Exon-intron chain reconstruction of circular RNA using RNA-Seq

Circular RNAs (circRNAs) are a class of RNA forming a covalently closed loop through back-splicing. A few well studied circRNAs indicate miRNA sponging and regulation of host gene transcription. CircRNAs can be identified in rRNA depleted RNA-Seq by detecting reads, which span a back-splice junction. CircRNA detection tools exist but none are able to summarize or characterize circRNAs. To perform accurate downstream analyses it is crucial to know the exact exon-intron structure of circRNAs. Here, I am presenting FUCHS and FUCHSdenovo to summarize circRNAs and reconstruct their exon-intron chain. In short: I developed a pipeline called FUCHS : FUll CHaracterization of circular RNA using RNA-Sequencing; that summarizes circRNAs, detects skipped exons, finds double-breakpoint fragments, generates coverage profiles, and clusters these profiles. I developed an additional module, FUCHSdenovo, to reconstruct the exon-intron structure based on linear-splice signals of back-spliced reads. I ran both programs on a dataset of young and old murine hearts and young and old murine livers. FUCHS revealed that heart circRNAs are less diverse but more abundant than liver circRNAs. From the obtained coverage profiles, I concluded that annotated gene models were not all matching the exon-intron structure of circRNAs. A de-novo reconstruction of circle structures using FUCHSdenovo showed a gain of information of 15%. Furthermore, FUCHSdenovo identified alternative splicing (AS) in 10% of circRNAs. Performing a differential motif enrichment analysis of the flanking introns of circRNAs with AS over circRNAs without AS identified FOXO as a potential transcription factor driving AS in circRNAs. Comparing the seed density of circRNAs and mRNAs showed that circRNAs were more densely populated with both. This suggests that circRNAs could form an additional layer in the gene-regulatory network by competing with their host genes for miRNA or RBP binding. https://github.com/dieterich-lab/FUCHS.gi

Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage

Summary Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.Peer reviewe

Corresponding authors Distributed under Creative Commons CC-BY 4.0 FUCHS-towards full circular RNA characterization using RNAseq

ABSTRACT Circular RNAs (circRNAs) belong to a recently re-discovered species of RNA that emerge during RNA maturation through a process called back-splicing. A downstream 5 splice site is linked to an upstream 3 splice site to form a circular transcript instead of a canonical linear transcript. Recent advances in next-generation sequencing (NGS) have brought circRNAs back into the focus of many scientists. Since then, several studies reported that circRNAs are differentially expressed across tissue types and developmental stages, implying that they are actively regulated and not merely a byproduct of splicing. Though functional studies have shown that some circRNAs could act as miRNA-sponges, the function of most circRNAs remains unknown. To expand our understanding of possible roles of circular RNAs, we propose a new pipeline that could fully characterizes candidate circRNA structure from RNAseq data-FUCHS: FUll CHaracterization of circular RNA using RNA-Sequencing. Currently, most computational prediction pipelines use back-spliced reads to identify circular RNAs. FUCHS extends this concept by considering all RNA-seq information from long reads (typically >150 bp) to learn more about the exon coverage, the number of double break point fragments, the different circular isoforms arising from one host-gene, and the alternatively spliced exons within the same circRNA boundaries. This new knowledge will enable the user to carry out differential motif enrichment and miRNA seed analysis to determine potential regulators during circRNA biogenesis. FUCHS is an easy-to-use Python based pipeline that contributes a new aspect to the circRNA research

A microRNA-129-5p/Rbfox crosstalk coordinates homeostatic downscaling of excitatory synapses

Synaptic downscaling is a homeostatic mechanism that allows neurons to reduce firing rates during chronically elevated network activity. Although synaptic downscaling is important in neural circuit development and epilepsy, the underlying mechanisms are poorly described. We performed small RNA profiling in picrotoxin (PTX)-treated hippocampal neurons, a model of synaptic downscaling. Thereby, we identified eight microRNAs (miRNAs) that were increased in response to PTX, including miR-129-5p, whose inhibition blocked synaptic downscaling in vitro and reduced epileptic seizure severity in vivo. Using transcriptome, proteome, and bioinformatic analysis, we identified the calcium pump Atp2b4 and doublecortin (Dcx) as miR-129-5p targets. Restoring Atp2b4 and Dcx expression was sufficient to prevent synaptic downscaling in PTX-treated neurons. Furthermore, we characterized a functional crosstalk between miR-129-5p and the RNA-binding protein (RBP) Rbfox1. In the absence of PTX, Rbfox1 promoted the expression of Atp2b4 and Dcx. Upon PTX treatment, Rbfox1 expression was downregulated by miR-129-5p, thereby allowing the repression of Atp2b4 and Dcx. We therefore identified a novel activitydependent miRNA/RBP crosstalk during synaptic scaling, with potential implications for neural network homeostasis and epileptogenesis