Modulatory role of RNA helicases in MBNL-dependent alternative splicing regulation.

Muscleblind-like splicing regulators (MBNLs) activate or repress the inclusion of alternative splicing (AS) events, enabling the developmental transition of fetal mRNA splicing isoforms to their adult forms. Herein, we sought to elaborate the mechanism by which MBNLs mediate AS related to biological processes. We evaluated the functional role of DEAD-box (DDX) RNA helicases, DDX5 and DDX17 in MBNL-dependent AS regulation. Whole-transcriptome analysis and validation approaches revealed a handful of MBNLs-dependent AS events to be affected by DDX5 and DDX17 in mostly an opposite manner. The opposite expression patterns of these two groups of factors during muscle development and coordination of fetal-to-adult splicing transition indicate the importance of these proteins at early stages of development. The identified pathways of how the helicases modulate MBNL splicing activity include DDX5 and DDX17-dependent changes in the ratio of MBNL splicing isoforms and most likely changes in accessibility of MBNL-binding sites. Another pathway involves the mode of action of the helicases independent of MBNL activity. These findings lead to a deeper understanding of the network of interdependencies between RNA-binding proteins and constitute a valuable element in the discussion on developmental homeostasis and pathological states in which the studied protein factors play a significant role.

Empirical testing of cryoconite granulation: Role of cyanobacteria in the formation of key biogenic structure darkening glaciers in polar regions.

Cryoconite, the dark sediment on the surface of glaciers, often aggregates into oval or irregular granules serving as biogeochemical factories. They reduce a glacier's albedo, act as biodiversity hotspots by supporting aerobic and anaerobic microbial communities, constitute one of the organic matter (OM) sources on glaciers, and are a feeder for micrometazoans. Although cryoconite granules have multiple roles on glaciers, their formation is poorly understood. Cyanobacteria are ubiquitous and abundant engineers of cryoconite hole ecosystems. This study tested whether cyanobacteria may be responsible for cryoconite granulation as a sole biotic element. Incubation of Greenlandic, Svalbard, and Scandinavian cyanobacteria in different nutrient availabilities and substrata for growth (distilled water alone and water with quartz powder, furnaced cryoconite without OM, or powdered rocks from glacial catchment) revealed that cyanobacteria bind mineral particles into granules. The structures formed in the experiment resembled those commonly observed in natural cryoconite holes: they contained numerous cyanobacterial filaments protruding from aggregated mineral particles. Moreover, all examined strains were confirmed to produce extracellular polymeric substances (EPS), which suggests that cryoconite granulation is most likely due to EPS secretion by gliding cyanobacteria. In the presence of water as the only substrate for growth, cyanobacteria formed mostly carpet-like mats. Our data empirically prove that EPS-producing oscillatorialean cyanobacteria isolated from the diverse community of cryoconite microorganisms can form granules from mineral substrate and that the presence of the mineral substrate increases the probability of the formation of these important and complex biogeochemical microstructures on glaciers.

Cyclic mismatch binding ligands interact with disease-associated CGG trinucleotide repeats in RNA and suppress their translation.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by a limited expansion of CGG repeats in the FMR1 gene. Degeneration of neurons in FXTAS cell models can be triggered by accumulation of polyglycine protein (FMRpolyG), a by-product of translation initiated upstream to the repeats. Specific aims of our work included testing if naphthyridine-based molecules could (i) block FMRpolyG synthesis by binding to CGG repeats in RNA, (ii) reverse pathological alterations in affected cells and (iii) preserve the content of FMRP, translated from the same FMR1 mRNA. We demonstrate that cyclic mismatch binding ligand CMBL4c binds to RNA structure formed by CGG repeats and attenuates translation of FMRpolyG and formation of nuclear inclusions in cells transfected with vectors expressing RNA with expanded CGG repeats. Moreover, our results indicate that CMBL4c delivery can reduce FMRpolyG-mediated cytotoxicity and apoptosis. Importantly, its therapeutic potential is also observed once the inclusions are already formed. We also show that CMBL4c-driven FMRpolyG loss is accompanied by partial FMRP reduction. As complete loss of FMRP induces FXS in children, future experiments should aim at evaluation of CMBL4c therapeutic intervention in differentiated tissues, in which FMRpolyG translation inhibition might outweigh adverse effects related to FMRP depletion.

