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In-vitro Mutagenesis

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To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

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PIWI-interacting RNAs, or piRNAs, are the most abundant short non-coding RNAs. More than 20,000 genes have been found in humans that code for piRNAs while only 2000 genes have been found for miRNAs. piRNAs can act at the transcriptional and post-transcriptional levels and have a vital role in silencing transposable elements present in germ cells. They are also involved in epigenetic silencing and activation. Previously, they were thought to function only in germ cells but new evidence suggests...

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Related Experiment Video

Updated: Aug 14, 2025

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

Published on: December 28, 2021

In vivo PIWI slicing in mouse testes deviates from rules established in vitro.

Mark Dowling¹, David Homolka¹, Nicole Raad¹

¹Department of Molecular Biology, University of Geneva, CH-1211 Geneva 4, Switzerland.

RNA (New York, N.Y.)

|January 8, 2023

Summary

This summary is machine-generated.

PIWI Argonautes can cleave mismatched RNA targets in vivo, contradicting established in vitro rules. This PIWI slicing does not initiate new piRNA production from target mRNAs.

Keywords:

MILI MIWI PIWI cleavage-site mismatch pachytene piRNA slicer

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Area of Science:

Molecular Biology
RNA Biology
Genetics

Background:

Argonaute proteins, including PIWI clade members, bind small RNAs and can cleave target nucleic acids.
PIWI-associated RNAs (piRNAs) are crucial for silencing transposable elements in animal gonads, ensuring fertility.
In vitro studies previously showed PIWI slicers cannot cleave targets with mismatches at the cleavage site.

Discussion:

This study challenges established in vitro PIWI slicing rules by demonstrating in vivo cleavage of targets with mismatches.
The findings reveal that PIWI slicing in vivo is more permissive than previously understood.
The precise slicing of mismatched targets in vivo mirrors that of perfectly matched targets.

Key Insights:

PIWI slicing occurs in vivo even with nucleotide mismatches between piRNA and target RNA at the cleavage site.
This in vivo PIWI slicing does not lead to phased piRNA production from the targeted mRNA.
Cleavage fragments are retained within PIWI proteins, suggesting a distinct fate from canonical piRNA biogenesis.

Outlook:

Re-evaluation of PIWI-mediated gene silencing mechanisms and their regulation in vivo.
Further investigation into the structural and biochemical basis for PIWI's altered substrate specificity in vivo.
Exploring the implications of PIWI slicing on piRNA biogenesis pathways and genome integrity.