Transcriptome-wide identification of miRNA targets and a TAS3-homologous gene in Populus by degradome sequencing

Degradome sequencing has been applied to identify miRNA-directed mRNA cleavage and understand the biological function of miRNAs and their target genes in plants defense to stress. miRNAs involved in the response to cold stress have been identified in Populus, however, there are few reports about the validated targets of miRNAs in Populus under cold stress.

The primary objective of this investigation was to globally identify and validate the targets of the miRNAs and regulatory components in Populus under cold stress.

Populus plantlets grown in vitro were treated with cold (4 °C for 8 h) and total RNA was extracted using Trizol reagent. Approximately 200 µg total RNA was used for the construction of the degradome library, and degradome sequencing was conducted on an Illumina HiSeq 2000. The sequences were mapped to Populus genome using SOAP 2.0 and then were collected for degradome analysis. Additionally, trans-acting siRNA sequences from transacting siRNA gene 3 sequences and mature miRNAs cleaved from precursor miRNAs of Populus were analyzed. 5' RNA ligase-mediated-RACE (5'-RACE) were further conducted.

80 genes were experimentally determined to be the target of 51 unique miRNAs, including three down-regulated miRNAs (pto-miR156k, pto-miR169i-m, and pto-miR394a-5p/b-5p) and two up-regulated miRNAs (pto-miR167a-d and pto-miR167f/g). The specificity and diversity of cleavage sites of miRNA targets were validated through 5'-RACE experiment and the results were similar with that of degradome sequencing, further supporting the empirical cleavage of miRNAs on targets in vivo in Populus. Interestingly, the TAS-homologous gene pto-TAS3 (EF146176.1) was identified and 11 potential ta-siRNAs [D1(+)-D11(+)] and their possible biogenesis sites within the pto-TAS3 transcript sequence were predicted in Populus. In addition, the biosynthesis of miRNA from precursor miRNA (pre-miRNA) was also validated through the detection of a total of 17 pre-miRNAs.

Our investigation expands the application of degradome sequencing for evaluating miRNA regulatory elements and evidence of the miRNA synthesis process, and provides empirical evidence of bona fide cleavage of target genes by miRNAs in Populus, which might be used for the research of miRNA-mediated regulation mechanism and molecular improvement of resistance to cold stress.