Abstract
Temporal and spatial control of transcript abundance for expressed genes is crucial for many biological processes and developmental programs. In eukaryotes, gene expression regulation occurs at many levels. At the post-transcriptional level, microRNAs (miRNAs) are emerging as an important class of sequence-specific, trans-acting endogenous small RNA molecules that modulate gene expression. It is well-established that many miRNAs are crucial for diverse plant development processes and responses to environmental challenges. However, three fundamental and inherently related questions wait answers: 1) how many miRNAs are there in plant species; 2) how does regulation from miRNAs cooperate with other gene regulation mechanisms; and 3) how does miRNAs as a group play roles in a specific pathway. This dissertation uses a combination of cutting-edge bioinformatic and experimental approaches to address these fundamental questions at a genomic level.
Ultra deep sampling of small RNA libraries by next generation sequencing has provided rich information on the miRNA transcriptome of various plant species. However, few computational tools have been developed to effectively de-convolute the complex information. I sought to employ the signature distribution of small RNA reads along the miRNA precursor as a model in plants to profile expression of known miRNA genes and to identify novel ones. A freely available package, miRDeep-P, was developed subsequently, which could comprehensively and accurately identify miRNAs from deeply sequenced small RNA libraries. Taking advantage of this method, I have examined miRNAs in 15 plant species and identified thousands of miRNAs. With this unprecedented dataset, several exciting findings were made, including that the number of miRNAs is strongly correlated with the number of protein-coding genes, and that plant miRNAs in a compact organization suggest several possible miRNA biogenesis mechanisms utilizing existing precursors, and many other clues on miRNA evolution.
Toward an understanding of the cooperation of miRNA-centered network with other gene regulation mechanisms, I investigated and discovered a novel genetic mechanism that links miRNAs with alternative mRNA splicing. By compiling a set of miRNA target genes and going through millions of pieces of RNA sequencing data, it was found for the first time in plants that mRNA isoforms produced by alternative splicing differ in the sequences encoding the miRNA binding sites. In collaboration with a postdoctoral researcher in my lab, it has been functionally shown that these alternative splicing events are relevant in controlling plant development.
Another piece of effort in this dissertation is to perform genetic experiments to test how miRNAs as a group interact with other pathways in response to external stimuli. Systemin-mediated pathway in tomato was selected as it has been largely understood in last decade. Genetic experiments combining with high-throughput data analyses indicate that changes in systemin-mediated pathway could affect the expression of many miRNAs and that a large number of miRNAs, in turn, are involved in this pathway. Examination of phenotypic changes in several transgenic lines further suggests that many miRNAs as a group potentially provides a buffering role when plants meet an intense stimulation.
