Regulation and function of R-loops at repetitive elements.

R-loops are atypical, three-stranded nucleic acid structures that contain a stretch of RNA:DNA hybrids and an unpaired, single stranded DNA loop. R-loops are physiological relevant and can act as regulators of gene expression, chromatin structure, DNA damage repair and DNA replication. However, unscheduled and persistent R-loops are mutagenic and can mediate replication-transcription conflicts, leading to DNA damage and genome instability if left unchecked. Detailed transcriptome analysis unveiled that 85% of the human genome, including repetitive regions, hold transcriptional activity. This anticipates that R-loops management plays a central role for the regulation and integrity of genomes. This function is expected to have a particular relevance for repetitive sequences that make up to 75% of the human genome. Here, we review the impact of R-loops on the function and stability of repetitive regions such as centromeres, telomeres, rDNA arrays, transposable elements and triplet repeat expansions and discuss their relevance for associated pathological conditions.

Stabilization of 14-3-3 protein-protein interactions with Fusicoccin-A decreases alpha-synuclein dependent cell-autonomous death in neuronal and mouse models.

Synucleinopathies are a group of neurodegenerative diseases without effective treatment characterized by the abnormal aggregation of alpha-synuclein (aSyn) protein. Changes in levels or in the amino acid sequence of aSyn (by duplication/triplication of the aSyn gene or point mutations in the encoding region) cause familial cases of synucleinopathies. However, the specific molecular mechanisms of aSyn-dependent toxicity remain unclear. Increased aSyn protein levels or pathological mutations may favor abnormal protein-protein interactions (PPIs) that could either promote neuronal death or belong to a coping response program against neurotoxicity. Therefore, the identification and modulation of aSyn-dependent PPIs can provide new therapeutic targets for these diseases. To identify aSyn-dependent PPIs we performed a proximity biotinylation assay based on the promiscuous biotinylase BioID2. When expressed as a fusion protein, BioID2 biotinylates by proximity stable and transient interacting partners, allowing their identification by streptavidin affinity purification and mass spectrometry. The aSyn interactome was analyzed using BioID2-tagged wild-type (WT) and pathological mutant E46K aSyn versions in HEK293 cells. We found the 14-3-3 epsilon isoform as a common protein interactor for WT and E46K aSyn. 14-3-3 epsilon correlates with aSyn protein levels in brain regions of a transgenic mouse model overexpressing WT human aSyn. Using a neuronal model in which aSyn cell-autonomous toxicity is quantitatively scored by longitudinal survival analysis, we found that stabilization of 14-3-3 protein-proteins interactions with Fusicoccin-A (FC-A) decreases aSyn-dependent toxicity. Furthermore, FC-A treatment protects dopaminergic neuronal somas in the substantia nigra of a Parkinson's disease mouse model. Based on these results, we propose that the stabilization of 14-3-3 epsilon interaction with aSyn might reduce aSyn toxicity, and highlight FC-A as a potential therapeutic compound for synucleinopathies.