ABSTRACT
BACKGROUND: Alternative splicing, which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing splicing pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses. RESULTS: RNA immunoprecipitation in tandem (RIPiT) of exon junction complex components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compare exon junction complex RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that captured by RNA-Seq. Our RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identify thousands of previously unannotated splicing events; while many can be attributed to splicing noise, others are evolutionarily conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis. CONCLUSIONS: Deep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the nonsense-mediated decay-linked alternative splicing pathway predominates. Exon junction complex RIPiT-Seq also revealed numerous conserved but previously unannotated AS-NMD events.
Subject(s)
Alternative Splicing , Biological Evolution , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Nonsense Mediated mRNA Decay , Ribonucleoproteins/metabolism , Computational Biology/methods , Gene Library , HEK293 Cells , Humans , Molecular Sequence Annotation , RNA Processing, Post-TranscriptionalABSTRACT
Selective Inhibitor of Nuclear Export (SINE) compounds are a family of small-molecules that inhibit nuclear export through covalent binding to cysteine 528 (Cys528) in the cargo-binding pocket of Exportin 1 (XPO1/CRM1) and promote cancer cell death. Selinexor is the lead SINE compound currently in phase I and II clinical trials for advanced solid and hematological malignancies. In an effort to understand selinexor-XPO1 interaction and to establish whether cancer cell response is a function of drug-target engagement, we developed a quantitative XPO1 occupancy assay. Biotinylated leptomycin B (b-LMB) was utilized as a tool compound to measure SINE-free XPO1. Binding to XPO1 was quantitated from SINE compound treated adherent and suspension cells in vitro, dosed ex vivo human peripheral blood mononuclear cells (PBMCs), and PBMCs from mice dosed orally with drug in vivo. Evaluation of a panel of selinexor sensitive and resistant cell lines revealed that resistance was not attributed to XPO1 occupancy by selinexor. Administration of a single dose of selinexor bound XPO1 for minimally 72 hours both in vitro and in vivo. While XPO1 inhibition directly correlates with selinexor pharmacokinetics, the biological outcome of this inhibition depends on modulation of pathways downstream of XPO1, which ultimately determines cancer cell responsiveness.
Subject(s)
Cell Nucleus/drug effects , Hydrazines/pharmacology , Karyopherins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Triazoles/pharmacology , Acrylamides/chemistry , Acrylamides/pharmacology , Acrylates/chemistry , Acrylates/pharmacology , Active Transport, Cell Nucleus/drug effects , Animals , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacokinetics , Antibiotics, Antineoplastic/pharmacology , Biotinylation , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Survival/drug effects , Cells, Cultured , Drug Evaluation, Preclinical/methods , Fatty Acids, Unsaturated/chemistry , Fatty Acids, Unsaturated/pharmacokinetics , Fatty Acids, Unsaturated/pharmacology , HCT116 Cells , Humans , Hydrazines/chemistry , Hydrazines/pharmacokinetics , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Mice , Molecular Structure , Reproducibility of Results , Thiazoles/chemistry , Thiazoles/pharmacology , Triazoles/chemistry , Triazoles/pharmacokinetics , Exportin 1 ProteinABSTRACT
Direct lineage conversion is a promising approach to generate therapeutically important cell types for disease modeling and tissue repair. However, the survival and function of lineage-reprogrammed cells in vivo over the long term has not been examined. Here, using an improved method for in vivo conversion of adult mouse pancreatic acinar cells toward beta cells, we show that induced beta cells persist for up to 13 months (the length of the experiment), form pancreatic islet-like structures and support normoglycemia in diabetic mice. Detailed molecular analyses of induced beta cells over 7 months reveal that global DNA methylation changes occur within 10 d, whereas the transcriptional network evolves over 2 months to resemble that of endogenous beta cells and remains stable thereafter. Progressive gain of beta-cell function occurs over 7 months, as measured by glucose-regulated insulin release and suppression of hyperglycemia. These studies demonstrate that lineage-reprogrammed cells persist for >1 year and undergo epigenetic, transcriptional, anatomical and functional development toward a beta-cell phenotype.
