miR-1/206 downregulates splicing factor Srsf9 to promote C2C12 differentiation

Background Myogenesis is driven by specific changes in the transcriptome that occur during the different stages of muscle differentiation. In addition to controlled transcriptional transitions, several other post-transcriptional mechanisms direct muscle differentiation. Both alternative splicing and miRNA activity regulate gene expression and production of specialized protein isoforms. Importantly, disruption of either process often results in severe phenotypes as reported for several muscle diseases. Thus, broadening our understanding of the post-transcriptional pathways that operate in muscles will lay the foundation for future therapeutic interventions. Methods We employed bioinformatics analysis in concert with the well-established C2C12 cell system for predicting and validating novel miR-1 and miR-206 targets engaged in muscle differentiation. We used reporter gene assays to test direct miRNA targeting and studied C2C12 cells stably expressing one of the cDNA candidates fused to a heterologous, miRNA-resistant 3&prime; UTR. We monitored effects on differentiation by measuring fusion index, myotube area, and myogenic gene expression during time course differentiation experiments. Results Gene ontology analysis revealed a strongly enriched set of putative miR-1 and miR-206 targets associated with RNA metabolism. Notably, the expression levels of several candidates decreased during C2C12 differentiation. We discovered that the splicing factor Srsf9 is a direct target of both miRNAs during myogenesis. Persistent Srsf9 expression during differentiation impaired myotube formation and blunted induction of the early pro-differentiation factor myogenin as well as the late differentiation marker sarcomeric myosin, Myh8. Conclusions Our data uncover novel miR-1 and miR-206 cellular targets and establish a functional link between the splicing factor Srsf9 and myoblast differentiation. The finding that miRNA-mediated clearance of Srsf9 is a key myogenic event illustrates the coordinated and sophisticated interplay between the diverse components of the gene regulatory network. </div

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

Ex vivo correction of selenoprotein N deficiency in rigid spine muscular dystrophy caused by a mutation in the selenocysteine codon

Premature termination of translation due to nonsense mutations is a frequent cause of inherited diseases. Therefore, many efforts were invested in the development of strategies or compounds to selectively suppress this default. Selenoproteins are interesting candidates considering the idiosyncrasy of the amino acid selenocysteine (Sec) insertion mechanism. Here, we focused our studies on SEPN1, a selenoprotein gene whose mutations entail genetic disorders resulting in different forms of muscular diseases. Selective correction of a nonsense mutation at the Sec codon (UGA to UAA) was undertaken with a corrector tRNASec that was engineered to harbor a compensatory mutation in the anticodon. We demonstrated that its expression restored synthesis of a full-length selenoprotein N both in HeLa cells and in skin fibroblasts from a patient carrying the mutated Sec codon. Readthrough of the UAA codon was effectively dependent on the Sec insertion machinery, therefore being highly selective for this gene and unlikely to generate off-target effects. In addition, we observed that expression of the corrector tRNASec stabilized the mutated SEPN1 transcript that was otherwise more subject to degradation. In conclusion, our data provide interesting evidence that premature termination of translation due to nonsense mutations is amenable to correction, in the context of the specialized selenoprotein synthesis mechanism

Inhibidores de bomba de protones vs. antagonistas del receptor 2 de histamina en la protección gástrica en el paciente anticoagulado: ¿qué ha definido la evidencia?

Objective: to evaluate the latest evidence on the difference in gastroprotection generated by proton pump inhibitors (PPIs) and histamine receptor 2 antagonists (H2RAs) in anticoagulated patients, as well as the associated risk of gastrointestinal bleeding. Methods: narrative review. A literature search was conducted in the PubMed, ScienceDirect, Web of Science, and MEDLINE databases. Results: Among the described mechanisms explaining bleeding in the gastrointestinal tract are mucosal damage, alterations in local pH, platelet inhibition, coagulation factor alteration, and vascular lesions. Depending on the pharmacological group, anticoagulants can act on different targets in the coagulation pathway, altering the factors involved in this pathway and potentially triggering bleeding. Antisecretory drugs, on the other hand, aim to inhibit or reduce gastric acid secretion through interaction with enzymatic systems, altering the local pH. Thus, these drugs influence the risk of bleeding. However, the latest evidence shows that PPIs reduce the likelihood of gastrointestinal bleeding by up to 33% compared to H2RAs, without impacting other outcomes such as mortality or hospitalization. Conclusion: Although the evidence is scarce and heterogeneous, it was observed that there is greater scientific support and gastroprotective benefit against the frequency of gastrointestinal bleeding with the use of PPIs compared to H2RAs in anticoagulated patients who concurrently use gastric acid suppressants.Objetivo: evaluar la evidencia más reciente sobre la diferencia de la gastroprotección generada por los inhibidores de bomba de protones (IBP) y antagonistas del receptor de histamina 2 (ARH2) en el paciente anticoagulado, así como del riesgo de sangrado gastrointestinal asociado. Métodos: revisión narrativa. Se realizó una búsqueda bibliográfica en las bases de datos PubMed, ScienceDirect, Web of Science, y MEDLINE. Resultados: dentro de los mecanismos descritos que explican un sangrado en el tracto gastrointestinal, se encuentran el daño a mucosa, alteraciones en el pH local, inhibición plaquetaria y alteración de factores de la coagulación, y lesiones vasculares. Dependiendo del grupo farmacológico, los anticoagulantes pueden actúan a nivel de diferentes dianas de la vía de la coagulación, alterando los factores involucrados en esta vía, y pudiendo desencadenar sangrado. Por su parte, los antisecretores gástricos, tienen como objetivo inhibir o reducir la secreción de ácido gástrico por medio de la interacción con sistemas enzimáticos, alterando el pH local. De esta forma, estos fármacos influyen sobre el riesgo de sangrado. No obstante, la evidencia más reciente demuestra que los IBP reducen la probabilidad de sangrado gastrointestinal hasta en un 33%, comparado con los ARH2, sin impactar en otros desenlaces como mortalidad u hospitalización. Conclusión: aunque la evidencia es escasa y heterogénea, se pudo observar mayor respaldo científico y beneficio gastroprotector contra la frecuencia de sangrado gastrointestinal, con el uso de IBP comparado a ARH2, en pacientes anticoagulados que concomitantemente, utilizan antisecretores gástricos

