# Dissection of the piRNA biogenesis pathway in germ cells

> **NIH NIH R01** · THOMAS JEFFERSON UNIVERSITY · 2020 · $373,760

## Abstract

Project Summary and Abstract
The small regulatory RNA pathway centered on Piwi (P-element-induced wimpy testis) proteins and their bound
Piwi-interacting RNAs (piRNAs) silence transposons and other targets to maintain genome integrity in animal
germlines. Disruption of the piRNA pathway causes elevation of transposon levels and defects in germline
development, eventually resulting in sterility of the animals. The piRNA pathway thus functions as guardians of
the germline genome to ensure genetic information is passed onto the next generation. Although the crucial
function of piRNAs in germline development is evident, the factors and mechanisms mediating piRNA biogenesis
remain elusive. Biogenesis of the piRNAs starts by transcription of long single-stranded RNAs from defined
genomic regions termed piRNA clusters. Precursors transcripts are likely processed into shorter primary
precursors. However, profiles and mechanisms of the processing for primary piRNA precursors are not well
elucidated. The primary precursors are further processed by the mitochondria-associated enzyme Zucchini to
generate pre-piRNAs, which are loaded onto Piwi proteins where they undergo 3′-end processing and
methylation to become mature piRNAs. Toward our research goal to clarify the biogenesis pathway of piRNAs,
we have been utilizing Bombyx mori ovary-derived BmN4 cells which endogenously express Piwi proteins and
piRNAs. The scarcity of piRNA-expressing culturable cells make BmN4 cells suitable and well used for piRNA
biogenesis research. In the cells, piRNAs are produced abundantly from tRNAs, and we recently revealed the
biogenesis mechanism of the tRNA-derived piRNAs, in which 5′-tRNA halves, not mature tRNAs, become direct
precursors for piRNAs. Our finding of a 2′,3′-cyclic phosphate (cP) at the 3′-end of 5′-tRNA halves prompted us
to hypothesize that cP-containing RNAs (cP-RNAs) form piRNA precursors. Our hypothesis was strengthened
by our further analyses showing that, in BmN4 cells, cP-RNAs are abundantly accumulated at the outer
membrane of mitochondria where piRNA biogenesis takes place, and that these cP-RNA sequences are
extensively overlapped with piRNA sequences. cP-RNAs form a hidden layer of the transcriptome because
standard RNA-seq is unable to capture them. We have developed a cP-RNA-seq method that can selectively
sequence cP-RNAs, and we will utilize it to comprehensively identify the cP-RNAs expressed in Bombyx BmN4
cells, Drosophila Kc167 cells, and mouse testes in order to investigate their expressional relationship with
piRNAs (Aim 1). We will further elucidate the interaction of cP-RNAs with Piwi proteins and a piRNA biogenesis
factor BmPapi (Aim 2), and characterize an RNase producing cP-RNAs in piRNA biogenesis (Aim 3). The
proposed studies will fuel our novel research efforts to understand the mechanism of piRNA biogenesis by
focusing on currently-uncharacterized cP-RNAs, and support biomedical goals to understand the pathogenesis
of reproduct...

## Key facts

- **NIH application ID:** 9985135
- **Project number:** 5R01GM106047-07
- **Recipient organization:** THOMAS JEFFERSON UNIVERSITY
- **Principal Investigator:** Yohei Kirino
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $373,760
- **Award type:** 5
- **Project period:** 2013-08-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9985135

## Citation

> US National Institutes of Health, RePORTER application 9985135, Dissection of the piRNA biogenesis pathway in germ cells (5R01GM106047-07). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9985135. Licensed CC0.

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