N6-Methyladenine Progressively Accumulates in Mitochondrial DNA during Aging.

N6-methyladenine (6mA) in the DNA is a conserved epigenetic mark with various cellular, physiological and developmental functions. Although the presence of 6mA was discovered a few years ago in the nuclear genome of distantly related animal taxa and just recently in mammalian mitochondrial DNA (mtDNA), accumulating evidence at present seriously questions the presence of N6-adenine methylation in these genetic systems, attributing it to methodological errors. In this paper, we present a reliable, PCR-based method to determine accurately the relative 6mA levels in the mtDNA of Caenorhabditis elegans, Drosophila melanogaster and dogs, and show that these levels gradually increase with age. Furthermore, daf-2(-)-mutant worms, which are defective for insulin/IGF-1 (insulin-like growth factor) signaling and live twice as long as the wild type, display a half rate at which 6mA progressively accumulates in the mtDNA as compared to normal values. Together, these results suggest a fundamental role for mtDNA N6-adenine methylation in aging and reveal an efficient diagnostic technique to determine age using DNA.

Downregulation of transposable elements extends lifespan in Caenorhabditis elegans.

Mobility of transposable elements (TEs) frequently leads to insertional mutations in functional DNA regions. In the potentially immortal germline, TEs are effectively suppressed by the Piwi-piRNA pathway. However, in the genomes of ageing somatic cells lacking the effects of the pathway, TEs become increasingly mobile during the adult lifespan, and their activity is associated with genomic instability. Whether the progressively increasing mobilization of TEs is a cause or a consequence of ageing remains a fundamental problem in biology. Here we show that in the nematode Caenorhabditis elegans, the downregulation of active TE families extends lifespan. Ectopic activation of Piwi proteins in the soma also promotes longevity. Furthermore, DNA N6-adenine methylation at TE stretches gradually rises with age, and this epigenetic modification elevates their transcription as the animal ages. These results indicate that TEs represent a novel genetic determinant of ageing, and that N6-adenine methylation plays a pivotal role in ageing control.

How does maternal age affect genomic stability in the offspring?

In high-income countries, women tend to give birth at increasingly advanced ages. Despite its physiological, developmental, and medical consequences, why this tendency significantly affects genetic stability of the offspring remains largely unresolved. Accumulating evidence indicates that the higher the age of the mother at fertilization, the more intense the activity of transposable elements causing insertional mutations in functional DNA stretches in her oocyte involved in zygote formation.

Highly efficient RNAi and Cas9-based auto-cloning systems for C. elegans research.

RNA interference (RNAi) technology used for the functional analysis of Caenorhabditis elegans genes frequently leads to phenotypes with low penetrance or even proves completely ineffective. The methods previously developed to solve this problem were built on mutant genetic backgrounds, such as those defective for rrf-3, in which endogenous RNAi pathways are overexpressed. These mutations, however, interferes with many other genetic pathways so that the detected phenotype cannot always be clearly linked to the RNAi-exposed gene. In addition, using RNAi-overexpressing mutant backgrounds requires time-consuming genetic crossing. Here, we present an improved RNAi vector that produces specific double-stranded RNA species only, and thereby significantly stronger phenotypes than the standard gene knockdown vector. The further advantage of the new RNAi vector is that the detected phenotype can be specifically linked to the gene silenced. We also created a new all-in-one C. elegans Cas9 vector whose spacer sequence is much easier to replace. Both new vectors include a novel CRISPR/Cas9-based auto-cloning vector system rendering needless the use of restriction and ligase enzymes in generating DNA constructs. This novel, efficient RNAi and auto-cloning Cas9 systems can be easily adapted to any other genetic model.

Prediction and characterization of human ageing-related proteins by using machine learning.

