Abstract
Purpose :
Piwi-interacting RNAs (piRNAs) have been shown to play a substantial regulatory role in neurons. Earlier, we demonstrated that loss of piRNAs in retinal pigment epithelial cells (ARPE19) downregulated specific targets implicated in circadian clock, ciliary transport and retinal degeneration. Intriguingly, we also observed that piR-62011 displayed seed sequence similarity to the microRNA cluster, miR-183/96/182. Therefore, the present study aims to further characterize the functional interaction between piR-62011 and miR-182.
Methods :
Stem-loop primers were designed to specifically reverse transcribe mature miR-182, piR-62011 and U6 snRNA and the relative quantification was done by two-step qRT-PCR. The methylation of piR-62011 was confirmed by RTL-PCR. TOPO cloning and sequencing was performed to validate piR-62011 and miR-182 sequences. Immunoprecipitation was carried out using antibody against piRNA binding protein PIWIL4 in ARPE19 cells and RNA was isolated for further analysis. siRNA mediated knockdown of PIWIL4 was done in ARPE19 and Y79 cells. RNA hybrid tool was used to predict the target binding sites. Drosophila PIWI mutants were obtained from Bloomington Drosophila Stock Centre (BDSC), USA for behavioural studies.
Results :
The copy number of piR-62011 was relatively lower than miR-182 in ARPE19 cells. piR-62011 was enriched in PIWIL4 immunoprecipitated RNA fraction. We speculated that piR-62011 and miR-182 could exhibit additive or co-operative regulation of mRNA targets. Subsequent experiments in PIWIL4 knockdown retinal cells showed that piR-62011 levels positively correlated with miR-182. Furthermore, we examined the common targets of piR-62011 and miR-182 to predict the optimal free energy estimate of piRNA:mRNA and miRNA:mRNA duplexes using RNA hybrid tool. Interestingly, one of the targets, circadian rhythm regulator protein CLOCK showed binding sites for piR-62011 and miR-182 at a distance which is favourable for co-operative regulation. Moreover, deletion of PIWI in drosophila lead to arrhythmic circadian pattern during light-dark adaptation.
Conclusions :
Thus, our data indicates that piRNAs can regulate mRNA targets which are specific for the crucial function of retina in concert with miRNAs.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.