Post-transcriptional regulation of the steady-state levels of mitochondrial tRNAs in HeLa cells

In human mitochondrial DNA (mtDNA), the tRNA genes are located in three different transcription units that are transcribed at three different rates. To analyze the regulation of tRNA formation by the three transcription units, we have examined the steady-state levels and metabolic properties of the tRNAs of HeLa cell mitochondria. DNA excess hybridization experiments utilizing separated strands of mtDNA and purified tRNA samples from exponential cells long term labeled with [32P]orthophosphate have revealed a steady-state level of 6 x 10(5) tRNA molecules/cell, with three-fourths being encoded in the H-strand and one-fourth in the L-strand. Hybridization of the tRNAs with a panel of M13 clones of human mtDNA containing, in most cases, single tRNA genes and a quantitation of two-dimensional electrophoretic fractionations of the tRNAs have shown that the steady-state levels of tRNA(Phe) and tRNA(Val) are two to three times higher than the average level of the other H-strand-encoded tRNAs and three to four times higher than the average level of the L-strand-encoded tRNAs. Similar experiments carried out with tRNAs isolated from cells labeled with very short pulses of [5-3H]uridine have indicated that the rates of formation of the individual tRNA species are proportional to their steady-state amounts. Therefore, the approximately 25-fold higher rate of transcription of the tRNA(Phe) and tRNA(Val) genes relative to the other H-strand tRNA genes and the 10-16-fold higher rate of transcription of the L-strand tRNA genes relative to the H-strand tRNA genes are not reflected in the steady-state levels or the rates of formation of the corresponding tRNAs. A comparison of the steady-state levels of the individual tRNAs with the corresponding codon usage for protein synthesis, as determined from the DNA sequence and the rates of synthesis of the various polypeptides, has not revealed any significant correlation between the two parameters

Back to Water: Signature of Adaptive Evolution in Cetacean Mitochondrial tRNAs

Abstract The mitochondrion is the power plant of the eukaryotic cell, and tRNAs are the fundamental components of its translational machinery. In the present paper, the evolution of mitochondrial tRNAs was investigated in the Cetacea, a clade of Cetartiodactyla that retuned to water and thus had to adapt its metabolism to a different medium than that of its mainland ancestors. Our analysis focussed on identifying the factors that influenced the evolution of Cetacea tRNA double-helix elements, which play a pivotal role in the formation of the secondary and tertiary structures of each tRNA and consequently manipulate the whole translation machinery of the mitochondrion. Our analyses showed that the substitution pathways in the stems of different tRNAs were influenced by various factors, determining a molecular evolution that was unique to each of the 22 tRNAs. Our data suggested that the composition, AT-skew, and GC-skew of the tRNA stems were the main factors influencing the substitution process. In particular, the range of variation and the fluctuation of these parameters affected the fate of single tRNAs. Strong heterogeneity was observed among the different species of Cetacea. Finally, it appears that the evolution of mitochondrial tRNAs was also shaped by the environments in which the Cetacean taxa differentiated. This latter effect was particularly evident in toothed whales that either live in freshwater or are deep divers

MINTmap: fast and exhaustive profiling of nuclear and mitochondrial tRNA fragments from short RNA-seq data.

Transfer RNA fragments (tRFs) are an established class of constitutive regulatory molecules that arise from precursor and mature tRNAs. RNA deep sequencing (RNA-seq) has greatly facilitated the study of tRFs. However, the repeat nature of the tRNA templates and the idiosyncrasies of tRNA sequences necessitate the development and use of methodologies that differ markedly from those used to analyze RNA-seq data when studying microRNAs (miRNAs) or messenger RNAs (mRNAs). Here we present MINTmap (for MItochondrial and Nuclear TRF mapping), a method and a software package that was developed specifically for the quick, deterministic and exhaustive identification of tRFs in short RNA-seq datasets. In addition to identifying them, MINTmap is able to unambiguously calculate and report both raw and normalized abundances for the discovered tRFs. Furthermore, to ensure specificity, MINTmap identifies the subset of discovered tRFs that could be originating outside of tRNA space and flags them as candidate false positives. Our comparative analysis shows that MINTmap exhibits superior sensitivity and specificity to other available methods while also being exceptionally fast. The MINTmap codes are available through https://github.com/TJU-CMC-Org/MINTmap/ under an open source GNU GPL v3.0 license

Difference between Mitochondrial RNase P and Nuclear RNase P [Letters to the Editor]

[Discussion of: Puranam, R. S., and G. Attardi. 2001. The RNase P associated with HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P. Mol. Cell. Biol. 21:548-561.

Evolution Meets Disease: Penetrance and Functional Epistasis of Mitochondrial tRNA Mutations

About half of the mitochondrial DNA (mtDNA) mutations causing diseases in humans occur in tRNA genes. Particularly intriguing are those pathogenic tRNA mutations than can reach homoplasmy and yet show very different penetrance among patients. These mutations are scarce and, in addition to their obvious interest for understanding human pathology, they can be excellent experimental examples to model evolution and fixation of mitochondrial tRNA mutations. To date, the only source of this type of mutations is human patients. We report here the generation and characterization of the first mitochondrial tRNA pathological mutation in mouse cells, an m.3739G>A transition in the mitochondrial mt-Ti gene. This mutation recapitulates the molecular hallmarks of a disease-causing mutation described in humans, an m.4290T>C transition affecting also the human mt-Ti gene. We could determine that the pathogenic molecular mechanism, induced by both the mouse and the human mutations, is a high frequency of abnormal folding of the tRNAIle that cannot be charged with isoleucine. We demonstrate that the cells harboring the mouse or human mutant tRNA have exacerbated mitochondrial biogenesis triggered by an increase in mitochondrial ROS production as a compensatory response. We propose that both the nature of the pathogenic mechanism combined with the existence of a compensatory mechanism can explain the penetrance pattern of this mutation. This particular behavior can allow a scenario for the evolution of mitochondrial tRNAs in which the fixation of two alleles that are individually deleterious can proceed in two steps and not require the simultaneous mutation of both

