The piwi-interacting RNAs (piRNAs) are small non-coding RNAs, mostly 24–32 nucleotides in length. The piRNAs are not known to have any conserved secondary structure or sequence motifs. Using bioinformatics analysis, we discovered the presence of putative G-quadruplex (GQ) forming sequences in human piRNAs. We studied human piR-48164/piR-GQ containing a potential GQ forming sequence and using biochemical and biophysical techniques confirmed its ability to form a GQ. Using EMSA, we discovered that the formation of GQ structure led to inhibition of the piRNA binding to the HIWI-PAZ domain as well as the complementary base pairing to a target RNA. The inability of the piR-GQ to interact with the PIWI protein might be detrimental to the function of the piRNA. To investigate if the formation of a GQ structure in piRNA prevents its target gene silencing in vivo, we used a reporter assay. The piR-GQ failed to inhibit the reporter gene expression while a mutated version that lacked the ability to form GQ inhibited reporter gene expression indicating that the presence of GQ in piRNA is detrimental to its function. These studies unraveled the dependence of a piRNA's functionality on an RNA secondary structure and added a new layer of regulation to their function.

piwi相互作用RNA（piRNA）是小的非编码RNA，长度通常为24-32个核苷酸。未知piRNA具有任何保守的二级结构或序列基序。使用生物信息学分析，我们发现了人类piRNA中假定的G-四链体（GQ）形成序列的存在。我们研究了含有潜在GQ形成序列的人piR-48164 / piR-GQ，并使用生物化学和生物物理技术证实了其形成GQ的能力。使用EMSA，我们发现GQ结构的形成导致抑制piRNA与HIWI-PAZ域的结合以及与靶RNA的互补碱基配对。piR-GQ无法与PIWI蛋白相互作用可能对piRNA的功能有害。研究piRNA中GQ结构的形成是否会阻止其靶基因沉默在体内，我们使用了一种报告基因检测。piR-GQ不能抑制报告基因的表达，而缺乏形成GQ能力的突变型抑制了报告基因的表达，表明piRNA中GQ的存在对其功能是有害的。这些研究揭示了piRNA功能对RNA二级结构的依赖性，并为其功能增加了新的调节层。