Catherine Florentz

MITOS: improved de novo metazoan mitochondrial genome annotation.

About 2000 completely sequenced mitochondrial genomes are available from the NCBI RefSeq data base together with manually curated annotations of their protein-coding genes, rRNAs, and tRNAs. This annotation information, which has accumulated over two decades, has been obtained with a diverse set of computational tools and annotation strategies. Despite all efforts of manual curation it is still plagued by misassignments of reading directions, erroneous gene names, and missing as well as false positive annotations in particular for the RNA genes. Taken together, this causes substantial problems for fully automatic pipelines that aim to use these data comprehensively for studies of animal phylogenetics and the molecular evolution of mitogenomes. The MITOS pipeline is designed to compute a consistent de novo annotation of the mitogenomic sequences. We show that the results of MITOS match RefSeq and MitoZoa in terms of annotation coverage and quality. At the same time we avoid biases, inconsistencies of nomenclature, and typos originating from manual curation strategies. The MITOS pipeline is accessible online at http://mitos.bioinf.uni-leipzig.de.

Mitochondrial aspartyl-tRNA synthetase deficiency causes leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation

Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) has recently been defined based on a highly characteristic constellation of abnormalities observed by magnetic resonance imaging and spectroscopy. LBSL is an autosomal recessive disease, most often manifesting in early childhood. Affected individuals develop slowly progressive cerebellar ataxia, spasticity and dorsal column dysfunction, sometimes with a mild cognitive deficit or decline. We performed linkage mapping with microsatellite markers in LBSL families and found a candidate region on chromosome 1, which we narrowed by means of shared haplotypes. Sequencing of genes in this candidate region uncovered mutations in DARS2, which encodes mitochondrial aspartyl-tRNA synthetase, in affected individuals from all 30 families. Enzyme activities of mutant proteins were decreased. We were surprised to find that activities of mitochondrial complexes from fibroblasts and lymphoblasts derived from affected individuals were normal, as determined by different assays.

Human mitochondrial tRNAs in health and disease

AbstractThe human mitochondrial genome encodes 13 proteins, all subunits of the respiratory chain complexes and thus involved in energy metabolism. These genes are translated by 22 transfer RNAs (tRNAs), also encoded by the mitochondrial genome, which form the minimal set required for reading all codons. Human mitochondrial tRNAs gained interest with the rapid discovery of correlations between point mutations in their genes and various neuromuscular and neurodegenerative disorders. In this review, emerging fundamental knowledge on the structure/function relationships of these particular tRNAs and an overview of the large variety of mechanisms within translation, affected by mutations, are summarized. Also, initial results on wide-ranging molecular consequences of mutations outside the frame of mitochondrial translation are highlighted. While knowledge of mitochondrial tRNAs in both health and disease increases, deciphering the intricate network of events leading different genotypes to the variety of phenotypes requires further investigation using adapted model systems.

tRNA structure and aminoacylation efficiency.

Publisher Summary This chapter discusses the role of tRNA structure in the recognition process with synthetases and on the implications for aminoacylation efficiency. Many examples are taken from our own research on several specific aminoacylation systems, for example aspartate, histidine, valine, but concepts are presented more globally with reference to the complete set of aminoacylation systems. It emphasizes on the importance of tRNA-like structures for understanding the interaction of canonical tRNAs with synthetase. Although tRNA-like molecules found in some plant viral RNAs do not participate in protein synthesis, they represent interesting natural mutants to be compared to canonical tRNAs. This is also the case of tRNAlike structures found in some messenger RNAs as well as of bizarre tRNAs from mitochondria . In addition, competition and kinetic effects may also contribute to the overall specificity of the various aminoacylation systems; the balance between the concentration of tRNAs and synthetases would be essential for ensuring optimal specificity. According to this view, individual aminoacylation systems do not work at their optimal chemical efficiency, but work instead to assure optimal discrimination among the different aminoacylation systems. Such a balance may be perturbed under certain physiological or pathological conditions. Finally, this chapter discusses a comparison of recent results with previous observations, and show how old concepts established phenomenologically can now be tested more explicitly.

