Zusammenspiel von posttranskriptionellen Modifikationen und Strukturen von humanen mitochondrialen tRNAs

Zusammenfassung Mitochondrien sind Organellen eukaryotischer Zellen, die für die Energieproduktion der Zellen verantwortlich sind, sowie über ein eigenes Genom verfügen. Das menschliche mitochondriale Genom kodiert 13 Proteine für die Untereinheiten der Atmungskettenkomplexe und 22 transfer RNAs (tRNAs). Mehr als 80 verschiedene Punktmutationen in den humanen mitochondrialen tRNA Genen werden mit Krankheiten wie Kardiopathie, Enzephalopathie und Myopathien assoziert. Die Bildung von alternativen sekundären und tertiären tRNA-Strukturen könnte die Pathogenität dieser Mutationen erklären. Um Informationen über die strukturelle Dynamik der humanen mitochondrialen tRNAs und über pathogene Mutanten (tRNALys-WT, -A8344G, tRNASer(UCN)-WT, -G7497A, -T7512C, tRNAGln-WT, -T4336C, tRNALeu(CUN)-WT, -A12320G, -G12315A) zu bekommen, wurde die Struktur von in vitro Transkripten mit folgenden Methoden genauer untersucht: chemische und enzymatische Kartierung, UV-Schmelzkurvenanalyse und elektrophoretische Mobilität auf nativen Gelen, sowie posttranskriptionelle Modifikation mit Enzymen aus humanen mitochondrialen Extrakten und aus Saccharomyces cerevisiae (scPus1p und scPus4p). Folgende Ergebnisse wurden dabei gewonnen: enzymatische und chemische Kartierungsdaten und UV-Schmelzkurvenanalyse zeigen, dass die A12320G und G12315A Mutanten der tRNALeu(CUN) eine ungewöhnliche Struktur besitzen, die sich vom Wildtyp deutlich unterscheidet. In humaner mitochondrialer tRNASer(UCN) führt die G7497A Mutation zu einer stark kompaktierten Struktur, was durch temperaturabhängige Strukturanalyse gezeigt wurde. Das durch diese Mutation eingeführte G•U Wobble-Paar im D-Stem hat einen negativen Einfluss auf die Erkennung sowohl durch humane mitochondriale Enzyme, als auch durch Pus4 und Pus1 aus S.cerevisiae. Die strukturelle Dynamik von nicht-modifizierten und teilmodifizierten Transkripten, die mit UV-Schmelzkurven analysiert wurden, zeigt, dass posttranskriptionelle Modifikationen eine wichtige Rolle in der strukturellen und wahrscheinlich auch in der metabolischen Stabilität dieser tRNA spielen. Die Studien der pathogenen G7497A Punktmutation in Osteosarcoma Zellen zeigen, dass untermodifizierte tRNAs instabil sind und in der Zelle schnell abgebaut werden. Es wurde eine Reduktion zur Verfügung stehender funktioneller tRNAs in Mitochondrien auf 10 % gefunden, was zu einer Verminderung der Proteinsynthese um 40 % führt [Mollers et al., 2005]. Insgesamt konnte in der vorliegenden Arbeit gezeigt werden, dass die meisten pathogenen Mutationen im Kernbereich der tRNA deren Struktur signifikant beeinflussen. Mit Ausnahme einer Mutation T4336C in tRNAGln, welche die Aktivität von Pseudouridin-Synthase im Vergleich zu dem Wildtyp erhöhte, zeigten die meisten pathogenen Mutationen eine Verringerung der Aktivität von den Modifikationsenzymen

Contemporary community composition, spatial distribution patterns, and biodiversity characteristics of zooplankton in large alpine Lake Sevan, Armenia

We studied the quantitative composition, spatial distribution, and temporal dynamics of the zooplankton community of the alpine Lake Sevan, Armenia, the largest surface water in the Caucasus region. This article is providing a long-term information and fills the research gap of multiyear data on zooplankton, as the previous research on zooplankton provided only snapshots of the community, and a consistent assessment over multiple years was missing. However, an initial mini-review of historical studies indicated that zooplankton biomass and fish abundance were undergoing large fluctuations, indicating the importance of top-down control. We analysed 239 samples from the period 2016-2019 from 32 sampling sites in Lake Sevan and recorded 37 species of meso- and macrozooplankton (Rotifers, Copepods, Cladocera). Biomass fluctuations were high with peaking biomasses in 2016 and lowest biomasses in 2018, yearly averaged biomass varied about one order of magnitude. Variability over time was hence much higher than spatial variability. The pelagic habitat at the deepest part of the lake showed the highest diversity and biomasses but contrasts between sampling sites remained smaller than changes from year to year or seasonally. Many samples were dominated by a single species, and these key species explain observed biomass dynamics to a wide extent. We applied hierarchical clustering in order to identify phenological groups that appear to show similar patterns of occurrence. This clustering resulted in 6 groups where of 5 groups just consisting of one single species and these 5 key species were the Cladocerans Daphnia magna, Daphnia hyalina, Diaphanosoma sp. as well as the calanoids Arctodiaptomus bacillifer and Acanthodiaptomus denticornis. The most important species in Lake Sevan’s zooplankton during the observation period was D. magna, which reached high biomasses in 2016 and 2017 but then suddenly almost disappeared in 2018 and 2019. When there were more D. magna present, the water became clearer, which was measured using Secchi depth. This shows that these large water fleas effectively controlled the amount of phytoplankton in the water. Daphnia magna, in turn, managed to dominate zooplankton community only during times of extremely low fish biomass indicating strong top-down control of this large Cladoceran by fish. Both observations together imply a strong trophic linkage between fish, zooplankton, and phytoplankton and provide evidence for trophic cascades in Lake Sevan. Besides the novel insights into zooplankton community dynamics of this unique lake of high socio-economical, cultural, and ecological importance, our study also points to potential management opportunities for eutrophication control by biomanipulation, as well as our investigation allows us to conclude that probably biotic factors were more important than abiotic factors in explaining the observed changes and dynamics within the plankton community

