MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that post-transcriptionally regulate gene expression of more than half of human messenger RNAs (mRNAs); aberrant miRNA levels are linked to disease, including cancer. miRNAs associate with Argonaute (Ago) proteins and guide them to complementary sequences on mRNAs resulting either in mRNA suppression upon partial base-paring, or in Ago2-mediated mRNA cleavage when perfect base-pairing occurs. Recently Dr. Steitz?s group and others discovered yet another outcome of miRNA base-pairing for three herpesviral RNAs that exhibit extensive, yet imperfect, complementarity to host miRNAs, and result in degradation of the miRNAs. This process, of a still uncharacterized mechanism, is known as target-induced miRNA degradation (TIMD) and the miRNA-binding site is called the miRNA-degradation element (miR-DE). For example, HSUR1 ? a ncRNA encoded by Herpesvirus saimiri (HVS) binds to and mediates decay of host miR-27a. miR-27a is a repressor of T-cell activation and its decreased levels contribute to HVS-induced lymphomas. Importantly, while the biogenesis of miRNAs has been extensively studied, their turnover is poorly understood. The presence of miR-DE-containing viral RNAs alone is sufficient to induce miRNA decay, suggesting that TIMD is an undescribed cellular miRNA decay mechanism. In addition, other known herpesviral miR-DEs reside in mRNAs, implying the existence of analogous elements in other viral and human transcripts. In this study, I intend to delineate the novel miRNA degradation machinery (Aim 1), as well as to identify yet unknown transcripts derived from oncogenic viruses and their hosts that induce miRNA decay (Aim 2). In the K99 phase, I will identify TIMD intermediates by adapting TimeLapse ? a method developed in the lab of our collaborator Dr. Matthew Simon that assesses RNA turnover using single time point high-throughput sequencing. I will gain insight into TIMD mechanism by obtaining a crystal structure of Ago2-miR-27a-HSUR1 complexes in collaboration with Dr. Ian MacRae. I will validate predicted miR-DEs encoded by oncogenic Epstein-Barr virus that target tumor suppressor miRNAs (miR-150 and miR-125b) by analyzing their ability to induce miRNA decay in B cells. I will construct a fluorescent protein-based reporter responding to changes in miRNA levels to perform a loss-of- function genetic screen to identify cellular enzymes participating in TIMD. In the R00 phase, I will characterize the candidates for TIMD obtained in the genetic screen. I will search for additional RNA motifs necessary for miR-DE function by using bioinformatics and mutagenesis. I will then use a refined bioinformatic pipeline to search for human miR-DEs. This study will delineate the miRNA turnover mechanism, which could be linked to disease, and will bioinformatically identify potentially oncogenic viral and human transcripts. The Pathway to Independence Award will allow me to gain necessary expertise (i.e. in bioinformatics) and professional skills (i.e. in teaching, writing) to become a competitive candidate for an academic position at a top-tier university.
Recent identification of three herpesviral transcripts that selectively induce degradation of host microRNAs will enable delineation of a yet unclear cellular microRNA decay machinery. Discovery of additional viral transcripts with microRNA decay-inducing properties will advance knowledge of how human herpesviruses induce cancers and will contribute to development of new diagnostic and therapeutic approaches.
