Functional dissection of the Tug1 long noncoding RNA locus
Biochemistry
Final Report Abstract
In recent years, long noncoding RNAs (lncRNAs) have emerged as fundamental regulators of various biological pathways and gene expression programs. However, their target genes and mechanisms of action remain largely obscure. Tug1 is a lncRNA with a distinctive gene locus: It contains a repressive DNA element that functions in cis, transcribes a lncRNA that works in trans, and encodes a putative small peptide. We reported a sterility phenotype in male mice with a possible contribution of DNA, RNA, and protein functionalities. My initial goal was to dissect the functionality of the Tug1 locus with respect to its role in gene regulation in mouse ES cells. After technical difficulties and a change in lab priorities, I instead set out to functionally dissect the Firre lncRNA locus. While there is neither evidence for a cis-acting DNA element nor for a small peptide encoded in the Firre locus, it does encode a trans-acting lncRNA. Here I developed an inducible Firre overexpression system in mouse ES cells to identify Firre target genes and study the mechanism of their regulation. We defined a list of Firre targets that interestingly are all upregulated upon Firre induction. The upregulation of most Firre targets is accompanied by an increase in chromatin accessibility, suggesting a possible mechanism for transcription factors or other proteins to be recruited to those sites. Further experiments will determine the DNA-binding sites and protein interaction partners of Firre and shed more light on Firre’s mechanism in gene regulation. Together, these results propose Firre as a transcriptional activator of gene expression. A question that arises from the fact that lncRNAs are rapidly evolving and thus are usually poorly conserved at the sequence level is whether lncRNAs from different species serve the same function. The function of a lncRNA is typically determined by its subcellular localization and abundance. Here my colleagues and I show that the syntenically conserved lncRNA Firre displays distinct expression and localization patterns in human and mouse. In a range of cell lines, mouse Firre is predominantly nuclear, while human FIRRE is distributed between the cytoplasm and nucleus. This localization pattern is maintained in human/mouse hybrid cells expressing both human and mouse Firre, implying that the localization of the lncRNA is sequence-driven. The majority of human FIRRE transcripts in the cytoplasm is comprised of isoforms that are enriched in RRD repeats. Furthermore, mouse Firre is more highly expressed than its human counterpart in various tissues. These data illustrate that the rapid evolution of syntenic lncRNAs can lead to variations in lncRNA localization and abundance, which in turn may result in disparate lncRNA functions even in closely related species.
Publications
- The Tug1 lncRNA locus is essential for male fertility. Genome Biol 21 (2020)
Lewandowski, J.P., Dumbović, G., Watson, A.R., Hwang T., Jacobs-Palmer E., Chang, N., Much, C., Turner, K.M., Kirby, C., Rubinstein, N.D., Groff, A.F., Liapis, S.C., Gerhardinger, C., Bester, A., Pandolfi, P.P., Clohessy, J.G., Hoekstra, H.E., Sauvageau, M., Rinn, J.L.
(See online at https://doi.org/10.1186/s13059-020-02081-5)