Ageing has a huge impact on human health and economy, but its molecular basis - regulation and mechanism - is still poorly understood. By today, more than three hundred genes (almost all of them function as protein-coding genes) have been related to human ageing. Although individual ageing-related genes or some small subsets of these genes have been intensively studied, their analysis as a whole has been highly limited. To fill this gap, for each human protein we extracted 21000 protein features from various databases, and using these data as an input to state-of-the-art machine learning methods, we classified human proteins as ageing-related or non-ageing-related. We found a simple classification model based on only 36 protein features, such as the "number of ageing-related interaction partners", "response to oxidative stress", "damaged DNA binding", "rhythmic process" and "extracellular region". Predicted values of the model quantify the relevance of a given protein in the regulation or mechanisms of the human ageing process. Furthermore, we identified new candidate proteins having strong computational evidence of their important role in ageing. Some of them, like Cytochrome b-245 light chain (CY24A) and Endoribonuclease ZC3H12A (ZC12A) have no previous ageing-associated annotations.

A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a 'self-cleaving' GFP-expression plasmid.

The efficacies of guide RNAs (gRNAs), the short RNA molecules that bind to and determine the sequence specificity of the Streptococcus pyogenes Cas9 nuclease, to mediate DNA cleavage vary dramatically. Thus, the selection of appropriate target sites, and hence spacer sequence, is critical for most applications. Here, we describe a simple, unparalleled method for experimentally pre-testing the efficiencies of various gRNAs targeting a gene. The method explores NHEJ-cloning, genomic integration of a GFP-expressing plasmid without homologous arms and linearized in-cell. The use of 'self-cleaving' GFP-plasmids containing universal gRNAs and corresponding targets alleviates cloning burdens when this method is applied. These universal gRNAs mediate efficient plasmid cleavage and are designed to avoid genomic targets in several model species. The method combines the advantages of the straightforward FACS detection provided by applying fluorescent reporter systems and of the PCR-based approaches being capable of testing targets in their genomic context, without necessitating any extra cloning steps. Additionally, we show that NHEJ-cloning can also be used in mammalian cells for targeted integration of donor plasmids up to 10 kb in size, with up to 30% efficiency, without any selection or enrichment.

The Piwi-piRNA pathway: road to immortality.

Despite its medical, social, and economic significance, understanding what primarily causes aging, that is, the mechanisms of the aging process, remains a fundamental and fascinating problem in biology. Accumulating evidence indicates that a small RNA-based gene regulatory machinery, the Piwi-piRNA pathway, represents a shared feature of nonaging (potentially immortal) biological systems, including the germline, somatic cancer stem cells, and certain 'lower' eukaryotic organisms like the planarian flatworm and freshwater hydra. The pathway primarily functions to repress the activity of mobile genetic elements, also called transposable elements (TEs) or 'jumping genes', which are capable of moving from one genomic locus to another, thereby causing insertional mutations. TEs become increasingly active and multiply in the genomes of somatic cells as the organism ages. These characteristics of TEs highlight their decisive mutagenic role in the progressive disintegration of genetic information, a molecular hallmark associated with aging. Hence, TE-mediated genomic instability may substantially contribute to the aging process.

The mechanism of ageing: primary role of transposable elements in genome disintegration.

Understanding the molecular basis of ageing remains a fundamental problem in biology. In multicellular organisms, while the soma undergoes a progressive deterioration over the lifespan, the germ line is essentially immortal as it interconnects the subsequent generations. Genomic instability in somatic cells increases with age, and accumulating evidence indicates that the disintegration of somatic genomes is accompanied by the mobilisation of transposable elements (TEs) that, when mobilised, can be mutagenic by disrupting coding or regulatory sequences. In contrast, TEs are effectively silenced in the germ line by the Piwi-piRNA system. Here, we propose that TE repression transmits the persistent proliferation capacity and the non-ageing phenotype (e.g., preservation of genomic integrity) of the germ line. The Piwi-piRNA pathway also operates in tumorous cells and in somatic cells of certain organisms, including hydras, which likewise exhibit immortality. However, in somatic cells lacking the Piwi-piRNA pathway, gradual chromatin decondensation increasingly allows the mobilisation of TEs as the organism ages. This can explain why the mortality rate rises exponentially throughout the adult life in most animal species, including humans.