GCLiPP: global crosslinking and protein purification method for constructing high-resolution occupancy maps for RNA...

Area of Science:

• Molecular Biology
• Genomics
• RNA Biology

Background:

• RNA-binding proteins (RBPs) play crucial roles in gene regulation.
• Understanding RBP occupancy transcriptome-wide is essential for deciphering gene expression control.
• Existing methods for mapping RBP binding sites have limitations in resolution and scope.

Purpose of the Study:

• To introduce and validate Global RNA interactome capture (GCLiPP), a novel method for transcriptome-wide RBP occupancy mapping.
• To compare RBP occupancy patterns in human and mouse T cells.
• To identify conserved cis-regulatory elements and their functional implications.

Main Methods:

• GCLiPP was employed to capture and sequence RNA bound by RBPs across the transcriptome.
• GCLiPP data was compared with known RBP binding sites and crosslinking immunoprecipitation (CLIP) data.
• Comparative analysis was performed between human Jurkat T cells and mouse primary T cells.
• GCLiPP signal was analyzed for overlap with single nucleotide polymorphisms (SNPs) in immune-related genes.

Main Results:

• GCLiPP provides high-resolution mapping of RBP-occupied sites, surpassing phase separation-based techniques.
• GCLiPP sequence tags correlate with established RBP binding sites and are enriched for abundant cytosolic RBPs.
• Shared GCLiPP signal peaks were identified in homologous 3' UTRs of human and mouse T cells, revealing a conserved mRNA-destabilizing element.
• GCLiPP signal overlapping with immune-related SNPs highlighted stabilizing cis-regulatory regions in CD5, STAT6, and IKZF1.

Conclusions:

• GCLiPP is a powerful tool for high-resolution transcriptome-wide RBP occupancy mapping.
• The study identified conserved cis-regulatory elements and their roles in mRNA stability.
• GCLiPP can uncover functional consequences of genetic variations, such as SNPs, on gene regulation in immune cells.