A new mechanism for mtDNA pathogenesis: impairment of post-transcriptional maturation leads to severe depletion of mitochondrial tRNA(Ser(UCN)) caused by T7512C and G7497A point mutations

We have studied the consequences of two homoplasmic, pathogenic point mutations (T7512C and G7497A) in the tRNA(Ser(UCN)) gene of mitochondrial (mt) DNA using osteosarcoma cybrids. We identified a severe reduction of tRNA(Ser(UCN)) to levels below 10% of controls for both mutations, resulting in a 40% reduction in mitochondrial protein synthesis rate and in a respiratory chain deficiency resembling that in the patients muscle. Aminoacylation was apparently unaffected. On non-denaturating northern blots we detected an altered electrophoretic mobility for G7497A containing tRNA molecules suggesting a structural impact of this mutation, which was confirmed by structural probing. By comparing in vitro transcribed molecules with native RNA in such gels, we also identified tRNA(Ser(UCN)) being present in two isoforms in vivo, probably corresponding to the nascent, unmodified transcripts co-migrating with the in vitro transcripts and a second, faster moving isoform corresponding to the mature tRNA. In cybrids containing either mutations the unmodified isoforms were severely reduced. We hypothesize that both mutations lead to an impairment of post-transcriptional modification processes, ultimately leading to a preponderance of degradation by nucleases over maturation by modifying enzymes, resulting in severely reduced tRNA(Ser(UCN)) steady state levels. We infer that an increased degradation rate, caused by disturbance of tRNA maturation and, in the case of the G7497A mutant, alteration of tRNA structure, is a new pathogenic mechanism of mt tRNA point mutations

Molecular dysfunction associated with the human mitochondrial 3302A>G mutation in the MTTL1 (mt-tRNA(Leu(UUR))) gene

The gene encoding mt-tRNA(Leu(UUR)), MT-TL1, is a hotspot for pathogenic mtDNA mutations. Amongst the first to be described was the 3302A>G transition which resulted in a substantial accumulation in patient muscle of RNA19, an unprocessed RNA intermediate including mt-16S rRNA, mt-tRNA(Leu(UUR)) and MTND1. We have now been able to further assess the molecular aetiology associated with 3302A>G in transmitochondrial cybrids. Increased steady-state levels of RNA19 was confirmed, although not to the levels previously reported in muscle. This data was consistent with an increase in RNA19 stability. The mutation resulted in decreased mt-tRNA(Leu(UUR)) levels, but its stability was unchanged, consistent with a defect in RNA19 processing responsible for low tRNA levels. A partial defect in aminoacylation was also identified, potentially caused by an alteration in tRNA structure. These deficiencies lead to a severe defect in respiration in the transmitochondrial cybrids, consistent with the profound mitochondrial disorder originally associated with this mutation

Effect of a quaternary pentamine on RNA stabilization and enzymatic methylation.

International audienceAbstract Extreme thermophiles produce unusually long polyamines, including the linear caldopentamine (Cdp) and the branched pentamine tetrakis(3-aminopropyl)-ammonium (Taa), the latter containing a central quaternary ammonium moiety. Here, we compare the interaction of these two pentamines with RNA by studying the heat denaturation, the behavior in electrophoresis, and the ability of tRNA to be methylated in vitro by purified tRNA methyltransferases under various salt conditions. At concentrations in the micromolar range, the branched Taa causes a considerable increase of the T(m) of yeast tRNA(Phe) transcript by over 20 degrees C, significantly higher than stabilization by the linear counterpart Cdp. In non-denaturing gel electrophoresis, a strong and specific binding of branched Taa, but not of linear Cdp, to tRNA(Phe) was clearly evident. In both types of experiments, polyamines and monovalent metal ions compete with each other for binding sites. Structural probing showed no significant conformational changes of the tRNA upon Taa binding. In post-transcriptional in vitro methylation reactions, formation of m(2)G/m(2)(2)G by the methyltransferase Trm1p and of m(1)A by TrmIp were unaffected or slightly stimulated by polyamines. In contrast, Taa specifically inhibited Trm4p-dependent formation of m(5)C only in tRNA(Phe), likely by occupying sites that are relevant to RNA recognition by the methyltransferase