Our current understanding of the toxicity of altered mito-ribosomal fidelity during mitochondrial protein synthesis: What can it tell us about human disease?

Altered mito-ribosomal fidelity is an important and insufficiently understood causative agent of mitochondrial dysfunction. Its pathogenic effects are particularly well-known in the case of mitochondrially induced deafness, due to the existence of the, so called, ototoxic variants at positions 847C (m.1494C) and 908A (m.1555A) of 12S mitochondrial (mt-) rRNA. It was shown long ago that the deleterious effects of these variants could remain dormant until an external stimulus triggered their pathogenicity. Yet, the link from the fidelity defect at the mito-ribosomal level to its phenotypic manifestation remained obscure. Recent work with fidelity-impaired mito-ribosomes, carrying error-prone and hyper-accurate mutations in mito-ribosomal proteins, have started to reveal the complexities of the phenotypic manifestation of mito-ribosomal fidelity defects, leading to a new understanding of mtDNA disease. While much needs to be done to arrive to a clear picture of how defects at the level of mito-ribosomal translation eventually result in the complex patterns of disease observed in patients, the current evidence indicates that altered mito-ribosome function, even at very low levels, may become highly pathogenic. The aims of this review are three-fold. First, we compare the molecular details associated with mito-ribosomal fidelity to those of general ribosomal fidelity. Second, we gather information on the cellular and organismal phenotypes associated with defective translational fidelity in order to provide the necessary grounds for an understanding of the phenotypic manifestation of defective mito-ribosomal fidelity. Finally, the results of recent experiments directly tackling mito-ribosomal fidelity are reviewed and future paths of investigation are discussed.

Structural analysis of mitochondrial rRNA gene variants identified in patients with deafness.

The last few years have witnessed dramatic advances in our understanding of the structure and function of the mammalian mito-ribosome. At the same time, the first attempts to elucidate the effects of mito-ribosomal fidelity (decoding accuracy) in disease have been made. Hence, the time is right to push an important frontier in our understanding of mitochondrial genetics, that is, the elucidation of the phenotypic effects of mtDNA variants affecting the functioning of the mito-ribosome. Here, we have assessed the structural and functional role of 93 mitochondrial (mt-) rRNA variants thought to be associated with deafness, including those located at non-conserved positions. Our analysis has used the structural description of the human mito-ribosome of the highest quality currently available, together with a new understanding of the phenotypic manifestation of mito-ribosomal-associated variants. Basically, any base change capable of inducing a fidelity phenotype may be considered non-silent. Under this light, out of 92 previously reported mt-rRNA variants thought to be associated with deafness, we found that 49 were potentially non-silent. We also dismissed a large number of reportedly pathogenic mtDNA variants, 41, as polymorphisms. These results drastically update our view on the implication of the primary sequence of mt-rRNA in the etiology of deafness and mitochondrial disease in general. Our data sheds much-needed light on the question of how mt-rRNA variants located at non-conserved positions may lead to mitochondrial disease and, most notably, provide evidence of the effect of haplotype context in the manifestation of some mt-rRNA variants.

Heterologous Inferential Analysis (HIA) and Other Emerging Concepts: In Understanding Mitochondrial Variation In Pathogenesis: There is no More Low-Hanging Fruit.

Here we summarize our latest efforts to elucidate the role of mtDNA variants affecting the mitochondrial translation machinery, namely variants mapping to the mt-rRNA and mt-tRNA genes. Evidence is accumulating to suggest that the cellular response to interference with mitochondrial translation is different from that occurring as a result of mutations in genes encoding OXPHOS proteins. As a result, it appears safe to state that a complete view of mitochondrial disease will not be obtained until we understand the effect of mt-rRNA and mt-tRNA variants on mitochondrial protein synthesis. Despite the identification of a large number of potentially pathogenic variants in the mitochondrially encoded rRNA (mt-rRNA) genes, we lack direct methods to firmly establish their pathogenicity. In the absence of such methods, we have devised an indirect approach named heterologous inferential analysis (HIA ) that can be used to make predictions concerning the disruptive potential of a large subset of mt-rRNA variants. We have used HIA to explore the mutational landscape of 12S and 16S mt-rRNA genes. Our HIA studies include a thorough classification of all rare variants reported in the literature as well as others obtained from studies performed in collaboration with physicians. HIA has also been used with non-mammalian mt-rRNA genes to elucidate how mitotypes influence the interaction of the individual and the environment. Regarding mt-tRNA variations, rapidly growing evidence shows that the spectrum of mutations causing mitochondrial disease might differ between the different mitochondrial haplogroups seen in human populations.

