Transcriptome-wide high-throughput mapping of protein-RNA occupancy profiles using POP-seq.

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Protein-RNA interactions are crucial for gene regulation.
• Current methods for mapping these interactions have limitations, including biases and inability to detect interactions with non-poly-A RNAs.

Purpose of the Study:

• To introduce and validate Protein Occupancy Profile-Sequencing (POP-seq), a novel method for unbiased mapping of protein-RNA interactions.
• To assess the ability of POP-seq to identify interaction sites across the whole transcriptome, including non-poly-A RNAs.

Main Methods:

• Developed three versions of POP-seq, a phase separation-based technique.
• One POP-seq variant eliminates the need for crosslinking, enabling unbiased profiling.
• Applied POP-seq to map protein occupancy across the whole cell transcriptome.

Main Results:

• POP-seq identified protein occupancy profiles with significant overlap with existing RBP interaction data.
• POP-seq successfully captured interactions on diverse RNA types, including transcription factors, RBPs, and lncRNAs.
• Enrichment of POP-seq peaks with GWAS SNPs and cancer-related variants suggests functional significance of protein-occupied RNA sites.

Conclusions:

• POP-seq is a robust, cost-effective, and unbiased method for mapping global protein-RNA interactions.
• The method can be applied to primary tissues for comprehensive analysis.
• Findings highlight the role of lncRNAs in sponging RBPs and potentially rewiring the interactome in cancer.