A recent paper published in Molecular Cell by Dimitrova et al. from the lab of Dr. Tyler Jacks at MIT, used Stellaris® RNA FISH to help answer questions regarding whether a pro-apoptotic lncRNA (lncRNA-p21) can regulate nearby genes via cis- or trans-acting mechanisms1. Long non-coding RNA-p21 and p21 expression is stimulated in response to the activation of the p53 pathway. This pathway is an important area of study because it is involved in tumor suppression and is elicited in response to DNA damage and cellular stress.
The researchers wanted to investigate the mechanisms whereby lncRNA-p21 acts to influence the p53 mediated transcriptional response. Long non-coding RNAs that act in cis control the expression of genes that are positioned near the lncRNA’s transcription sites and sometimes spread their effects longer distances on the same chromosome. In contrast, trans-acting lncRNAs either repress or activate expression at independent loci 2.
Using Stellaris RNA FISH for localization studies, the authors concluded that lncRNA-p21 acts in cis for the regulation of p21 expression. First, Dimitrova et al. showed that lncRNA-p21 localized to the nucleus using Stellaris RNA FISH probes labeled with TAMRA. Additionally, the authors performed an important negative control experiment in cells lacking lncRNA-p21, where over 90% of the lncRNA-p21 -/- cells did not contain lncRNA-p21 fluorescent Stellaris RNA FISH signals. Next, the authors performed a single gene iceFISH™ assay by designing Stellaris FISH probes labeled with Quasar® 670 dye to the intron of lncRNA-p21 in order to help identify the genomic locus from which it is expressed. Since the intronic probes were labeled in a different fluorophore than the exonic probe set, this allowed the authors to observe a tight co-localization between exonic and intronic lncRNA-p21 signal (see figure below for an example diagram of how this works). This result suggested that this particular lncRNA may be acting on the p21 gene via cis mechanisms. Further evidence for the cis mechanism of action was also gathered using Stellaris RNA FISH. Overexpression of lncRNA-21 failed to rescue p21 levels in lncrRNA-p21 -/-MEFs, which was attributed to the improper localization and exportation of the overexpressed RNA to the cytoplasm.
iceFISH: Uses Stellaris probes to target RNA introns. Because introns degrade rapidly after splicing, this allows the measurement of actively transcribing genes.
- LincRNA-p21 Activates p21 In cis to Promote Polycomb Target Gene Expression and to Enforce the G1/S Checkpoint. Dimitrova, N.; Zamudio, JR.; Jong, RM.; Soukup, D.; Resnick, R.; Sarma, K.; Ward, AJ.; Raj, A.; Lee, JT.; Sharp, PA.; Jacks, T. Mol Cell. 2014 Jun 5;54(5):777-90, doi: 10.1016/j.molcel.2014.04.025.
- Long non-coding RNAs: New Players in Cell Differentiation and Development. Fatica A1, Bozzoni I2. Nat Rev Genet. 2014 Jan;15(1):7-21. doi: 10.1038/nrg3606.