Mitochondrial DNA mutations induce mitochondrial biogenesis and increase the tumorigenic potential of Hodgkin and Reed-Sternberg cells.

Functioning mitochondria are crucial for cancer metabolism, but aerobic glycolysis is still considered to be an important pathway for energy production in many tumor cells. Here we show that two well established, classic Hodgkin lymphoma (cHL) cell lines harbor deleterious variants within mitochondrial DNA (mtDNA) and thus exhibit reduced steady-state levels of respiratory chain complexes. However, instead of resulting in the expected bioenergetic defect, these mtDNA variants evoke a retrograde signaling response that induces mitochondrial biogenesis and ultimately results in increased mitochondrial mass as well as function and enhances proliferation in vitro as well as tumor growth in mice in vivo. When complex I assembly was impaired by knockdown of one of its subunits, this led to further increased mitochondrial mass and function and, consequently, further accelerated tumor growth in vivo. In contrast, inhibition of mitochondrial respiration in vivo by the mitochondrial complex I inhibitor metformin efficiently slowed down growth. We conclude that, as a new mechanism, mildly deleterious mtDNA variants in cHL cancer cells cause an increase of mitochondrial mass and enhanced function as a compensatory effect using a retrograde signaling pathway, which provides an obvious advantage for tumor growth.

Minority Gene Expression Profiling: Probing the Genetic Signatures of Pathogenesis Using Ribosome Profiling.

Minority Gene Expression Profiling (MGEP) refers to a scenario where the expression profiles of specific genes of interest are concentrated in a small cellular pool that is embedded within a larger, non-expressive pool. An example of this is the analysis of disease-related genes within sub-populations of blood or biopsied tissues. These systems are characterized by low signal-to-noise ratios that make it difficult, if not impossible, to uncover the desired signatures of pathogenesis in the absence of lengthy, and often problematic, technical manipulations. We have adapted ribosome profiling (RP) workflows from the Illumina to the Ion Proton platform and used them to analyze signatures of pathogenesis in an MGEP model system consisting of human cells eliciting <3% productive dengue infection. We find that RP is powerful enough to identify relevant responses of differentially expressed genes, even in the presence of significant noise. We discuss how to deal with sources of unwanted variation, and propose ways to further improve this powerful approach to the study of pathogenic signatures within MGEP systems.

Precision Medicine and Precision Public Health in the Era of Pathogen Next-Generation Sequencing.

This brief report serves as an introduction to a supplement of the Journal of Infectious Diseases entitled "Next-Generation Sequencing (NGS) Technologies to Advance Global Infectious Disease Research." We briefly discuss the history of NGS technologies and describe how the techniques developed during the past 40 years have impacted our understanding of infectious diseases. Our focus is on the application of NGS in the context of pathogen genomics. Beyond obvious clinical and public health applications, we also discuss the challenges that still remain within this rapidly evolving field.

Outer membrane protein FrpA, the siderophore piscibactin receptor of Photobacterium damselae subsp. piscicida, as a subunit vaccine against photobacteriosis in sole (Solea senegalensis).

Photobacteriosis caused by Photobacterium damselae subsp. piscicida (Pdp) remains one of the main infectious diseases affecting cultured fish in Mediterranean countries. Diverse vaccine formulations based in the use of inactivated bacterial cells have been used with unsatisfactory results, especially in newly cultured species like sole (Solea senegalensis). In this work, we describe the use of the outer membrane receptor (FrpA) of the siderophore piscibactin produced by Pdp as a novel subunit vaccine against photobacteriosis. FrpA has been cloned and expressed in Escherichia coli under an arabinose-inducible promoter. A recombinant protein (rFrpA) containing the pelB localization signal and a His tag was constructed to obtain a pure native form of the protein from E. coli outer membranes. The immunogenicity of rFrpA, and its protective effect against photobacteriosis, was tested by i.p. injection of 30  µg of the protein, mixed with Freund's adjuvant, in sole fingerlings with two immunizations separated by 30 days. Results showed that using either pure rFrpA or whole cells as immobilized antigens in ELISA assays, rFrpA induces the production of specific antibodies in sole. An experimental infection using fish vaccinated with rFrpA or formalin-killed whole cells of Pdp showed that both groups were protected against Pdp infection at similar levels, with no significant differences, reaching RPS values of 73% and 79%, respectively. Thus, FrpA constitutes a promising antigen candidate for the development of novel more effective vaccines against fish photobacteriosis.

Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness.

Diet may be modified seasonally or by biogeographic, demographic or cultural shifts. It can differentially influence mitochondrial bioenergetics, retrograde signalling to the nuclear genome, and anterograde signalling to mitochondria. All these interactions have the potential to alter the frequencies of mtDNA haplotypes (mitotypes) in nature and may impact human health. In a model laboratory system, we fed four diets varying in Protein: Carbohydrate (P:C) ratio (1:2, 1:4, 1:8 and 1:16 P:C) to four homoplasmic Drosophila melanogaster mitotypes (nuclear genome standardised) and assayed their frequency in population cages. When fed a high protein 1:2 P:C diet, the frequency of flies harbouring Alstonville mtDNA increased. In contrast, when fed the high carbohydrate 1:16 P:C food the incidence of flies harbouring Dahomey mtDNA increased. This result, driven by differences in larval development, was generalisable to the replacement of the laboratory diet with fruits having high and low P:C ratios, perturbation of the nuclear genome and changes to the microbiome. Structural modelling and cellular assays suggested a V161L mutation in the ND4 subunit of complex I of Dahomey mtDNA was mildly deleterious, reduced mitochondrial functions, increased oxidative stress and resulted in an increase in larval development time on the 1:2 P:C diet. The 1:16 P:C diet triggered a cascade of changes in both mitotypes. In Dahomey larvae, increased feeding fuelled increased ß-oxidation and the partial bypass of the complex I mutation. Conversely, Alstonville larvae upregulated genes involved with oxidative phosphorylation, increased glycogen metabolism and they were more physically active. We hypothesise that the increased physical activity diverted energy from growth and cell division and thereby slowed development. These data further question the use of mtDNA as an assumed neutral marker in evolutionary and population genetic studies. Moreover, if humans respond similarly, we posit that individuals with specific mtDNA variations may differentially metabolise carbohydrates, which has implications for a variety of diseases including cardiovascular disease, obesity, and perhaps Parkinson's Disease.

Recruitment of RNA molecules by connexin RNA-binding motifs: Implication in RNA and DNA transport through microvesicles and exosomes.

Connexins (Cxs) are integral membrane proteins that form high-conductance plasma membrane channels, allowing communication from cell to cell (via gap junctions) and from cells to the extracellular environment (via hemichannels). Initially described for their role in joining excitable cells (nerve and muscle), gap junctions (GJs) are found between virtually all cells in solid tissues and are essential for functional coordination by enabling the direct transfer of small signalling molecules, metabolites, ions, and electrical signals from cell to cell. Several studies have revealed diverse channel-independent functions of Cxs, which include the control of cell growth and tumourigenicity. Connexin43 (Cx43) is the most widespread Cx in the human body. The myriad roles of Cx43 and its implication in the development of disorders such as cancer, inflammation, osteoarthritis and Alzheimer's disease have given rise to many novel questions. Several RNA- and DNA-binding motifs were predicted in the Cx43 and Cx26 sequences using different computational methods. This review provides insights into new, ground-breaking functions of Cxs, highlighting important areas for future work such as transfer of genetic information through extracellular vesicles. We discuss the implication of potential RNA- and DNA-binding domains in the Cx43 and Cx26 sequences in the cellular communication and control of signalling pathways.

The presence of highly disruptive 16S rRNA mutations in clinical samples indicates a wider role for mutations of the mitochondrial ribosome in human disease.

