RESUMO
Alternative splicing transitions occur during organ development, and, in numerous diseases, splicing programs revert to fetal isoform expression. We previously found that extensive splicing changes occur during postnatal mouse heart development in genes encoding proteins involved in vesicle-mediated trafficking. However, the regulatory mechanisms of this splicing-trafficking network are unknown. Here, we found that membrane trafficking genes are alternatively spliced in a tissue-specific manner, with striated muscles exhibiting the highest levels of alternative exon inclusion. Treatment of differentiated muscle cells with chromatin-modifying drugs altered exon inclusion in muscle cells. Examination of several RNA-binding proteins revealed that the poly-pyrimidine tract binding protein 1 (PTBP1) and quaking regulate splicing of trafficking genes during myogenesis, and that removal of PTBP1 motifs prevented PTBP1 from binding its RNA target. These findings enhance our understanding of developmental splicing regulation of membrane trafficking proteins which might have implications for muscle disease pathogenesis.
Assuntos
Processamento Alternativo , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Animais , Éxons , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Camundongos , Desenvolvimento Muscular/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismoRESUMO
Brincidofovir (BCV) is an investigational lipid conjugate of the nucleotide analog cidofovir (CDV), which is being developed as a medical countermeasure for the treatment of smallpox. BCV is active against double-stranded DNA viruses including BK and JC viruses. Here, we validated procedures for quantifying BCV and its pharmacologically active moiety cidofovir diphosphate (CDV-PP) in mouse kidney, brain and spleen tissue homogenates. Following homogenization, BCV and CDV-PP were extracted from the tissues by protein precipitation with their stable, isotopically labeled internal standards, BCV-d6 and 13 C3 15 N2 -CDV-PP. Then, samples were analyzed for BCV by reverse-phase chromatography on a Waters Xterra MS C18 (50 × 2.1 mm, 3.5 µm particle size) column while CDV-PP was analyzed on a Thermo BioBasic AX (50 × 2.1 mm, 5 µm particle size) column using anion exchange chromatography. Detection was achieved by electrospray ionization in positive ion mode on an AB Sciex API-5000 triple quadrupole mass spectrometer. The calibration curves were linear over a range of 1.00-1,000 ng/ml homogenate and 0.050-50.0 ng/ml homogenate for BCV and CDV-PP, respectively. These methods were validated according to US Food and Drug Administration guidance for industry and may be used to characterize the tissue pharmacology of both analytes to advance its preclinical development.