# Target specificity of human RNA-induced silencing complex

> **NIH NIH R01** · OHIO STATE UNIVERSITY · 2020 · $299,134

## Abstract

PROJECT SUMMARY
In eukaryotic cells, gene expression is controlled at multiple different layers. One of them is post-
transcriptional gene silencing where microRNAs (miRNAs) bind target mRNAs in a sequence complementary
manner and cause translational repression and/or deadenylation. In humans, miRNAs are loaded onto one of
four Argonaute proteins (AGOs), forming a ribonucleoprotein complex terms `RNA-induced silencing complex
(RISC)'. The target specificity of the RISC has been defined solely by the base complementarity between the
miRNA (guide) and target strands. The loaded guide strand occupies part of the nucleic acid-binding
channel between the AGO N-terminal and C-terminal lobes, while the remaining space serves as the
composite target-binding channel. In this study, we hypothesize that the target specificity of the RISC is
defined by the structure of the composite channel rather than just base complementarity, and thus that four
human AGOs possess different target specificities due to their unique local structures. To validate this
hypothesis, we will pursue the following specific aims. In Aim 1, cleavage assay and chemical probing will be
used to determine how differently target strands are recognized in the presence and absence of the N-
terminal lobe. In Aim 2, X-ray crystallography will be used to solve the structure of human AGO3-RISC. This
structure, along with the previously determined ones, will enable us to identify local structures making their
target-binding channels different from each other. RNA bind-n-seq experiments using wild type and its
mutant lacking the identified unique local structure(s) will determine the target specificities conferred by the
characteristic target-binding channel. In Aim 3, filter-binding assays and chemical probing will be used to
elucidate the molecular mechanism by which a miRNA, miR-3191-5p, activates only AGO4 for binding to the
internal ribosome entry site (IRES) of CACNA1A mRNA, and blocks its IRES-driven translation to prevent the
neurological disease. Outcomes from this study will provide a new concept on the target specificity of the
RISC, which is significant because beyond canonical gene silencing, many different cellular bioprocesses are
regulated by miRNAs.

## Key facts

- **NIH application ID:** 9980454
- **Project number:** 5R01GM124320-04
- **Recipient organization:** OHIO STATE UNIVERSITY
- **Principal Investigator:** Kotaro Nakanishi
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $299,134
- **Award type:** 5
- **Project period:** 2017-09-15 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9980454

## Citation

> US National Institutes of Health, RePORTER application 9980454, Target specificity of human RNA-induced silencing complex (5R01GM124320-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9980454. Licensed CC0.

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