Traditionally, chimeric RNAs were considered to be exclusive to cancer cells. When occasionally observed in normal samples, they were usually considered to be transcriptional 'noises,' or artifacts due to template switching during the reverse transcription and/or Polymerase chain reaction (PCR) steps of experimentation. However, with the advances being made in next generation sequencing technologies and software tools, as well as the accumulation of new experimental evidences, increasing numbers of chimeric transcripts are being identified in noncancerous tissues and cells. Recent studies have also demonstrated functional relevance, for at least a subset of chimeric RNAs in normal physiology. The advances have resulted in an influx of knowledge; this knowledge indicates that chimeric RNAs are a component of basic biology, and thus challenging traditional dogma. In addition to chromosomal rearrangement, chimeric RNAs can also be formed via different molecular mechanisms including cis-splicing of adjacent genes (cis-SAGe) and trans-splicing, as well as others. Little is known about the details of these noncanonical splicing processes. However, research in this new field promises to not only advance our basic understanding of the human genome and gene regulation, but also lead to improvements in clinical practice, especially in the areas of cancer diagnostics and treatment. WIREs RNA 2017, 8:e1427. doi: 10.1002/wrna.1427 For further resources related to this article, please visit the WIREs website.
Duchenne muscular dystrophy (DMD) is a genetic disease evoked by a mutation in the dystrophin gene. It is associated with progressive muscle degeneration and increased inflammation. Up to this date, mainly anti-inflammatory treatment is available for patients suffering from DMD. miR-146a is known to diminish inflammation and fibrosis in different tissues by downregulating the expression of proinflammatory cytokines. However, its role in DMD has not been studied so far. In our work, we have generated mice globally lacking both dystrophin and miR-146a (miR-146a −/− mdx ) and examined them together with wild-type, single miR-146a knockout and dystrophic ( mdx —lacking dystrophin) mice in a variety of aspects associated with DMD pathophysiology (muscle degeneration, inflammatory reaction, muscle satellite cells, muscle regeneration, and fibrosis). We have shown that miR-146a level is increased in dystrophic muscles in comparison to wild-type mice. Its deficiency augments the expression of proinflammatory cytokines (IL-1β, CCL2, TNFα). However, muscle degeneration was not significantly worsened in mdx mice lacking miR-146a up to 24 weeks of age, although some aggravation of muscle damage and inflammation was evident in 12-week-old animals, though no effect of miR-146a deficiency was visible on quantity, proliferation, and in vitro differentiation of muscle satellite cells isolated from miR-146a −/− mdx mice vs. mdx . Similarly, muscle regeneration and collagen deposition were not changed by miR-146a deficiency. Nevertheless, the lack of miR-146a is associated with decreased Vegfa and increased Tgfb1 . Overall, the lack of miR-146a did not aggravate significantly the dystrophic conditions in mdx mice, but its effect on DMD in more severe conditions warrants further investigation. Electronic supplementary material The online version of this article (10.1186/s13395-019-0207-0) contains supplementary material, which is available to authorized users.
Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of neuromuscular disease such as Duchenne muscular dystrophy has been an advance for the splice-switching field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. To overcome limitations of existing technologies, we engineered chimeric stereopure oligonucleotides with phosphorothioate (PS) and phosphoryl guanidine-containing (PN) backbones. We demonstrate that these chimeric stereopure oligonucleotides have markedly improved pharmacology and efficacy compared with PS-modified oligonucleotides, preventing premature death and improving median survival from 49 days to at least 280 days in a dystrophic mouse model with an aggressive phenotype. These data demonstrate that chemical optimization alone can profoundly impact oligonucleotide pharmacology and highlight the potential for continued innovation around the oligonucleotide backbone. More specifically, we conclude that chimeric stereopure oligonucleotides are a promising splice-switching modality with potential for the treatment of neuromuscular and other genetic diseases impacting difficult to reach tissues such as the skeletal muscle and heart.
Chimeric RNAs generated by cis-splicing between adjacent genes (cis-SAGe) are increasingly recognized as a widespread phenomenon. These chimeric messenger RNAs are present in normal human cells, and are also detected in various cancers. The mechanisms for how this group of chimeras is formed are not yet clear, in part due to the lack of a tractable system for their experimental investigation. Here we developed a fast, easy and versatile cell-based reporter system to identify regulators of cis-SAGe. The reporter, consisting of four main cassettes, simultaneously measures the effects of a candidate regulator on cis-SAGe and canonical splicing. Using this cell-based assay, we screened 102 candidate factors involved in RNA pol II cleavage and termination, elongation, splicing, alternative splicing and R-loop formation. We discovered that two factors, SRRM1 and SF3B1, affect not only cis-SAGe chimeras, but also other types of chimeric RNAs in a genome-wide fashion. This system can be used for studying trans-acting factors and cis-acting sequence elements and factors, as well as for screening small molecule inhibitors.
cis-Splicing between adjacent genes (cis-SAGe) is being recognized as one way to produce chimeric fusion RNAs. However, its detail mechanism is not clear. Recent study revealed induction of transcriptions downstream of genes (DoGs) under osmotic stress. Here, we investigated the influence of osmotic stress on cis-SAGe chimeric RNAs and their connection to DoGs. We found, the absence of induction of at least some cis-SAGe fusions and/or their corresponding DoGs at early time point(s). In fact, these DoGs and their cis-SAGe fusions are inversely correlated. This negative correlation was changed to positive at a later time point. These results suggest a direct competition between the two categories of transcripts when total pool of readthrough transcripts is limited at an early time point. At a later time point, DoGs and corresponding cis-SAGe fusions are both induced, indicating that total readthrough transcripts become more abundant. Finally, we observed overall enhancement of cis-SAGe chimeric RNAs in KCl-treated samples by RNA-Seq analysis.
Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by mutations in the survival motor neuron 1 ( SMN1 ) gene. All patients have at least one copy of a paralog, SMN2 , but a C-to-T transition in this gene results in exon 7 skipping in a majority of transcripts. Approved treatment for SMA involves promoting exon 7 inclusion in the SMN2 transcript or increasing the amount of full-length SMN by gene replacement with a viral vector. Increasing the pool of SMN2 transcripts and increasing their translational efficiency can be used to enhance splice correction. We sought to determine whether the 5′ untranslated region (5′ UTR) of SMN2 contains a repressive feature that can be targeted to increase SMN levels. We found that antisense oligonucleotides (ASOs) complementary to the 5′ end of SMN2 increase SMN mRNA and protein levels and that this effect is due to inhibition of SMN2 mRNA decay. Moreover, use of the 5′ UTR ASO in combination with a splice-switching oligonucleotide (SSO) increases SMN levels above those attained with the SSO alone. Our results add to the current understanding of SMN regulation and point toward a new therapeutic target for SMA.
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