Differential protein occupancy profiling of the mRNA transcriptome.

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• RNA-binding proteins (RBPs) are crucial regulators of mRNA processing, translation, and decay.
• We previously developed a next-generation sequencing method to map transcriptome-wide RBP contacts on polyadenylated transcripts.
• Comparing these profiles across conditions can reveal dynamic changes in protein-contacted mRNA regions without prior knowledge of the specific RBP.

Purpose of the Study:

• To perform a global comparison of protein occupancy profiles between MCF7 and HEK293 cells.
• To identify differential protein-RNA contact sites and understand their regulatory implications.
• To introduce POPPI, a bioinformatics workflow for analyzing protein occupancy profiling data.

Main Methods:

• Developed a bioinformatics workflow to identify differential crosslinking sites in 4-thiouridine crosslinked polyadenylated RNA samples.
• Compared protein occupancy profiles of polyadenylated transcripts in MCF7 and HEK293 cells.
• Utilized RNA-seq data to distinguish protein-RNA contacts from changes in exon usage.

Main Results:

• Identified 30,000 differential crosslinking sites between MCF7 and HEK293 cells with a 10% false discovery rate.
• 73% of differential protein-RNA contact sites were not explained by exon usage changes.
• Most differential sites were in 3' UTRs, exhibited specific secondary structures, and overlapped with known RBP binding sites (e.g., ELAVL1).
• mRNA transcripts with significant occupancy changes showed prolonged half-lives in MCF7 cells.

Conclusions:

• Presented a global comparison of protein occupancy profiles across different cell types.
• Provided evidence for altered mRNA metabolism due to differential protein-RNA contacts.
• Demonstrated the utility of protein occupancy profiling for studying RNA sequence space dynamics in biological processes.