Related Experiment Video
Updated: Oct 4, 2025
Enhanced Crosslinking Immunoprecipitation eCLIP Method for Efficient Identification of Protein-bound RNA in Mouse Testis
Published on: May 10, 2019
Identification of Protein-RNA Interactions in Mouse Testis Tissue Using fRIP
Alexis S Bailey1, Pedro J Batista2, Howard Y Chang3
1Department of Developmental Biology, Stanford University School of Medicine, Stanford, United States.
Abstract:
During development, cells must quickly switch from one cell state to the next to execute precise and timely differentiation. One method to ensure fast transitions in cell states is by controlling gene expression at the post-transcriptional level through action of RNA-binding proteins on mRNAs. The ability to accurately identify the RNA targets of RNA-binding proteins at specific stages is key to understanding the functional role of RNA-binding proteins during development. Here we describe an adapted formaldehyde RNA immunoprecipitation (fRIP) protocol to identify the in vivo RNA targets of a cytoplasmic RNA-binding protein, YTHDC2, from testis, during the first wave of spermatogenesis, at the stage when germ cells are shutting off the proliferative program and initiating terminal differentiation ( Bailey et al., 2017 ). This protocol enables quick and efficient identification of endogenous RNAs bound to an RNA-binding protein, and facilitates the monitoring of stage-specific changes during development.
Insights
Researchers developed a new method to track RNA targets of RNA-binding proteins during cell development. This technique helps understand how proteins like YTHDC2 control gene expression for precise cell differentiation.
Area of Science:
- Developmental biology
- Molecular biology
- Genetics
Background:
- Cell differentiation requires precise gene expression control.
- Post-transcriptional regulation by RNA-binding proteins is crucial for rapid cell state transitions.
- Identifying RNA targets of RNA-binding proteins is key to understanding their developmental roles.
Purpose of the Study:
- To adapt and describe a formaldehyde RNA immunoprecipitation (fRIP) protocol.
- To identify the in vivo RNA targets of the RNA-binding protein YTHDC2.
- To analyze stage-specific RNA targets during the first wave of spermatogenesis.
Main Methods:
- Adapted formaldehyde RNA immunoprecipitation (fRIP) protocol.
- Immunoprecipitation of YTHDC2 protein from testis cells.
- Identification of bound endogenous RNAs using sequencing.
Main Results:
- Successfully identified in vivo RNA targets of YTHDC2 during early spermatogenesis.
- Demonstrated the protocol's efficiency in capturing stage-specific RNA-protein interactions.
- Provided insights into YTHDC2's role in germ cell differentiation.
Conclusions:
- The adapted fRIP protocol is effective for identifying endogenous RNA targets of RNA-binding proteins.
- This method facilitates the study of stage-specific regulatory events during development.
- Understanding YTHDC2-RNA interactions is vital for comprehending germ cell differentiation.