Mitochondrial DNA mutations are well recognized as an important cause of disease, with over two hundred variants in the protein encoding and mt-tRNA genes associated with human disorders. In contrast, the two genes encoding the mitochondrial rRNAs (mt-rRNAs) have been studied in far less detail. This is because establishing the pathogenicity of mt-rRNA mutations is a major diagnostic challenge. Only two disease causing mutations have been identified at these loci, both mapping to the small subunit (SSU). On the large subunit (LSU), however, the evidence for the presence of pathogenic LSU mt-rRNA changes is particularly sparse. We have previously expanded the list of deleterious SSU mt-rRNA mutations by identifying highly disruptive base changes capable of blocking the activity of the mitoribosomal SSU. To do this, we used a new methodology named heterologous inferential analysis (HIA). The recent arrival of near-atomic-resolution structures of the human mitoribosomal LSU, has enhanced the power of our approach by permitting the analysis of the corresponding sites of mutation within their natural structural context. Here, we have used these tools to determine whether LSU mt-rRNA mutations found in the context of human disease and/or ageing could disrupt the function of the mitoribosomal LSU. Our results clearly show that, much like the for SSU mt-rRNA, LSU mt-rRNAs mutations capable of compromising the function of the mitoribosomal LSU are indeed present in clinical samples. Thus, our work constitutes an important contribution to an emerging view of the mitoribosome as an important element in human health.

Heterologous inferential analysis (HIA) as a method to understand the role of mitochondrial rRNA mutations in pathogenesis.

Despite the identification of a large number of potentially pathogenic variants in the mitochondrially encoded rRNA (mt-rRNA) genes, we lack direct methods to firmly establish their pathogenicity. In the absence of such methods, we have devised an indirect approach named heterologous inferential analysis or HIA that can be used to make predictions on the disruptive potential of a large subset of mt-rRNA variants. First, due to the high evolutionary conservation of the rRNA fold, comparison of phylogenetically derived secondary structures of the human mt-rRNAs and those from model organisms allows the location of structurally equivalent residues. Second, visualization of the heterologous equivalent residue in high-resolution structures of the ribosome allows a preliminary structural characterization of the residue and its neighboring region. Third, an exhaustive search for biochemical and genetic information on the residue and its surrounding region is performed to understand their degree of involvement in ribosomal function. Additional rounds of visualization in biochemically relevant high-resolution structures will lead to the structural and functional characterization of the residue's role in ribosomal function and to an assessment of the disruptive potential of mutations at this position. Notably, in the case of certain mitochondrial variants for which sufficient information regarding their genetic and pathological manifestation is available; HIA data alone can be used to predict their pathogenicity. In other cases, HIA will serve to prioritize variants for additional investigation. In the context of a scoring system specifically designed for these variants, HIA could lead to a powerful diagnostic tool.

The role of the mitochondrial ribosome in human disease: searching for mutations in 12S mitochondrial rRNA with high disruptive potential.

Mutations of mitochondrial DNA are linked to many human diseases. Despite the identification of a large number of variants in the mitochondrially encoded rRNA (mt-rRNA) genes, the evidence supporting their pathogenicity is, at best, circumstantial. Establishing the pathogenicity of these variations is of major diagnostic importance. Here, we aim to estimate the disruptive effect of mt-rRNA variations on the function of the mitochondrial ribosome. In the absence of direct biochemical methods to study the effect of mt-rRNA variations, we relied on the universal conservation of the rRNA fold to infer their disruptive potential. Our method, named heterologous inferential analysis or HIA, combines conservational information with functional and structural data obtained from heterologous ribosomal sources. Thus, HIA's predictive power is superior to the traditional reliance on simple conservation indexes. By using HIA, we have been able to evaluate the disruptive potential for a subset of uncharacterized 12S mt-rRNA variations. Our analysis revealed the existence of variations in the rRNA component of the human mitoribosome with different degrees of disruptive power. In cases where sufficient information regarding the genetic and pathological manifestation of the mitochondrial phenotype is available, HIA data can be used to predict the pathogenicity of mt-rRNA mutations. In other cases, HIA analysis will allow the prioritization of variants for additional investigation. Eventually, HIA-inspired analysis of potentially pathogenic mt-rRNA variations, in the context of a scoring system specifically designed for these variants, could lead to a powerful diagnostic tool.

Tertiary interactions between helices h13 and h44 in 16S RNA contribute to the fidelity of translation.

