Profiling of RNA-binding protein binding sites by in situ reverse transcription-based sequencing

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• RNA-binding proteins (RBPs) are crucial regulators of cellular functions through dynamic interactions with RNA.
• Existing methods struggle to capture both stable and transient RBP-RNA interactions, particularly with limited sample sizes or dynamic processes.

Purpose of the Study:

• To develop a novel assay for efficient and specific identification of RBP binding sites.
• To enable the capture of dynamic RBP-RNA interactions, even from limited cellular material.

Main Methods:

• Developed ARTR-seq (assay of reverse transcription-based RBP binding site sequencing).
• ARTR-seq utilizes in situ reverse transcription guided by antibodies to map RBP binding sites.
• The method avoids ultraviolet crosslinking and immunoprecipitation, employing rapid formaldehyde fixation.

Main Results:

• ARTR-seq successfully identifies RBP binding sites from as few as 20 cells or a tissue section.
• The assay captures dynamic RBP-RNA interactions on short timescales (e.g., 10 minutes).
• Demonstrated profiling of dynamic RNA binding of G3BP1 during stress granule assembly.

Conclusions:

• ARTR-seq provides an efficient and sensitive method for mapping RBP binding sites.
• This technique overcomes limitations of existing assays, especially for dynamic interactions and limited samples.
• ARTR-seq facilitates the study of RBP-RNA interactions in various biological contexts.