3 research outputs found

    LET-Dependent Low Dose and Synergistic Inhibition of Human Angiogenesis by Charged Particles: Validation of miRNAs that Drive Inhibition

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    Space radiation inhibits angiogenesis by two mechanisms depending on the linear energy transfer (LET). Using human 3D micro-vessel models, blockage of the early motile stage of angiogenesis was determined to occur after exposure to low LET ions (/AMU), whereas inhibition of the later stages occurs after exposure to high LET ions (\u3e8 KeV/AMU). Strikingly, the combined effect is synergistic, detectible as low as 0.06 Gy making mixed ion space radiation more potent. Candidates for bystander transmission are microRNAs (miRNAs), and analysis on miRNA-seq data from irradiated mice shows that angiogenesis would in theory be downregulated. Further analysis of three previously identified miRNAs showed downregulation of their targets associated with angiogenesis and confirmed their involvement in angiogenesis pathways and increased health risks associated with cardiovascular disease. Finally, synthetic molecules (antagomirs) designed to inhibit the predicted miRNAs were successfully used to reverse the inhibition of angiogenesis

    Cooperative-Binding and Splicing-Repressive Properties of hnRNP A1▿ †

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    hnRNP A1 binds to RNA in a cooperative manner. Initial hnRNP A1 binding to an exonic splicing silencer at the 3′ end of human immunodeficiency virus type 1 (HIV-1) tat exon 3, which is a high-affinity site, is followed by cooperative spreading in a 3′-to-5′ direction. As hnRNP A1 propagates toward the 5′ end of the exon, it antagonizes binding of a serine/arginine-rich (SR) protein to an exonic splicing enhancer, thereby inhibiting splicing at that exon's alternative 3′ splice site. tat exon 3 and the preceding intron of HIV-1 pre-mRNA can fold into an elaborate RNA secondary structure in solution, which could potentially influence hnRNP A1 binding. We report here that hnRNP A1 binding and splicing repression can occur on an unstructured RNA. Moreover, hnRNP A1 can effectively unwind an RNA hairpin upon binding, displacing a bound protein. We further show that hnRNP A1 can also spread in a 5′-to-3′ direction, although when initial binding takes place in the middle of an RNA, spreading preferentially proceeds in a 3′-to-5′ direction. Finally, when two distant high-affinity sites are present on the same RNA, they facilitate cooperative spreading of hnRNP A1 between the two sites

    Antisense Masking of an hnRNP A1/A2 Intronic Splicing Silencer Corrects SMN2 Splicing in Transgenic Mice

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    survival of motor neuron 2, centromeric (SMN2) is a gene that modifies the severity of spinal muscular atrophy (SMA), a motor-neuron disease that is the leading genetic cause of infant mortality. Increasing inclusion of SMN2 exon 7, which is predominantly skipped, holds promise to treat or possibly cure SMA; one practical strategy is the disruption of splicing silencers that impair exon 7 recognition. By using an antisense oligonucleotide (ASO)-tiling method, we systematically screened the proximal intronic regions flanking exon 7 and identified two intronic splicing silencers (ISSs): one in intron 6 and a recently described one in intron 7. We analyzed the intron 7 ISS by mutagenesis, coupled with splicing assays, RNA-affinity chromatography, and protein overexpression, and found two tandem hnRNP A1/A2 motifs within the ISS that are responsible for its inhibitory character. Mutations in these two motifs, or ASOs that block them, promote very efficient exon 7 inclusion. We screened 31 ASOs in this region and selected two optimal ones to test in human SMN2 transgenic mice. Both ASOs strongly increased hSMN2 exon 7 inclusion in the liver and kidney of the transgenic animals. Our results show that the high-resolution ASO-tiling approach can identify cis-elements that modulate splicing positively or negatively. Most importantly, our results highlight the therapeutic potential of some of these ASOs in the context of SMA
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