The A-minor interaction, formed between single-stranded adenosines and the minor groove of a receptor helix, is among the most common motifs found in rRNA. Among the A-minors found in 16S rRNA are a set of interactions between adenosines at positions 1433, 1434 and 1468 in helix 44 (h44) and their receptors in the nucleotide 320-340 region of helix 13 (h13). These interactions have been implicated in the maintenance of translational accuracy, because base substitutions at the adjacent C1469 increase miscoding errors. We have tested their functional significance through mutagenesis of h13 and h44. Mutations at the h44 A residues, or the A-minor receptors in h13, increase a variety of translational errors and a subset of the mutants show decreased association between 30S and 50S ribosomal subunits. These results are consistent with the involvement of h13-h44 interactions in the alignment and packing of these helices in the 30S subunit and the importance of this helical alignment for tRNA selection and subunit-subunit interaction.

Mutations in the intersubunit bridge regions of 16S rRNA affect decoding and subunit-subunit interactions on the 70S ribosome.

The small and large subunits of the ribosome are held together by a series of bridges, involving RNA-RNA, RNA-protein and protein-protein interactions. Some 12 bridges have been described for the Escherichia coli 70S ribosome. In this work, we have targeted for mutagenesis, some of the 16S rRNA residues involved in the formation of intersubunit bridges B3, B5, B6, B7b and B8. In addition to effects on subunit association, the mutant ribosomes also affect the fidelity of translation; bridges B5, B6 and B8 increase decoding errors during elongation, while disruption of bridges B3 and B7b alters the stringency of start codon selection. Moreover, mutations in the bridge B5, B6 and B8 regions of 16S rRNA also correct the growth and decoding defects associated with alterations in ribosomal protein S12. These results link bridges B5, B6 and B8 with the decoding process and are consistent with the recently described location of translation factor EF-Tu on the ribosome and the proposed involvement of h14 in activating Guanosine-5'-triphosphate (GTP) hydrolysis by aminoacyl-tRNA • EF-Tu • GTP. These observations are consistent with a model in which bridges B5, B6 and B8 contribute to the fidelity of translation by modulating GTP hydrolysis by aminoacyl-tRNA • EF-Tu • GTP ternary complexes during the elongation phase of protein synthesis.

Phylogenetic and multivariate analyses to determine the effects of different tillage and residue management practices on soil bacterial communities.

Bacterial communities are important not only in the cycling of organic compounds but also in maintaining ecosystems. Specific bacterial groups can be affected as a result of changes in environmental conditions caused by human activities, such as agricultural practices. The aim of this study was to analyze the effects of different forms of tillage and residue management on soil bacterial communities by using phylogenetic and multivariate analyses. Treatments involving zero tillage (ZT) and conventional tillage (CT) with their respective combinations of residue management, i.e., removed residue (-R) and kept residue (+R), and maize/wheat rotation, were selected from a long-term field trial started in 1991. Analysis of bacterial diversity showed that soils under zero tillage and crop residue retention (ZT/+R) had the highest levels of diversity and richness. Multivariate analysis showed that beneficial bacterial groups such as fluorescent Pseudomonas spp. and Burkholderiales were favored by residue retention (ZT/+R and CT/+R) and negatively affected by residue removal (ZT/-R). Zero-tillage treatments (ZT/+R and ZT/-R) had a positive effect on the Rhizobiales group, with its main representatives related to Methylosinus spp. known as methane-oxidizing bacteria. It can be concluded that practices that include reduced tillage and crop residue retention can be adopted as safer agricultural practices to preserve and improve the diversity of soil bacterial communities.

Mutagenesis of the modified bases, m(5)U1939 and psi2504, in Escherichia coli 23S rRNA.

Ribosomal RNAs (rRNAs) from all kingdoms contain a variety of post-transcriptional modifications and these are typically clustered in the functional centers of the ribosome. The functions of two bases in the 23S rRNA of Escherichia coli that are post-transcriptionally modified, m(5)U1939 and psi2504, were examined by mutagenesis of the rRNA bases and by inactivation of the RumA methylase that methylates U1939. Base substitutions at U1939 had little effect on growth or the fidelity of translation, but altered the sensitivity of the ribosomes to the antibiotics fusidic acid and capreomycin. Strains lacking the RumA methylase were gradually out-competed by wild type strains in growth competition experiments, suggesting that the m(5)U methylation improves ribosome performance. Base changes at psi2504 had dramatic effects on growth and resistance to several peptidyltransferase inhibitor antibiotics and increased the levels of translational errors. The results link these sites of post-transcriptional modification with the ribosome's response to antibiotics and the control of translational fidelity.

