The incredible complexity of RNA splicing

Alternative splice isoforms are common and important and have been shown to impact many human diseases. A new study by Nellore et al. offers a comprehensive study of splice junctions in humans by re-analyzing over 21,500 public human RNA sequencing datasets

Chemical treatment enhances skipping of a mutated exon in the dystrophin gene

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by a loss of the dystrophin protein. Control of dystrophin mRNA splicing to convert severe DMD to a milder phenotype is attracting much attention. Here we report a dystrophinopathy patient who has a point mutation in exon 31 of the dystrophin gene. Although the mutation generates a stop codon, a small amount of internally deleted, but functional, dystrophin protein is produced in the patient cells. An analysis of the mRNA reveals that the mutation promotes exon skipping and restores the open reading frame of dystrophin. Presumably, the mutation disrupts an exonic splicing enhancer and creates an exonic splicing silencer. Therefore, we searched for small chemicals that enhance exon skipping, and found that TG003 promotes the skipping of exon 31 in the endogenous dystrophin gene in a dose-dependent manner and increases the production of the dystrophin protein in the patient's cells

Over-expression of adenosine deaminase in mouse podocytes does not reverse puromycin aminonucleoside resistance

<p>Abstract</p> <p>Background</p> <p>Edema in nephrotic syndrome results from renal retention of sodium and alteration of the permeability properties of capillaries. Nephrotic syndrome induced by puromycin aminonucleoside (PAN) in rats reproduces the biological and clinical signs of the human disease, and has been widely used to identify the cellular mechanisms of sodium retention. Unfortunately, mice do not develop nephrotic syndrome in response to PAN, and we still lack a good mouse model of the disease in which the genetic tools necessary for further characterizing the pathophysiological pathway could be used. Mouse resistance to PAN has been attributed to a defect in glomerular adenosine deaminase (ADA), which metabolizes PAN. We therefore attempted to develop a mouse line sensitive to PAN through induction of normal adenosine metabolism in their podocytes.</p> <p>Methods</p> <p>A mouse line expressing functional ADA under the control of the podocyte-specific podocin promoter was generated by transgenesis. The effect of PAN on urinary excretion of sodium and proteins was compared in rats and in mice over-expressing ADA and in littermates.</p> <p>Results</p> <p>We confirmed that expression of ADA mRNAs was much lower in wild type mouse than in rat glomerulus. Transgenic mice expressed ADA specifically in the glomerulus, and their ADA activity was of the same order of magnitude as in rats. Nonetheless, ADA transgenic mice remained insensitive to PAN treatment in terms of both proteinuria and sodium retention.</p> <p>Conclusions</p> <p>Along with previous results, this study shows that adenosine deaminase is necessary but not sufficient to confer PAN sensitivity to podocytes. ADA transgenic mice could be used as a background strain for further transgenesis.</p

Interplay between Exonic Splicing Enhancers, mRNA Processing, and mRNA Surveillance in the Dystrophic Mdx Mouse

BACKGROUND: Pre-mRNA splicing, the removal of introns from RNA, takes place within the spliceosome, a macromolecular complex composed of five small nuclear RNAs and a large number of associated proteins. Spliceosome assembly is modulated by the 5′ and 3′ splice site consensus sequences situated at the ends of each intron, as well as by exonic and intronic splicing enhancers/silencers recognized by SR and hnRNP proteins. Nonsense mutations introducing a premature termination codon (PTC) often result in the activation of cellular quality control systems that reduce mRNA levels or alter the mRNA splicing pattern. The mdx mouse, a commonly used genetic model for Duchenne muscular dystrophy (DMD), lacks dystrophin by virtue of a premature termination codon (PTC) in exon 23 that also severely reduces the level of dystrophin mRNA. However, the effect of the mutation on dystrophin RNA processing has not yet been described. METHODOLOGY/PRINCIPAL FINDING: Using combinations of different biochemical and cellular assays, we found that the mdx mutation partially disrupts a multisite exonic splicing enhancer (ESE) that is recognized by a 40 kDa SR protein. In spite of the presence of an inefficient intron 22 3′ splice site containing the rare GAG triplet, the mdx mutation does not activate nonsense-associated altered splicing (NAS), but induces exclusively nonsense-mediated mRNA decay (NMD). Functional binding sites for SR proteins were also identified in exon 22 and 24, and in vitro experiments show that SR proteins can mediate direct association between exon 22, 23, and 24. CONCLUSIONS/SIGNIFICANCE: Our findings highlight the complex crosstalk between trans-acting factors, cis-elements and the RNA surveillance machinery occurring during dystrophin mRNA processing. Moreover, they suggest that dystrophin exon–exon interactions could play an important role in preventing mdx exon 23 skipping, as well as in facilitating the pairing of committed splice sites

Design concepts for the Cherenkov Telescope Array CTA: an advanced facility for ground-based high-energy gamma-ray astronomy

Ground-based gamma-ray astronomy has had a major breakthrough with the impressive results obtained using systems of imaging atmospheric Cherenkov telescopes. Ground-based gamma-ray astronomy has a huge potential in astrophysics, particle physics and cosmology. CTA is an international initiative to build the next generation instrument, with a factor of 5-10 improvement in sensitivity in the 100 GeV-10 TeV range and the extension to energies well below 100 GeV and above 100 TeV. CTA will consist of two arrays (one in the north, one in the south) for full sky coverage and will be operated as open observatory. The design of CTA is based on currently available technology. This document reports on the status and presents the major design concepts of CTA

Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice

It is often challenging for the clinician interested in cystic fibrosis (CF) to interpret molecular genetic results, and to integrate them in the diagnostic process. The limitations of genotyping technology, the choice of mutations to be tested, and the clinical context in which the test is administered can all influence how genetic information is interpreted. This paper describes the conclusions of a consensus conference to address the use and interpretation of CF mutation analysis in clinical settings

Tests of the low beta cavities and cryomodules for the SPIRAL 2 Linac

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