Project description:Alternative splicing-induced inclusion of poison exons containing in-frame stop codons is a mechanism that can be used to attenuate gene expression. Poison exon have been implicated in cancer, but how they operate within the context of normal development and physiology is poorly understood. Several splicing regulator genes, including Tra2b, contain ultra-conserved poison exons that function within regulatory loops to fine-tune their activity. To investigate the physiological role of poison exons in vivo, we created mice lacking either Tra2b or its poison exon, specifically during spermatogenesis to reveal both are essential for male fertility. The mouse Tra2b gene is essential for mitotic proliferation of germ cells, whereas, in contrast, the Tra2b poison exon is critically required during meiosis and not needed by mitotically proliferating cell populations within the germline. Poison exon deletion causes infertility, with a block in male meiotic prophase where Tra2β protein expression levels normally increase. Deletion of the Tra2b poison exon changes expression patterns of genes important for meiosis and splicing patterns of Tra2β target exons, suggesting Tra2b poison exon splicing prevents meiotic cells accumulating toxic levels of Tra2b expression. Our data provide a new physiological explanation for Tra2b poison exon ultra-conservation and indicate the importance of evaluating poison exon function within a physiological context.
Project description:Alternative splicing-induced inclusion of poison exons containing in-frame stop codons is a mechanism that can be used to attenuate gene expression. Poison exon have been implicated in cancer, but how they operate within the context of normal development and physiology is poorly understood. Several splicing regulator genes, including Tra2b, contain ultra-conserved poison exons that function within regulatory loops to fine-tune their activity. To investigate the physiological role of poison exons in vivo, we created mice lacking either Tra2b or its poison exon, specifically during spermatogenesis to reveal both are essential for male fertility. The mouse Tra2b gene is essential for mitotic proliferation of germ cells, whereas, in contrast, the Tra2b poison exon is critically required during meiosis and not needed by mitotically proliferating cell populations within the germline. Poison exon deletion causes infertility, with a block in male meiotic prophase where Tra2β protein expression levels normally increase. Deletion of the Tra2b poison exon changes expression patterns of genes important for meiosis and splicing patterns of Tra2β target exons, suggesting Tra2b poison exon splicing prevents meiotic cells accumulating toxic levels of Tra2b expression. Our data provide a new physiological explanation for Tra2b poison exon ultra-conservation and indicate the importance of evaluating poison exon function within a physiological context.
Project description:Analysis of transcript changes with enough depth for evaluation of splicing changes between scrambled control CD8+ T or Tra2b-PE KO CD8+ T cells from mice
Project description:The ability to precisely control gene expression using small molecule drugs is a valuable tool in research and has significant therapeutic potential. However, existing systems are often limited by the toxicity of the drugs and the need to alter gene sequences or endogenous regulatory elements. Here, we introduce Cyclone (acyclovir-controlled poison exon), an acyclovir-controlled poison exon cassette that can be used for small molecule control of both transgene and endogenous gene expression. Cyclone is a portable “intron-poison exon-intron” element that can be inserted into nearly any gene and is completely removed upon acyclovir treatment, leaving the native transcript intact. Cyclone offers tunable, reversible gene expression with nearly undetectable background and a ~295-fold activation. We also present Pac-Cyclone, a cassette that simplifies the generation of cell lines with acyclovir-controlled endogenous gene expression. Finally, we demonstrate the programmability of Cyclone, underscoring its potential for developing diverse genetic circuits controlled by various ligands.