity of deeper structures like amygdala and hippocampus as the main components of the fear memory circuit in acquisition, storage, and expression of the memory

In Vitro-Derived Gametes from Stem Cells

Sperm and eggs are essential cells for reproduction and fertility in mammals. Lack of sperm production is one of the leading causes of infertility, a major and growing problem in the developed world affecting 13 to 18% of reproductive-age couples. The birth of the first test tube baby by in vitro fertilization marked an advance in infertility treatment. Later on, several important new techniques called assisted reproductive technologies were developed to help couples who experience infertility. One limiting factor is the requirement of reproductive cells (gametes) for use in in vitro fertilization. For azoospermic men lacking sperm cells, producing gametes in vitro could be a new window to overcome infertility. In the past few years, several reports have been published on generating germ cells from stem cells, one of the epitomes of which was the report on functional in vitro-derived (IVD) germ cells. These mature haploid sperm cells from mouse embryonic stem cells were capable of egg fertilization and producing live offspring. In tandem with previous advancements in germ cell research, development of new technologies based on IVD gametes will change the future of infertility and provide a new basis for the establishment of novel therapeutic approaches to cure more complicated conditions of infertility. In addition, IVD gametogenesis provides an accessible system for studying the specification and differentiation of sperm cells and related processes such as meiosis, morphogenesis, and motility