Quantitative Amplification of Cleaved Ends (qACE) to assay miRNA-directed target cleavage

Suresh Damodaran,Marie Turner,Sajag Adhikari,Senthil Subramanian

doi:10.12688/f1000research.5266.2

Abstract

microRNA (miRNA) regulation is crucial to achieve precise spatio-temporal expression patterns of their target genes. This makes it crucial to determine the levels of cleavage of a particular target mRNA in different tissues and under different conditions. We developed a quantitative PCR method “quantitative Amplification of Cleaved Ends (qACE)” to assay levels of specific cleavage products in order to determine the extent of miRNA-directed target cleavage of a specific target gene. qACE uses cDNA generated from adapter-ligated RNA molecules and relies on a carefully designed fusion primer that spans the adapter-cleaved RNA junction in qPCR to specifically amplify and quantify cleaved products. The levels of full-length transcripts can also be assayed in the same cDNA preparation using primers that span across the miRNA cleavage site. We used qACE to demonstrate that soybean roots over-expressing miR164 had increased levels of target cleavage and that miRNA deficient Arabidopsis thaliana hen1-1 mutants had reduced levels of target cleavage. We used qACE to discover that differential cleavage by miR164 in nodule vs. adjacent root tissue contributed to nodule-specific expression of NAC1 transcription factors in soybean. These experiments show that qACE can be used to discover and demonstrate tissue-specific cleavage by miRNAs to achieve specific spatio-temporal expression of target genes in plants.

Highlights

MiRNAs are short 21-22nt RNA molecules that regulate the expression of cognate target genes primarily through post-transcriptional mechanisms[1]
Oligo-dT primed cDNAs generated from adapter-ligated RNA are subject to real-time PCR where a primer that spans the adapter junction is used as a forward primer and a gene-specific reverse primer (Figure 1)
The forward primer had to be designed such that it does not amplify full-length cDNA molecules or bind non- to other adapter-ligated molecules. We achieved this by careful design such that this primer corresponds to the adapter-cleavage product junction with 6 nucleotides at the 3’end of the primer corresponding to the nucleotides downstream of the cleavage site in the target gene

Summary

Introduction

MiRNAs are short 21-22nt RNA molecules that regulate the expression of cognate target genes primarily through post-transcriptional mechanisms[1]. Polarized expression of miR166 and its target HD-ZIP IIIs is conserved in other plant species as well, e.g. maize[7], and soybean[9]. Another example is the nodule-meristem specific expression of MtHAP2-1 (alpha subunit of a CCAAT-binding NFY), which is spatially restricted by miR169, which is expressed in the infection zone adjacent to the meristem. PHO2 maintains proper levels of phosphate transporter activities[12] and its down regulation increases phosphate acquisition These examples demonstrate that mere transcriptional regulation is not sufficient to achieve proper spatial and temporal expression of target genes and miRNA regulation is crucial for additional finetuning of gene expression

Methods

Results

Conclusion