Short antisense oligonucleotides alleviate the pleiotropic toxicity of RNA harboring expanded CGG repeats.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an incurable neurodegenerative disorder caused by expansion of CGG repeats in the FMR1 5'UTR. The RNA containing expanded CGG repeats (rCGGexp) causes cell damage by interaction with complementary DNA, forming R-loop structures, sequestration of nuclear proteins involved in RNA metabolism and initiation of translation of polyglycine-containing protein (FMRpolyG), which forms nuclear insoluble inclusions. Here we show the therapeutic potential of short antisense oligonucleotide steric blockers (ASOs) targeting directly the rCGGexp. In nuclei of FXTAS cells ASOs affect R-loop formation and correct miRNA biogenesis and alternative splicing, indicating that nuclear proteins are released from toxic sequestration. In cytoplasm, ASOs significantly decrease the biosynthesis and accumulation of FMRpolyG. Delivery of ASO into a brain of FXTAS mouse model reduces formation of inclusions, improves motor behavior and corrects gene expression profile with marginal signs of toxicity after a few weeks from a treatment.

MEF2C shapes the microtranscriptome during differentiation of skeletal muscles.

Myocyte enhancer factor 2C (MEF2C) is a transcription factor that regulates heart and skeletal muscle differentiation and growth. Several protein-encoding genes were identified as targets of this factor; however, little is known about its contribution to the microtranscriptome composition and dynamics in myogenic programs. In this report, we aimed to address this question. Deep sequencing of small RNAs of human muscle cells revealed a set of microRNAs (miRNAs), including several muscle-specific miRNAs, that are sensitive to MEF2C depletion. As expected, in cells with knockdown of MEF2C, we found mostly downregulated miRNAs; nevertheless, as much as one-third of altered miRNAs were upregulated. The majority of these changes are driven by transcription efficiency. Moreover, we found that MEF2C affects nontemplated 3'-end nucleotide addition of miRNAs, mainly oligouridylation. The rate of these modifications is associated with the level of TUT4 which mediates RNA 3'-uridylation. Finally, we found that a quarter of miRNAs which significantly changed upon differentiation of human skeletal myoblasts is inversely altered in MEF2C deficient cells. We concluded that MEF2C is an essential factor regulating both the quantity and quality of the microtranscriptome, leaving an imprint on the stability and perhaps specificity of many miRNAs during the differentiation of muscle cells.

Global Increase in Circular RNA Levels in Myotonic Dystrophy.

Splicing aberrations induced as a consequence of the sequestration of muscleblind-like splicing factors on the dystrophia myotonica protein kinase transcript, which contains expanded CUG repeats, present a major pathomechanism of myotonic dystrophy type 1 (DM1). As muscleblind-like factors may also be important factors involved in the biogenesis of circular RNAs (circRNAs), we hypothesized that the level of circRNAs would be decreased in DM1. To test this hypothesis, we selected 20 well-validated circRNAs and analyzed their levels in several experimental systems (e.g., cell lines, DM muscle tissues, and a mouse model of DM1) using droplet digital PCR assays. We also explored the global level of circRNAs using two RNA-Seq datasets of DM1 muscle samples. Contrary to our original hypothesis, our results consistently showed a global increase in circRNA levels in DM1, and we identified numerous circRNAs that were increased in DM1. We also identified many genes (including muscle-specific genes) giving rise to numerous (>10) circRNAs. Thus, this study is the first to show an increase in global circRNA levels in DM1. We also provided preliminary results showing the association of circRNA level with muscle weakness and alternative splicing changes that are biomarkers of DM1 severity.