Monitored eCLIP: high accuracy mapping of RNA-protein interactions

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• CLIP-seq is crucial for mapping RNA-protein interactions in vivo.
• Reverse transcriptase read-throughs at cross-linked sites introduce errors in binding site identification.
• Existing methods struggle with the unpredictable rate of these read-through events.

Purpose of the Study:

• To introduce and validate the monitored enhanced CLIP (meCLIP) method.
• To improve the reliability and precision of RNA-protein binding site mapping.
• To provide a robust approach for analyzing transcriptome-wide RNA-protein interactions.

Main Methods:

• Ligation of a barcoded biotinylated linker to 5' ends of cross-linked RNA fragments.
• Purification of RNA fragments before reverse transcription.
• Bioinformatic filtering of cDNAs containing barcode sequences, indicative of read-through events.

Main Results:

• meCLIP effectively identifies and removes reverse transcription read-throughs.
• Filtering these reads significantly enhances the accuracy of protein binding site assignment.
• The proportion of read-throughs can be substantial, up to 25% of total reads.

Conclusions:

• meCLIP offers a more reliable and precise method for mapping RNA-protein interactions.
• This technique addresses a key limitation in standard CLIP-seq protocols.
• meCLIP is valuable for accurate transcriptome-wide analysis of RNA-binding proteins.