Mechanisms for modulation of miRNA-mediated gene silencing

点击翻译按钮获取中文摘要

Abstract In eukaryotic cells, gene expression is regulated at multiple levels. Post-transcriptional regulation is, in part, mediated by micro RNAs (miRNAs), which act within the miRNA-induced silencing complex (miRISC). However, susceptibility of mRNAs to miRNA-mediated silencing appears to depend on the cell type and on the features of mRNA target itself. Our recent studies indicate that the stability and translation efficiency of miRNA- targeted mRNAs are determined by interactions between miRISC and RNA-binding proteins (RBPs). In this proposal, we seek to elucidate the basic mechanism of miRISC-mediated translational repression and its modulation by RBPs and target mRNAs, in a cell-type-specific manner. In Aim 1, we intend to determine how miRNAs, as part of the miRISC, repress translation during the initiation phase. Specifically, we will seek to determine the role of eIF4A helicase and the eIF4F complex in miRNA-mediated translational repression during translation initiation and 43S ribosome scanning. To achieve this goal we will use massively parallel reporter assays (MPRA), CRISPR/Cas-9 engineered cells, and translation inhibitors. We will use the same approach to analyze the mechanism through which Pumilio and AU-rich (ARE) binding sites affect miRNA-mediated translational repression. In Aim 2, we will ascertain how AU-rich motifs modulate miRNA- mediated repression of target mRNAs. Specifically, we will determine the mechanism by which AREs and ARE-binding proteins (ARE-BPs) can up- or down-regulate miRNA-mediated gene silencing in a cell-specific manner. Lastly, we will use immunoprecipitation of ARE-BPs and target mRNA and test interaction between miRISC, ARE-BPs, and mRNA, to determine how ARE-BPs' binding to target mRNAs in a positionally biased manner interferes with mRISC-mRNA target interaction to modulate gene regulation. In Aim 3, we will elucidate how modulation of miRNA-mediated gene silencing by RBPs shapes the differentiation of mouse neurons and results in cell-specific effects distinguishing neurons and astrocytes in the mature CNS. Specifically, we will analyze individual and combinatorial effects of miRNAs and RBPs using reporter libraries, endogenous mRNAs, polysome profile analyses, and translating ribosome affinity purification (TRAP) to evaluate specialization of gene expression in murine brain cell types. Our central hypothesis is that cell-type- specific interactions between the target mRNAs, RBPs, and miRISC determine the extent of miRNA-mediated translational repression and mRNA decay. Our long-term goal is to define how miRNA-mediated gene expression control is established in a cell-type-specific manner by modulation of RBPs and features of target mRNAs. The nervous system, with an exquisite heterogeneity of cells, represents a great structure to test our hypotheses. Together, these experiments will reveal why specific mRNAs respond robustly to miRISC while others do not, and how the modulation of miRNA–mediated gene silencing is established in a cell-specific manner by the activity and presence of RBPs as well as sequence features of target mRNAs.