Inhibidores de bomba de protones vs. antagonistas del receptor 2 de histamina en la protección gástrica en el paciente anticoagulado: ¿qué ha definido la evidencia?

Objetivo: evaluar la evidencia más reciente sobre la diferencia de la gastroprotección generada por los inhibidores de bomba de protones (IBP) y antagonistas del receptor de histamina 2 (ARH2) en el paciente anticoagulado, así como del riesgo de sangrado gastrointestinal asociado. Métodos: revisión narrativa. Se realizó una búsqueda bibliográfica en las bases de datos PubMed, ScienceDirect, Web of Science, y MEDLINE. Resultados: dentro de los mecanismos descritos que explican un sangrado en el tracto gastrointestinal, se encuentran el daño a mucosa, alteraciones en el pH local, inhibición plaquetaria y alteración de factores de la coagulación, y lesiones vasculares. Dependiendo del grupo farmacológico, los anticoagulantes pueden actúan a nivel de diferentes dianas de la vía de la coagulación, alterando los factores involucrados en esta vía, y pudiendo desencadenar sangrado. Por su parte, los antisecretores gástricos, tienen como objetivo inhibir o reducir la secreción de ácido gástrico por medio de la interacción con sistemas enzimáticos, alterando el pH local. De esta forma, estos fármacos influyen sobre el riesgo de sangrado. No obstante, la evidencia más reciente demuestra que los IBP reducen la probabilidad de sangrado gastrointestinal hasta en un 33%, comparado con los ARH2, sin impactar en otros desenlaces como mortalidad u hospitalización. Conclusión: aunque la evidencia es escasa y heterogénea, se pudo observar mayor respaldo científico y beneficio gastroprotector contra la frecuencia de sangrado gastrointestinal, con el uso de IBP comparado a ARH2, en pacientes anticoagulados que concomitantemente, utilizan antisecretores gástricos

Phase I Clinical Trial of Systemically Administered TUSC2(FUS1)-Nanoparticles Mediating Functional Gene Transfer in Humans

Background: Tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of–function genetic abnormalities. We report the first in-human systemic gene therapy clinical trial of tumor suppressor gene TUSC2. Methods: Patients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP):cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) every 3 weeks. Results: Thirty-one patients were treated at 6 dose levels (range 0.01 to 0.09 milligrams per kilogram). The MTD was determined to be 0.06 mg/kg. Five patients achieved stable disease (2.6–10.8 months, including 2 minor responses). One patient had a metabolic response on positron emission tomography (PET) imaging. RT-PCR analysis detected TUSC2 plasmid expression in 7 of 8 post-treatment tumor specimens but not in pretreatment specimens and peripheral blood lymphocyte controls. Proximity ligation assay, performed on paired biopsies from 3 patients, demonstrated low background TUSC2 protein staining in pretreatment tissues compared with intense (10–25 fold increase) TUSC2 protein staining in posttreatment tissues. RT-PCR gene expression profiling analysis of apoptotic pathway genes in two patients with high posttreatmen

Interaction of hnRNP A1 with snRNPs and pre-mRNAs: evidence for a possible role of A1 RNA annealing activity in the first steps of spliceosome assembly.

The in vitro interaction of recombinant hnRNP A1 with purified snRNPs and with pre-mRNAs was investigated. We show that protein A1 can stably bind U2 and U4 snRNP but not U1. Oligo-RNAse H cleavage of U2 nucleotides involved in base pairing with the branch site, totally eliminates the A1-U2 interaction. RNase T1 protection and immunoprecipitation experiments demonstrate that recombinant protein A1 specifically binds the 3'-end regions of both beta-globin and Ad-2 introns. However, while on the beta-globin intron only binding to the polypyrimidine tract was observed, on the Ad-2 intron a 32 nt fragment encompassing the branch point and the AG splice-site dinucleotide was bound and protected. Such protection was drastically reduced in the presence of U2 snRNP. Altogether these results indicate that protein A1 can establish a different pattern of association with different pre-mRNAs and support the hypothesis that this protein could play a role in the annealing of U2 to the branch site and hence in the early events of pre-splicing complex assembly