Identity elements for specific aminoacylation of yeast tRNA(Asp) by cognate aspartyl-tRNA synthetase.

The nucleotides crucial for the specific aminoacylation of yeast tRNA(Asp) by its cognate synthetase have been identified. Steady-state aminoacylation kinetics of unmodified tRNA transcripts indicate that G34, U35, C36, and G73 are important determinants of tRNA(Asp) identity. Mutations at these positions result in a large decrease (19- to 530-fold) of the kinetic specificity constant (ratio of the catalytic rate constant kcat and the Michaelis constant Km) for aspartylation relative to wild-type tRNA(Asp). Mutation to G10-C25 within the D-stem reduced kcat/Km eightfold. This fifth mutation probably indirectly affects the presentation of the highly conserved G10 nucleotide to the synthetase. A yeast tRNA(Phe) was converted into an efficient substrate for aspartyl-tRNA synthetase through introduction of the five identity elements. The identity nucleotides are located in regions of tight interaction between tRNA and synthetase as shown in the crystal structure of the complex and suggest sites of base-specific contacts.

Conformation in solution of yeast tRNAAsp transcripts deprived of modified nucleotides

A synthetic gene of yeast aspartic acid tRNA with a promoter for phage T7 RNA polymerase was cloned in Escherichia coli. The in vitro transcribed tRNA(Asp) molecules are deprived of modified nucleotides and retain their aspartylation capacity. The solution conformation of these molecules was mapped with chemical structural probes and compared to that of fully modified molecules. Significant differences in reactivities were observed in Pb2+ cleavage of the RNAs and in modification of the bases with dimethyl sulphate. The most striking result concerns C56, which becomes reactive in unmodified tRNA(Asp), indicating the disruption of the C56-G19 base pair involved in the D- and T-loop interaction. The chemical data indicate that unmodified tRNA(Asp) transcripts possess a relaxed conformation compared to that of the native tRNA. This conclusion is confirmed by thermal melting experiments. Thus it can be proposed that post-transcriptional modifications of nucleotides in tRNA stabilize the biologically active conformations in these molecules.

Improved systematic tRNA gene annotation allows new insights into the evolution of mitochondrial tRNA structures and into the mechanisms of mitochondrial genome rearrangements

Transfer RNAs (tRNAs) are present in all types of cells as well as in organelles. tRNAs of animal mitochondria show a low level of primary sequence conservation and exhibit ‘bizarre’ secondary structures, lacking complete domains of the common cloverleaf. Such sequences are hard to detect and hence frequently missed in computational analyses and mitochondrial genome annotation. Here, we introduce an automatic annotation procedure for mitochondrial tRNA genes in Metazoa based on sequence and structural information in manually curated covariance models. The method, applied to re-annotate 1876 available metazoan mitochondrial RefSeq genomes, allows to distinguish between remaining functional genes and degrading ‘pseudogenes’, even at early stages of divergence. The subsequent analysis of a comprehensive set of mitochondrial tRNA genes gives new insights into the evolution of structures of mitochondrial tRNA sequences as well as into the mechanisms of genome rearrangements. We find frequent losses of tRNA genes concentrated in basal Metazoa, frequent independent losses of individual parts of tRNA genes, particularly in Arthropoda, and wide-spread conserved overlaps of tRNAs in opposite reading direction. Direct evidence for several recent Tandem Duplication-Random Loss events is gained, demonstrating that this mechanism has an impact on the appearance of new mitochondrial gene orders.

