Control of mammalian gene expression by modulation of polyA signal cleavage at 5' UTR.

The ability to control gene expression in mammalian cells is crucial for safe and efficacious gene therapies and for elucidating gene functions. Current gene regulation systems have limitations such as harmful immune responses or low efficiency. We describe the pA regulator, an RNA-based switch that controls mammalian gene expression through modulation of a synthetic polyA signal (PAS) cleavage introduced into the 5' UTR of a transgene. The cleavage is modulated by a 'dual-mechanism'-(1) aptamer clamping to inhibit PAS cleavage and (2) drug-induced alternative splicing that removes the PAS, both activated by drug binding. This RNA-based methodology circumvents the immune responses observed in other systems and achieves a 900-fold induction with an EC50 of 0.5 µg ml-1 tetracycline (Tc), which is well within the FDA-approved dose range. The pA regulator effectively controls the luciferase transgene in live mice and the endogenous CD133 gene in human cells, in a dose-dependent and reversible manner with long-term stability.

RNA-driven JAZF1-SUZ12 gene fusion in human endometrial stromal cells.

Oncogenic fusion genes as the result of chromosomal rearrangements are important for understanding genome instability in cancer cells and developing useful cancer therapies. To date, the mechanisms that create such oncogenic fusion genes are poorly understood. Previously we reported an unappreciated RNA-driven mechanism in human prostate cells in which the expression of chimeric RNA induces specified gene fusions in a sequence-dependent manner. One fundamental question yet to be addressed is whether such RNA-driven gene fusion mechanism is generalizable, or rather, a special case restricted to prostate cells. In this report, we demonstrated that the expression of designed chimeric RNAs in human endometrial stromal cells leads to the formation of JAZF1-SUZ12, a cancer fusion gene commonly found in low-grade endometrial stromal sarcomas. The process is specified by the sequence of chimeric RNA involved and inhibited by estrogen or progesterone. Furthermore, it is the antisense rather than sense chimeric RNAs that effectively drive JAZF1-SUZ12 gene fusion. The induced fusion gene is validated both at the RNA and the genomic DNA level. The ability of designed chimeric RNAs to drive and recapitulate the formation of JAZF1-SUZ12 gene fusion in endometrial cells represents another independent case of RNA-driven gene fusion, suggesting that RNA-driven genomic recombination is a permissible mechanism in mammalian cells. The results could have fundamental implications in the role of RNA in genome stability, and provide important insight in early disease mechanisms related to the formation of cancer fusion genes.

Validating Gene Fusion as the Source of Chimeric RNAs.

While chimeric RNAs may be generated by transcription-mediated mechanisms such as "trans-splicing" and "read-through/splicing" (Zhang et al., Cancer Discov 2:598-607, 2012; Zaphiropoulos, Front Genet 2:92, 2011; Li et al., Cell Cycle 8:218-222, 2009; Jividen and Li, Genes Chromosomes Cancer 53:963-971, 2014; Jia et al., Trends Cancer 2:475-484, 2016), most highly expressed chimeric RNA species identified so far are usually transcribed directly from fusion genes. Fusion genes, formed by joining two parental genes as a result of chromosomal rearrangements, are hallmarks of many types of cancer. Various methods can be deployed for confirming a particular fusion gene as the original source of transcribed chimeric RNA. In this chapter, we discuss commonly used methodologies such as genomic DNA breakpoint mapping and fluorescent in situ hybridization useful for confirming gene fusion events. In addition, we highlight the development of new technologies such as de novo whole-genome optical mapping suitable for global analysis of genomic arrangements. The advantages and disadvantages of each of these technologies are presented and compared.