Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon

Authors: JEONG, DONG-HOON - University Of Delaware; SCHMIDT, SKYE - University Of Delaware; RYMARQUIS, LINDA - University Of Delaware; PARK, SUNHEE - University Of Delaware; GANSSMANN, MATTHIAS - University Of Delaware; GERMAN, MARCELO - University Of Delaware; ACCERBI, MONICA - University Of Delaware; ZHAI, JIXIAN - University Of Delaware; DEPAOLI, EMANUELE - University Of Delaware; FAHLGREN, NOAH - Danforth Plant Science Center; FOX, SAMUEL - Oregon State University; Garvin, David; MOCKLER, TODD - Danforth Plant Science Center; CARRINGTON, JAMES - Danforth Plant Science Center; MEYERS, BLAKE - University Of Delaware; GREEN, PAMELA - University Of Delaware

Submitted to: Genome Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/24/2013
Publication Date: 12/24/2013
Citation: Jeong, D., Schmidt, S.A., Rymarquis, L.A., Park, S., Ganssmann, M., German, M.A., Accerbi, M., Zhai, J., Depaoli, E., Fahlgren, N., Fox, S.E., Garvin, D.F., Mockler, T.C., Carrington, J.C., Meyers, B.C., Green, P.J. 2013. Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon. Genome Biology. 14:R145.

Interpretive Summary: In crops, when and where genes are active in the plant is of paramount importance because this controls how a plant develops, adapts to environmental stresses, and ultimately produces a harvestable product. Understanding how to control key genes associated with crop productivity is a major goal in agricultural biotechnology. Small molecules called microRNA (miRNA) have been proposed to provide one level of control over the activity of genes. In this study, the small grass species Brachypodium distachyon, a model system used to accelerate biological discovery relevant to improving cereal crops and biofuel grasses to which it is related, was grown under a variety of stresses including drought, salt, cold, heat, submergence and nutrient deficiency. These plants were then used to identify 116 miRNAs, including both new and previously reported ones. Some of the miRNAs are present in other plant species, while a set of them appear to be unique to Brachypodium. Further, it was possible to identify nearly 250 genes that these miRNAs play a role in regulating. The results of this study will open new research avenues to enhance the productivity of important grass crop relatives of Brachypodium by improving their tolerance to environmental stresses that limit crop productivity.

Technical Abstract: The wild grass Brachypodium distachyon (Brachypodium) has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of microRNAs (miRNAs), molecules known to be a key for eukaryotic gene regulation, has been limited in Brachypodium to studies examining a few samples or that rely on computational predictions. The use of Parallel Analysis of RNA Ends (PARE) or other RNA degradome data to identify precisely cleaved miRNA targets has yet to be reported. Brachypodium small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in Brachypodium, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of ~70 million sequences. The ~5 million distinct genome-matched sequences that resulted represent an extensive dataset to analyze small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets that were characterized in more detail. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. Brachypodium miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insight about the roles of miRNAs and the regulation of their targets in Brachypodium and related plants.