Structure of transfer RNAs: similarity and variability

Transfer RNAs (tRNAs) are ancient molecules whose origin goes back to the beginning of life on Earth. Key partners in the ribosome‐translation machinery, tRNAs read genetic information on messenger RNA and deliver codon specified amino acids attached to their distal 3′‐extremity for peptide bond synthesis on the ribosome. In addition to this universal function, tRNAs participate in a wealth of other biological processes and undergo intricate maturation events. Our understanding of tRNA biology has been mainly phenomenological, but ongoing progress in structural biology is giving a robust physico‐chemical basis that explains many facets of tRNA functions. Advanced sequence analysis of tRNA genes and their RNA transcripts have uncovered rules that underly tRNA 2D folding and 3D L‐shaped architecture, as well as provided clues about their evolution. The increasing number of X‐ray structures of free, protein‐ and ribosome‐bound tRNA, reveal structural details accounting for the identity of the 22 tRNA families (one for each proteinogenic amino acid) and for the multifunctionality of a given family. Importantly, the structural role of post‐transcriptional tRNA modifications is being deciphered. On the other hand, the plasticity of tRNA structure during function has been illustrated using a variety of technical approaches that allow dynamical insights. The large range of structural properties not only allows tRNAs to be the key actors of translation, but also sustain a diversity of unrelated functions from which only a few have already been pinpointed. Many surprises can still be expected. WIREs RNA 2012, 3:37–61. doi: 10.1002/wrna.103

Towards understanding human mitochondrial leucine aminoacylation identity.

Specific recognition of tRNAs by aminoacyl-tRNA synthetases is governed by sets of aminoacylation identity elements, well defined for numerous prokaryotic systems and eukaryotic cytosolic systems. Only restricted information is available for aminoacylation of human mitochondrial tRNAs, despite their particularities linked to the non-classical structures of the tRNAs and their involvement in a growing number of human neurodegenerative disorders linked to mutations in the corresponding tRNA genes. A major difficulty to be overcome is the preparation of active in vitro transcripts enabling a rational mutagenic analysis, as is currently performed for classical tRNAs. Here, structural and aminoacylation properties of in vitro transcribed tRNA(Leu(UUR)) are presented. Solution probing using a combination of enzymatic and chemical tools revealed only partial folding into an L-shaped structure, with an acceptor branch but with a floppy anticodon branch. Optimization of aminoacylation conditions allowed charging of up to 75% of molecules, showing that, despite its partially relaxed structure, in vitro transcribed tRNA(Leu(UUR)) is able to adapt to the synthetase. In addition, mutational analysis demonstrates that the discriminator base as well as residue A14 are important leucine identity elements. Thus, human mitochondrial leucylation is dependent on rules similar to those that apply in Escherichia coli. The impact of a subset of pathology-related mutations on aminoacylation and on tRNA structure, has been explored. These variants do not show significant structural rearrangements and either do not affect aminoacylation (mutations T3250C, T3271C, C3303T) or lead to marked effects. Interestingly, two variants with a mutation at the same position (A3243G and A3243T) lead to markedly different losses in aminoacylation efficiencies (tenfold and 300-fold, respectively).

Novel features in the tRNA-like world of plant viral RNAs

Abstract. tRNA-like domains are found at the 3′ end of genomic RNAs of several genera of plant viral RNAs. Three groups of tRNA mimics have been characterized on the basis of their aminoacylation identity (valine, histidine and tyrosine) for aminoacyl-tRNA synthetases. Folding of these domains deviates from the canonical tRNA cloverleaf. The closest sequence similarities with tRNA are those found in valine accepting structures from tymoviruses (e.g. TYMV). All the viral tRNA mimics present a pseudoknotted amino acid accepting stem, which confers special structural and functional characteristics. In this review emphasis is given to newly discovered tRNA-like structures (e.g. in furoviruses) and to recent advances in the understanding of their three-dimensional architecture, which mimics L-shaped tRNA. Identity determinants in tRNA-like domains for aminoacylation are described, and evidence for their functional expression, as in tRNAs, is given. Properties of engineered tRNA-like domains are discussed, and other functional mimicries with tRNA are described (e.g. interaction with elongation factors and tRNA maturation enzymes). A final section reviews the biological role of the tRNA-like domains in amplification of viral genomes. In this process, in which the mechanisms can vary in specificity and efficiency according to the viral genus, function can be dependent on the aminoacylation properties of the tRNA-like domains and/or on structural properties within or outside these domains.