RNA modification landscape of the human mitochondrial tRNA(LYs) regulates protein synthesis

Post-transcriptional RNA modifications play a critical role in the pathogenesis of human mitochondrial disorders, but the mechanisms by which specific modifications affect mitochondrial protein synthesis remain poorly understood. Here we used a quantitative RNA sequencing approach to investigate, at nucleotide resolution, the stoichiometry and methyl modifications of the entire mitochondrial tRNA pool, and establish the relevance to human disease. We discovered that a N-1 -methyladenosine (m(1)A) modification is missing at position 58 in the mitochondrial tRNA(LYs) of patients with the mitochondrial DNA mutation m.8344 A > G associated with MERRF (myoclonus epilepsy, ragged-red fibers). By restoring the modification on the mitochondrial tRNA(LYs), we demonstrated the importance of the m(1)A58 to translation elongation and the stability of selected nascent chains. Our data indicates regulation of post-transcriptional modifications on mitochondrial tRNAs is finely tuned for the control of mitochondrial gene expression. Collectively, our findings provide novel insight into the regulation of mitochondrial tRNAs and reveal greater complexity to the molecular pathogenesis of MERRF.Peer reviewe

Human Mitochondrial tRNA Mutations in Maternally Inherited Deafness

AbstractMutations in mitochondrial tRNA genes have been shown to be associated with maternally inherited syndromic and non-syndromic deafness. Among those, mutations such as tRNALeu(UUR)3243A>G associated with syndromic deafness are often present in heteroplasmy, and the non-syndromic deafness-associated tRNA mutations including tRNASer(UCN)7445A>G are often in homoplasmy or in high levels of heteroplasmy. These tRNA mutations are the primary factors underlying the development of hearing loss. However, other tRNA mutations such as tRNAThr15927G>A and tRNASer(UCN)7444G>A are insufficient to produce a deafness phenotype, but always act in synergy with the primary mitochondrial DNA mutations, and can modulate their phenotypic manifestation. These tRNA mutations may alter the structure and function of the corresponding mitochondrial tRNAs and cause failures in tRNAs metabolism. Thereby, the impairment of mitochondrial protein synthesis and subsequent defects in respiration caused by these tRNA mutations, results in mitochondrial dysfunctions and eventually leads to the development of hearing loss. Here, we summarized the deafness-associated mitochondrial tRNA mutations and discussed the pathophysiology of these mitochondrial tRNA mutations, and we hope these data will provide a foundation for the early diagnosis, management, and treatment of maternally inherited deafness

Post-transcriptional nucleotide modification and alternative folding of RNA

Alternative foldings are an inherent property of RNA and a ubiquitous problem in scientific investigations. To a living organism, alternative foldings can be a blessing or a problem, and so nature has found both, ways to harness this property and ways to avoid the drawbacks. A simple and effective method employed by nature to avoid unwanted folding is the modulation of conformation space through post-transcriptional base modification. Modified nucleotides occur in almost all classes of natural RNAs in great chemical diversity. There are about 100 different base modifications known, which may perform a plethora of functions. The presumably most ancient and simple nucleotide modifications, such as methylations and uridine isomerization, are able to perform structural tasks on the most basic level, namely by blocking or reinforcing single base-pairs or even single hydrogen bonds in RNA. In this paper, functional, genomic and structural evidence on cases of folding space alteration by post-transcriptional modifications in native RNA are reviewed

Mitochondrial tRNA Valine in Cardiomyopathies

Mitochondrial respiratory chain disorders are a heterogeneous group of diseases that clinically involve multiple tissues although they tend to mainly affect nervous system and skeletal muscle. The predominance of neurologic and neuromuscular manifestations in mitochondrial diseases has generally masked the presence of other, but not less important, clinical phenotypes, such as cardiac complications. Nowadays, mitochondrial defects are being increasingly recognized to play an important role in the pathogenesis of a subgroup of cardiomyopathies produced by defects in the energetic metabolism (mitochondrial cardiomyopathies). These diseases can result from mutations in either nuclear or mitochondrial encoded genes although mitochondrial DNA mutations are more frequent. In fact, cardiac conduction abnormalities have been associated with different mtDNA rearrangements. In a same way, sporadic or inherited mutations in mitochondrial DNA specifically in the mitochondrial transfer ribonucleic acid genes (mostly in the tRNALeu(UUR) and tRNAIle) have also been associated with hypertrophic and dilated cardiomyopathy. Mitochondrial diseases caused by mutations in the mitochondrial tRNAVal gene (MT-TV) are not very frequent. However, a relatively high percentage of mutations in this gene have been associated with mitochondrial cardiomyopathy. Besides, functional and molecular analyses suggest that the MT-TV gene should be routinely considered in the diagnosis when there is a high suspicion of mitochondrial cardiomyopathy. Finally, the increasingly importance of the role that this gene has begun to play in the pathophysiology of mitochondrial cardiomyopathies indicates that future studies about the molecular mechanisms that could explain why the cardiomyopathy phenotype appears must be carried ouThis work was supported by grants of the Center for Biomedical Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III (grants PI 07/0167, PI 10/0703 to R.G. and PI06/0205, PS09/00941 to B.B.) and Comunidad de Madrid (grant number S2010/BMD-2402)