# Mechanism to regulate the length of small silencing RNAs

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2021 · $343,875

## Abstract

Abstract
Post-transcriptional gene silencing (PTGS) mediated by small silencing RNAs such as ~22-24 nt
microRNAs (miRNAs) and ~21 nt small interfering RNAs (siRNAs) produced by Dicer enzymes, is
important in every aspect of biology. Making small RNAs with an appropriate length is crucial for their
functions. Small RNAs with incorrect lengths, which can be produced if Dicer selects incorrect cleavage
sites in precursor RNAs, can cause detrimental effects in cells. Thus, understanding the molecular
mechanisms by which the length of small silencing RNAs produced by Dicer are determined and
regulated is significant and is the goal of research in this proposal.
 Drosophila Dicer-1 makes ~22-24 nt miRNAs from pre-miRNAs while Dicer-2 precisely produces
~21 nt siRNAs from long dsRNAs derived from virus and transposon. We recently reported that the
miRNA length produced by Drosophila Dicer-1 and mammalian Dicer can be changed by its binding
partner proteins Loquacious-PB (Loqs-PB) in Drosophila and TRBP in mammals, respectively, but not
by their alternative partners Loqs-PA and PACT. However, the molecular mechanism by which Loqs-
PB/TRBP, but not Loqs-PA/PACT, changes the length of miRNAs is unknown, and we will aim to better
understand it. We will test the hypothesis that Loqs-PB/TRBP, but not Loqs-PA/PACT, binds the
unpaired bases at the central part of the pre-miRNA stem and changes their conformation in order to
change the miRNA length.
 In contrast to miRNAs that have a broader length distribution (~22-24 nt), the length of ~21 nt
siRNAs is more precise. The difference in the length between miRNAs and siRNAs is important for their
respective functions. However, how Drosophila Dicer-2 can produce siRNAs with such high precision is
unknown. Based on our previous and preliminary results, we will test the hypothesis that the
phosphate-binding pocket in the PAZ domain of Dicer-2 plays crucial roles to produce precise ~21 nt
siRNAs. We also hypothesize that the C-terminal dsRNA-binding domain (dsRBD) of Dicer-2
additionally contributes to the precision of the ~21 nt siRNA production. We hypothesize that the
phosphate-binding pocket and C-terminal dsRBD anchors the terminal monophosphate and body of
long dsRNAs, respectively, thereby aligning the RNAs precisely along the RNaseIII active site.
 To achieve these aims, we will use Drosophila genetics, reconstituted in vitro biochemistry, and
high-throughput sequencing. The proposed studies will reveal the molecular mechanisms by which the
length of miRNAs and siRNAs are defined and regulated, which will advance our understanding of
sequence-specific post-transcriptional gene silencing.

## Key facts

- **NIH application ID:** 10236331
- **Project number:** 5R01GM116841-05
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Ryuya Fukunaga
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $343,875
- **Award type:** 5
- **Project period:** 2017-09-08 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10236331

## Citation

> US National Institutes of Health, RePORTER application 10236331, Mechanism to regulate the length of small silencing RNAs (5R01GM116841-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10236331. Licensed CC0.

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