Alternative mRNA splicing events and regulators in epidermal differentiation.

Alternative splicing (AS) of messenger RNAs occurs in ∼95% of multi-exon human genes and generates diverse RNA and protein isoforms. We investigated AS events associated with human epidermal differentiation, a process crucial for skin function. We identified 6,413 AS events, primarily involving cassette exons.

Knockdown Results in Reduction of mtDNA Copy Number, OXPHOS Deficiency and Abnormalities in Zebrafish Embryos.

High mitochondrial DNA (mtDNA) copy numbers are essential for oogenesis and embryogenesis and correlate with fertility of oocytes and viability of embryos. To understand the pathology and mechanisms associated with low mtDNA copy numbers, we knocked down mitochondrial transcription factor A (), a regulator of mtDNA replication, during early zebrafish development. Reduction of using a splice-modifying morpholino (MO) resulted in a 42 ± 17% decrease in mtDNA copy number in embryos at 4 days post fertilization.

Research Techniques Made Simple: CRISPR Genetic Screens.

CRISPR and Cas proteins, often referred to as CRISPR/Cas, are the components of a bacterial genome editing system that can be used to perturb genes in cells and tissues. A classic application is to use CRISPR/Cas to generate genetic loss-of-function. When performed at large scale and combined with deep sequencing techniques, CRISPR-based perturbations can be performed in a high throughput setting to screen many candidate genomic elements for their roles in a phenotype of interest.

A genome-wide long noncoding RNA CRISPRi screen identifies as a novel regulator of epidermal homeostasis.

Genome-wide association studies indicate that many disease susceptibility regions reside in non-protein-coding regions of the genome. Long noncoding RNAs (lncRNAs) are a major component of the noncoding genome, but their biological impacts are not fully understood. Here, we performed a CRISPR interference (CRISPRi) screen on 2263 epidermis-expressed lncRNAs and identified nine novel candidate lncRNAs regulating keratinocyte proliferation.

Expression of pathogenic SCN9A mutations in the zebrafish: A model to study small-fiber neuropathy.

Small-fiber neuropathy (SFN) patients experience a spectrum of sensory abnormalities, including attenuated responses to non-noxious temperatures in combination with a decreased density of the small-nerve fibers. Gain-of-function mutations in the voltage-gated sodium channels SCN9A, SCN10A and SCN11A have been identified as an underlying genetic cause in a subpopulation of patients with SFN. Based on clinical-diagnostic tests for SFN, we have set up a panel of two read-outs reflecting SFN in zebrafish, being nerve density and behavioral responses.

Replication Errors Made During Oogenesis Lead to Detectable De Novo mtDNA Mutations in Zebrafish Oocytes with a Low mtDNA Copy Number.

Of all pathogenic mitochondrial DNA (mtDNA) mutations in humans, ∼25% is de novo, although the occurrence in oocytes has never been directly assessed. We used next-generation sequencing to detect point mutations directly in the mtDNA of 3-15 individual mature oocytes and three somatic tissues from eight zebrafish females. Various statistical and biological filters allowed reliable detection of de novo variants with heteroplasmy ≥1.

Differences in Strength and Timing of the mtDNA Bottleneck between Zebrafish Germline and Non-germline Cells.

We studied the mtDNA bottleneck in zebrafish to elucidate size, timing, and variation in germline and non-germline cells. Mature zebrafish oocytes contain, on average, 19.0 × 10(6) mtDNA molecules with high variation between oocytes.

Evolutionary defined role of the mitochondrial DNA in fertility, disease and ageing.

Background: The endosymbiosis of an alpha-proteobacterium and a eubacterium a billion years ago paved the way for multicellularity and enabled eukaryotes to flourish. The selective advantage for the host was the acquired ability to generate large amounts of intracellular hydrogen-dependent adenosine triphosphate. The price was increased reactive oxygen species (ROS) inside the eukaryotic cell, causing high mutation rates of the mitochondrial DNA (mtDNA).