Functional analysis of conserved microRNAs in Arabidopsis thaliana
Creators: name: Li, Yanjiao
Publisher: name: The Australian National University
Year: 2015
Subjects: miR159-MYB pathway
miRNA decoy
Arabidopsis

description: MicroRNAs (miRNAs) are non-coding RNAs of approximately 21 nucleotides that negatively regulate gene expression. In plants, many miRNAs target key regulatory genes, such as transcription factors, which play critical roles in plant developmental processes and stress responses. Underscoring their importance, many of these miRNA-target relationships are highly conserved, such as the miR159-MYB regulatory module that appears present in all land plants. This regulatory relationship has been extensively studied in Arabidopsis. In seeds and flowers, where miR159 activity is weak, MYB activity promotes programmed cell death (PCD), facilitating seed germination and anther development respectively. Moreover, the generation of a loss-of-function mir159 mutant has been shown to result in pleiotropic vegetative defects (e.g. curled/rounded leaves and stunted growth), indicating a critical role of miR159 in controlling rosette development. However, what the funcational role of this regulatory module in rosettes remains unknown. Via transcript analysis, I found that miR159 was strongly and ubiquitously expressed throughout the rosette development, where it appeared to constitutively silence MYB activity, suggesting the miR159-MYB module is not developmentally responsive. However, constant miR159 activity is required for normal rosette growth, as an inducible inhibition of miR159 resulted in morphological abnormalities of the rosette. This led to the hypothesis that miR159 could be stress responsive, where if repressed under a particular condition this would enable MYB expression. However, under what environmental condition this occurs remains unclear, because miR159 silencing was found to be extremely robust: neither biotic stresses known to inhibit miRNA activity (a virus containing a silencing suppressor), nor a range of abiotic stresses was able to inhibit miR159 function sufficiently to activate MYB-related phenotypic defects. As a complementary approach to gain insights into the function of the MYB33/65 pathway, an ethyl methanesulfonate (EMS) mutagenesis screen of mir159 suppressor was undertaken. Interestingly, a high frequency of mir159 revertants was obtained, in which MYB expression was attenuated, suggesting the existence of multiple regulators of MYB expression, and that possibly the miR159-MYB module is extensively networked. The eventual identification of these repressor alleles may shed light on function of the miR159-MYB module in rosettes. Owing to the fact that most miRNAs belong to multigene families, to which traditional loss-of-function approaches cannot be applied due to genetic redundancy, the transgenic approach of using miRNA "SPONGEs (SPs)" was explored to determine their ability in generating loss-of-function mirna outcomes. SPs are transgenes that harbor multiple target sites complementary to miRNAs, leading to their sequestration, an approach that has been effective in animal systems, but not tested in plants. Here, ten miRNA SPs were designed to target different conserved plant miRNA families. Their efficacies in inhibiting the respective miRNAs varied dramatically, where some SPs induced a strong loss-of-function outcome, whereas others did not. What underpins this variability is unclear; neither SP expression level, the free energy (ΔG) of the miRNA-SP interaction or the predicted target accessibility correlated with inhibition efficacies. This likely highlights the complexity of miRNA-target interactions and that when a SP cannot induce any obvious phenotypic impact, alternative approaches will be required.