Engineering U1-Based Tetracycline-Inducible Riboswitches to Control Gene Expression in Mammals.

Synthetic riboswitches are promising regulatory devices due to their small size, lack of immunogenicity, and ability to fine-tune gene expression in the absence of exogenous trans-acting factors. Based on a gene inhibitory system developed at our lab, termed U1snRNP interference (U1i), we developed tetracycline (TC)-inducible riboswitches that modulate mRNA polyadenylation through selective U1 snRNP recruitment. First, we engineered different TC-U1i riboswitches, which repress gene expression unless TC is added, leading to inductions of gene expression of 3-to-4-fold. Second, we developed a technique called Systematic Evolution of Riboswitches by Exponential Enrichment (SEREX), to isolate riboswitches with enhanced U1 snRNP binding capacity and activity, achieving inducibilities of up to 8-fold. Interestingly, by multiplexing riboswitches we increased inductions up to 37-fold. Finally, we demonstrated that U1i-based riboswitches are dose-dependent and reversible and can regulate the expression of reporter and endogenous genes in culture cells and mouse models, resulting in attractive systems for gene therapy applications. Our work probes SEREX as a much-needed technology for the in vitro identification of riboswitches capable of regulating gene expression in vivo.

U1A is a positive regulator of the expression of heterologous and cellular genes involved in cell proliferation and migration.

Here, we show that direct recruitment of U1A to target transcripts can increase gene expression. This is a new regulatory role, in addition to previous knowledge showing that U1A decreases the levels of U1A mRNA and other specific targets. In fact, genome-wide, U1A more often increases rather than represses gene expression and many U1A-upregulated transcripts are directly bound by U1A according to individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) studies. Interestingly, U1A-mediated positive regulation can be transferred to a heterologous system for biotechnological purposes. Finally, U1A-bound genes are enriched for those involved in cell cycle and adhesion. In agreement with this, higher U1A mRNA expression associates with lower disease-free survival and overall survival in many cancer types, and U1A mRNA levels positively correlate with those of some oncogenes involved in cell proliferation. Accordingly, U1A depletion leads to decreased expression of these genes and the migration-related gene CCN2/CTGF, which shows the strongest regulation by U1A. A decrease in U1A causes a strong drop in CCN2 expression and CTGF secretion and defects in the expression of CTGF EMT targets, cell migration, and proliferation. These results support U1A as a putative therapeutic target for cancer treatment. In addition, U1A-binding sequences should be considered in biotechnological applications.

Targeting of epigenetic regulators in neuroblastoma.

Approximately 15,000 new cases of pediatric cancer are diagnosed yearly in Europe, with 8-10% corresponding to neuroblastoma, a rare disease with an incidence of 8-9 cases per million children <15 years of age. Although the survival rate for low-risk and intermediate-risk patients is excellent, half of children with high-risk, refractory, or relapsed tumors will be cured, and two-thirds of the other half will suffer major side effects and life-long disabilities. Epigenetic therapies aimed at reversing the oncogenic alterations in chromatin structure and function are an emerging alternative against aggressive tumors that are or will become resistant to conventional treatments. This approach proposes targeting epigenetic regulators, which are proteins that are involved in the creation, detection, and interpretation of epigenetic signals, such as methylation or histone post-translational modifications. In this review, we focused on the most promising epigenetic regulators for targeting and current drugs that have already reached clinical trials.