P68 RNA Helicase (DDX5) Required for the Formation of Various Specific and Mature miRNA Active RISC Complexes.

Abstract

Introduction: DEAD-box RNA helicases catalyze the ATP-dependent unwinding of doublestranded RNA. In addition, they are required for protein displacement and remodelling of RNA or RNA/protein complexes. P68 RNA helicase regulates the alternative splicing of the important protooncogene H-Ras, and numerous studies have shown that p68 RNA helicase is probably involved in miRNA biogenesis, mainly through Drosha and RISC/DICER complexes.

Objective: This study aimed to determine how p68 RNA helicase affects the activity of selected mature miRNAs, including miR-342, miR-330, miR-138 and miR-206, miR-126, and miR-335, and let-7a, which are known to be related to cancer processes.

Methods: The miRNA levels were analyzed in stable HeLa cells containing p68 RNA helicase RNAi induced by doxycycline (DOX). Relevant results were repeated using transient transfection with pSuper/ pSuper-p68 RNA helicase RNAi to avoid DOX interference.

Results: Herein, we reported that p68 RNA helicase downregulation increases the accumulation of the mature miRNAs, such as miR-126, let-7a, miR-206, and miR-138. Interestingly, the accumulation of these mature miRNAs does not downregulate their known protein targets, thus suggesting that p68 RNA helicase is required for mature miRNA-active RISC complex activity.

Conclusion: Furthermore, we demonstrated that this requirement is conserved, as drosophila p68 RNA helicase can complete the p68 RNA helicase depleted activity in human cells. Dicer and Drosha proteins are not affected by the downregulation of p68 RNA helicase despite the fact that Dicer is also localized in the nucleus when p68 RNA helicase activity is reduced.