# Mechanisms for modulation of miRNA-mediated gene silencing

> **NIH NIH R01** · WASHINGTON UNIVERSITY · 2020 · $340,673

## Abstract

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–mediate...

## Key facts

- **NIH application ID:** 10121629
- **Project number:** 2R01GM112824-06
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Sergej Djuranovic
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $340,673
- **Award type:** 2
- **Project period:** 2015-09-01 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10121629

## Citation

> US National Institutes of Health, RePORTER application 10121629, Mechanisms for modulation of miRNA-mediated gene silencing (2R01GM112824-06). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10121629. Licensed CC0.

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