IC4R008-miRNA-2011-21113019

Project Title

• Identification and analysis of seven H2O2-responsive miRNAs and 32 new miRNAs in the seedlings of rice (Oryza sativa L. ssp. indica)

The Background of This Project

• Plant microRNAs (miRNAs) have been shown to play critical roles in regulating gene expression at the post-transcriptional level. In this study, the researchers employed high throughput sequencing combined with computational analysis to survey miRNAomes from the seedlings of rice under normal conditions and treatments of H2O2 that result in oxidative stress.

Plant Culture & Treatment

• Rice (Oryza sativa L. ssp indica cv. 93-11) seedlings were grown in a growth chamber with 28/21℃ (16 h day/8 h night) and relative humidity of 70% as reported earlier (4). For H2O2 treatment, 12-day-old seedlings were treated with three H2O2 concentrations (0.6, 3.0 and 15.0mM) for 6 h in plastic containers, respectively (4). Seedlings immersed in dd H2O were used as a control. The rice seedlings were snap-frozen in liquid nitrogen, and then stored at -80℃ for RNA extraction.

Research Findings

• Comparison of the miRNAomes and subsequent northern blot analysis(Figure 1) identified seven miRNAs (miR169, miR397, miR528, miR827, miR1425, miR319a.2 and miR408-5p) can be considered to be H2O2-responsive miRNAs because they are expressed differentially between H2O2-treated and control samples in northern blot analysis and their predicted targets all have specific function information(Table 1).

Figure 1. Northern blot analysis of the known miRNA families.

Table 1. H2O2-responsive miRNA familes and their targets.

• Predicted and experimentally validated targets of these H2O2-responsive miRNAs are involved in different cellular responses and metabolic processes including transcriptional regulation, nutrient transport, auxin homeostasis, cell proliferation and programmed cell death. This indicates that diverse miRNAs form a complex regulatory network to coordinate plants’responses under oxidative stress.

• The researchers also discovered 32 new miRNAs in the seedlings of rice,18 from known miRNA loci and 14 from novel miRNA loci. Interestingly, of these new miRNAs, miR3981 was originally found to be a putative exonic miRNA located in the exon of AK106348(Figure 2), suggesting that plants may also use some exons as an miRNA source.

Table 2. (A) miR3981 is located in the last exon of AK106348. The hairpin represents the miR3981 stem-loop and the blue boxes indicate the exons. (B) Small RNAs are generated from the miR3981 precursor. miR3981-5p and miR3981-3p are shown in blue and red, respectively. Na, number of reads; Lb, length of sequences. (C) Mapping of the cleavage site in the AK106348 mRNA by RNA ligase-mediated 50 RACE. The arrow indicates the cleavage site and the number shows the frequency of clones sequenced.

Labs working on this Project

• Laboratory of Molecular Biology and MOE Laboratory of Protein Science, School of Life Sciences, Tsinghua University, Beijing 100084, P. R. China

Corresponding Author

• liujy@mail.tsinghua.edu.cn