Functional dissection of the Tug1 long noncoding RNA locus

• Applicant: Dr. Christian Much
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry
• Term: from 2019 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 424761055

Several long noncoding RNAs (lncRNAs) have been ascribed a function in the regulation of gene expression. But even though thousands of lncRNAs exist, many of them are merely transcriptional byproducts that lack an RNA-dependent role. Therefore, lncRNA genes of regulatory importance must preferentially act in different ways to exert their functions. We set out to study the lncRNA locus Tug1, which in contrast to many other lncRNAs is ubiquitously expressed and highly conserved between mouse and human. Remarkably, we found that the Tug1 locus plays an indispensable role in male fertility, as Tug1-deleted mice are completely sterile due to spermatogenesis defects. Our genetic and molecular analyses indicate that the Tug1 locus functions through multiple mechanisms, involving DNA-, RNA- and protein-mediated effects. We gathered experimental evidence that the Tug1 locus harbors a repressive DNA element acting on neighboring genes, that it produces an RNA that regulates the expression of distant genes, and that it might encode a peptide of unknown function. However, how these components contribute to the function and physiological significance of the Tug1 locus remains unknown.Here I propose to functionally dissect the Tug1 locus with the aim to understand its mechanisms of action. For this purpose, I will analyze the contributions of DNA, RNA and protein to the regulatory logic of the Tug1 locus using mouse embryonic stem cells as a model system. The repressive DNA element will be characterized by testing multiple sequences derived from the Tug1 locus for their gene silencing activity and their ability to recruit repressive protein complexes. In order to define how the Tug1 RNA regulates its distant gene targets, I will use loss- and gain-of-function experiments to define the true targets of Tug1 as well as identify specific RNA domains and RNA-binding proteins that are important for gene regulation. The protein-coding potential of the Tug1 gene will be tested through bioinformatic and experimental approaches, and identified TUG1 peptides will be investigated for their ability to regulate gene expression and other processes in the cell.Collectively, these experiments will define the multifaceted features of the Tug1 locus and elucidate whether they converge on a single function or act independently in different biological pathways. The complex characteristics of the Tug1 locus might be more common than previously thought and help explain the functionality of many other lncRNA genes.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor John L. Rinn, Ph.D.