Modification of the Ribosome and the Translational Machinery during Reduced Growth Due to Environmental Stress.

Escherichia coli strains normally used under laboratory conditions have been selected for maximum growth rates and require maximum translation efficiency. Recent studies have shed light on the structural and functional changes undergone by the translational machinery in E. coli during heat and cold shock and upon entry into stationary phase. In these situations both the composition and the partitioning of this machinery into the different pools of cellular ribosomes are modified. As a result, the translational capacity of the cell is dramatically altered. This review provides a comprehensive account of these modifications, regardless of whether or not their underlying mechanisms and their effects on cellular physiology are known. Not only is the composition of the ribosome modified upon entry into stationary phase, but the modification of other components of the translational machinery, such as elongation factor Tu (EFTu) and tRNAs, has also been observed. Hibernation-promoting factor (HPF), paralog protein Y (PY), and ribosome modulation factor (RMF) may also be related to the general protection against environmental stress observed in stationary-phase E. coli cells, a role that would not be revealed necessarily by the viability assays. Even for the best-characterized ribosome-associated factors induced under stress (RMF, PY, and initiation factors), we are far from a complete understanding of their modes of action.

Modulation of 16S rRNA function by ribosomal protein S12.

Ribosomal protein S12 is a critical component of the decoding center of the 30S ribosomal subunit and is involved in both tRNA selection and the response to streptomycin. We have investigated the interplay between S12 and some of the surrounding 16S rRNA residues by examining the phenotypes of double-mutant ribosomes in strains of Escherichia coli carrying deletions in all chromosomal rrn operons and expressing total rRNA from a single plasmid-borne rrn operon. We show that the combination of S12 and otherwise benign mutations at positions C1409-G1491 in 16S rRNA severely compromises cell growth while the level and range of aminoglycoside resistances conferred by the G1491U/C substitutions is markedly increased by a mutant S12 protein. The G1491U/C mutations in addition confer resistance to the unrelated antibiotic, capreomycin. S12 also interacts with the 912 region of 16S rRNA. Genetic selection of suppressors of streptomycin dependence caused by mutations at proline 90 in S12 yielded a C912U substitution in 16S rRNA. The C912U mutation on its own confers resistance to streptomycin and restricts miscoding, properties that distinguish it from a majority of the previously described error-promoting ram mutants that also reverse streptomycin dependence.

Structural analysis of kasugamycin inhibition of translation.

The prokaryotic ribosome is an important target of antibiotic action. We determined the X-ray structure of the aminoglycoside kasugamycin (Ksg) in complex with the Escherichia coli 70S ribosome at 3.5-A resolution. The structure reveals that the drug binds within the messenger RNA channel of the 30S subunit between the universally conserved G926 and A794 nucleotides in 16S ribosomal RNA, which are sites of Ksg resistance. To our surprise, Ksg resistance mutations do not inhibit binding of the drug to the ribosome. The present structural and biochemical results indicate that inhibition by Ksg and Ksg resistance are closely linked to the structure of the mRNA at the junction of the peptidyl-tRNA and exit-tRNA sites (P and E sites).

Structures of the bacterial ribosome at 3.5 A resolution.

We describe two structures of the intact bacterial ribosome from Escherichia coli determined to a resolution of 3.5 angstroms by x-ray crystallography. These structures provide a detailed view of the interface between the small and large ribosomal subunits and the conformation of the peptidyl transferase center in the context of the intact ribosome. Differences between the two ribosomes reveal a high degree of flexibility between the head and the rest of the small subunit. Swiveling of the head of the small subunit observed in the present structures, coupled to the ratchet-like motion of the two subunits observed previously, suggests a mechanism for the final movements of messenger RNA (mRNA) and transfer RNAs (tRNAs) during translocation.