Methods for Studying microRNA Functions During Stress

Abstract Constituting 5 % of the human genome, microRNAs represent a sizeable class of gene regulators that is predicted to control the expression of at least 60 % of all protein-coding RNAs. Dysregulation of microRNA function results in developmental defects and pathological diseases such as cancers and neurological disor -ders. Intriguingly, many phenotypes of microRNA deficiencies are subdued in normal condition but mani -fested apparently upon stress. Here, we outline experimental methods to monitor the level, targets, and activity of microRNAs as the first few steps to characterize how microRNA functions are altered upon stress. Key words microRNA, Argonaute, Real-time PCR, CLIP-seq, Dual-luciferase reporter assay 1 Introduction MicroRNAs (miRNAs) are a class of ~22 nucleotide short non-coding RNAs that play key roles in fundamental cellular processes, including how cells respond to changes in environment or stress [1–3]. Argonaute (Ago) proteins associate with miRNAs that bind to target messenger RNAs (mRNAs) through partial base-pairings to repress translation and/or induce mRNA decay. The level of target gene repression is dependent on the concentration of target mRNAs relative to the miRNA and the activity of miRNA-Ago complexes. Emerging data suggest that particular stress conditions can alter the biogenesis of miRNAs, the expression of mRNA tar-gets, and the activity/location of miRNA-Ago complexes [4–7].CrossLinking ImmunoPrecipitation followed by deep sequenc -ing of RNAs (CLIP-seq) revealed that Argonaute2 (Ago2) pro-teins can bind to target mRNAs on 3 ′ untranslated regions (UTRs) and coding sequences in both the presence and the absence of miRNAs [ 5, 8–10]. Ago2 proteins display a significant remodeling of its target occupancy upon stress in human cells [ 5]. For example, upon arsenite stress, Ago2 proteins increasingly associate with target mRNAs at binding sites for endogenous miRNAs, but they