Abstract
Although artificial microRNA (amiRNA) technology has been used frequently in gene silencing in plants, little research has been devoted to investigating the accuracy of amiRNA precursor processing. In this work, amiRNAchs1 (amiRchs1), based on the Arabidopsis miR319a precursor, was expressed in order to suppress the expression of CHS genes in petunia. The transgenic plants showed the CHS gene-silencing phenotype. A modified 5′ RACE technique was used to map small-RNA-directed cleavage sites and to detect processing intermediates of the amiRchs1 precursor. The results showed that the target CHS mRNAs were cut at the expected sites and that the amiRchs1 precursor was processed from loop to base. The accumulation of small RNAs in amiRchs1 transgenic petunia petals was analyzed using the deep-sequencing technique. The results showed that, alongside the accumulation of the desired artificial microRNAs, additional small RNAs that originated from other regions of the amiRNA precursor were also accumulated at high frequency. Some of these had previously been found to be accumulated at low frequency in the products of ath-miR319a precursor processing and some of them were accompanied by 3′-tailing variant. Potential targets of the undesired small RNAs were discovered in petunia and other Solanaceae plants. The findings draw attention to the potential occurrence of undesired target silencing induced by such additional small RNAs when amiRNA technology is used. No appreciable production of secondary small RNAs occurred, despite the fact that amiRchs1 was designed to have perfect complementarity to its CHS-J target. This confirmed that perfect pairing between an amiRNA and its targets is not the trigger for secondary small RNA production. In conjunction with the observation that amiRNAs with perfect complementarity to their target genes show high efficiency and specificity in gene silencing, this finding has an important bearing on future applications of amiRNAs in gene silencing in plants.
Highlights
MicroRNAs are a class of small RNAs of around 21 nt in length, which are generated from imperfect fold-back regions of long endogenous primary transcripts
CHS gene silencing by an artificial microRNA (amiRNA) in Petunia hybrida In order to suppress the expression of genes in petunia using an amiRNA, the amiRchs1 molecule was designed and its precursor was synthesized as described by Schwab et al [24]
An artificial microRNA was designed for the silencing of petunia CHS genes, following the rules set by Schwab et al [24]
Summary
MicroRNAs (miRNAs) are a class of small RNAs of around 21 nt (nucleotides) in length, which are generated from imperfect fold-back regions of long endogenous primary transcripts (primiRNAs). MiRNAs repress gene expression at the transcriptional, post-transcriptional and translational levels [1]. For the processing of Arabidopsis pri-miR172a, the 14- to 15-base-pair stem region below the miRNA/miRNA* duplex is essential, small unpaired bulges that do not damage its linear structure are tolerated; and a loop is required, mutations in the terminal loop are mostly neutral [16,17]. For the ‘long fold-back’ pri-miRNAs, pri-miR159 and pri-miR319, the processing mechanism is different from that undergone by animal miRNAs and most plant conserved miRNAs; it begins with a cleavage next to the terminal loop, and DCL1 cuts three more times at 20–
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