DIVIDE AND CONQUER: STUDYING MICRORNA REGULATORY NETWORKS BY SEPARATION OF MICRORNA DIRECT TARGETS FROM INDIRECT CHANGES
Patel, Ravi Krishnakant
Post-transcriptional regulation of gene expression is essential for cell function. MicroRNAs (miRNAs), small (~22nt) non-coding RNAs, are negative regulators of post-transcriptional expression. MiRNAs recruit the RNA induced silencing complex (RISC), which includes an Argonaute (AGO) protein, to partially complementary target sites located in 3' untranslated regions (UTRs), and induce accelerated mRNA decay and translational inhibition of target mRNAs. Each miRNA has potential to regulate many hundreds of mRNAs via this pathway. Although, miRNAs typically elicit a modest effect per mRNA, hundreds of such small changes result in a substantial cumulative impact on the transcriptome. Additionally, miRNA regulatory networks incorporate master regulators such as transcription factors downstream of miRNAs, enabling miRNAs to trigger substantial changes in gene regulatory networks. Understanding the biological impact of miRNAs requires knowledge of their targets, and robust distinction of miRNA direct targets from cascading downstream regulatory changes remains challenging. In this work, I developed a simple experimental approach to robustly uncouple post-transcriptional and transcriptional changes using RNA-seq and Precision Run-On sequencing (PRO-seq), a method for profiling actively transcribing RNA polymerases. The net change in mRNA abundance results from changes in synthesis and decay. I demonstrated that by subtracting the changes in mRNA synthesis (PRO-seq) from the changes in mRNA abundance (RNA-seq), a robust estimate of post-transcriptional regulation by miRNAs could be derived. I refer to this approach as CARP: Combined Analysis of RNA-seq and PRO-seq. Using CARP, I successfully separated true direct targets of specific miRNAs from the downstream indirect changes, which I validated using orthogonal assays such as Argonaute eCLIP-seq and ribosome profiling. Additionally, CARP analysis revealed that the majority of miRNAs used in the study elicited sizable indirect targeting at both transcriptional and post-transcriptional levels, which are often disregarded. Using motif enrichment analysis, I found candidate transcription factors underlying the miRNA-mediated indirect regulation at transcriptional level. I also demonstrated that CARP facilitates effective dissection of complex regulatory changes triggered by miRNAs. Furthermore, my analysis revealed that many miRNAs elicit discernible repression of target sites located in open reading frames (ORFs); the significance of ORF target sites is a potentially important aspect of miRNA biology, but the extent to which it occurs has been controversial. My data demonstrated that while ORF sites to certain miRNAs often mediate subtle repression, their likely role is in assisting the miRNA-mediated regulation of weaker 3'UTR sites to collectively elicit significant post-transcriptional repression of the target mRNA. Overall, the tools I developed in my graduate work facilitate robust distinction of direct target from indirect regulatory changes aiding in the study of miRNA regulatory networks at the systems-level. Finally, I apply CARP and other genomic approaches to better understand biological roles of miRNAs in the immune response of CD8+ T-cells in mouse models.
CARP; CD8+ T-cells; Gene regulatory networks; Genomics; miRNA targets; Post-transcriptional regulation
Grimson, Andrew W.
Yu, Haiyuan; Lis, John T.
Genetics, Genomics and Development
Ph. D., Genetics, Genomics and Development
Doctor of Philosophy
Attribution-NonCommercial-NoDerivatives 4.0 International
dissertation or thesis
Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivatives 4.0 International