MicroRNA-directed cleavage of targets: mechanism and experimental approaches

Abstract

MicroRNAs (miRNAs) are a large family of post-transcriptional regulators, which are 21-24 nt in length and play a role in a wide variety of biological processes in eukaryotes. The past few years have seen rapid progress in our understanding of miRNA biogenesis and the mechanism of action, which commonly entails a combination of target degradation and translational repression. The target degradation mediated by Argonaute-catalyzed endonucleolytic cleavage exerts a significant repressive effect on target mRNA expression, particularly during rapid developmental transitions. This review outlines the current understanding of the mechanistic aspects of this important process and discusses several different experimental approaches to identify miRNA cleavage targets.

Fig. 1.. Experimental approaches for microRNA-directed cleavage target identification. (A) 5'-RLM-RACE. In this modified protocol, particular steps (dephosphorylation and cap-removal reactions) from the original 5'-RACE protocol are omitted. A 5'-RNA adaptor is directly ligated to the 3'-end of miRNA-directed cleavage products that contain ligation-competent 5'-monophosphate. The ligated RNAs are reverse-transcribed, PCR-amplified, cloned, and sequenced, allowing determination of their mRNA cleavage sites. (B) Northern blot analysis. Shown are typical molecular phenotypes of transient or stable overexpression of miRNAs in plants, which often lead to reduced accumulation of the full-length target RNA and increased accumulation of cleavage product [miRNA (+); left lane on the blot]. miRNA-deficient mutant, such as dcl-1 (right lane on the blot), served as a negative control. (C) qRT-PCR. Shown is a typical qRT-PCR output of relative target mRNA expression in wild-type and miRNA mutants [miRNA (-): knockout / miRNA (+): overexpression]. (D) Reporter assay. Reporter constructs containing WT or a mutant 3'-UTR were cotransfected with a miRNA of interest [miRNA (+)] or a non-cognate miRNA [miRNA (-)]. To support the direct interaction, mutation of the miRNA-binding site is often introduced in the reporter construct, which should eliminate miRNA-target interaction. The level of repression for cleavage targets is usually expected to be higher than that are observed for seed-matched targets. (E) In vitro cleavage assay. The DNA fragment encoding a target RNA is in vitro transcribed under the control of a T7 promoter. This target RNA is cap-radiolabeled and incubated with lysates containing an endogenous miRNA or an exogenously assembled miRNA of interest. When such a target RNA substrate is cleaved, its 5'-end cleavage product remains detectable by autoradiography. A time-course in vitro RNAi reaction is performed and analyzed in a denaturing gel, with the wild-type and target site-mutated substrates as a negative control. (F) Degradome sequencing. Shown is the schematic description of degradome library construction and data analysis. Poly(A)-tailed RNAs are purified from total RNA with oligo-dT magnetic beads. A 5'-adaptor is ligated to the 5'-end of the cleaved molecules with 5'-monophosphate. Reverse transcription and subsequent alkaline hydrolysis of the RNA templates are performed to construct the first-strand cDNA templates. Primer extension is carried out to generate the second-strand cDNAs. Restriction enzyme MmeI cleaves ∼20-nt within the double-stranded cDNAs, which are ligated with a dsDNA 3'-adaptor. The resulting products are PCR-amplified and submitted for high-throughput sequencing. The pre-processed 20-nt sequences are matched to the transcriptome. The miRNAs, extracted from small RNA sequencing data from the same tissue or samples for degradome library construction, are aligned to find miRNA-target transcript base pairing.