Comprehensive RNP profiling in cells identifies U1 snRNP complexes with cleavage and polyadenylation factors active...

Area of Science:

• Molecular Biology
• Gene Regulation
• RNA Processing

Background:

• Full-length transcription in eukaryotes relies on U1 snRNP (U1) to prevent premature 3'-end cleavage and polyadenylation (PCPA).
• U1's role, termed telescripting, involves base-pairing with nascent RNA to block cryptic polyadenylation signals (PASs).

Purpose of the Study:

• To elucidate the molecular mechanism of U1 telescripting.
• To map the binding sites of U1 and cleavage and polyadenylation factors (CPAFs) relative to PCPA sites.
• To identify the interactomes of U1 and CPAFs.

Main Methods:

• Utilized rapid reversible protein-RNA and protein-protein chemical crosslinking (XLIP).
• Performed immunoprecipitations (XLIPs) of U1 and CPAFs.
• Employed RNA-seq and quantitative proteomic mass spectrometry to analyze crosslinked complexes.

Main Results:

• Captured U1-CPAFs complexes within cells, offering insights into the telescripting mechanism.
• Mapped U1 and CPAFs binding locations in relation to PCPA sites.
• Identified U1 and CPAFs interactomes.

Conclusions:

• XLIP profiling provides a method for comprehensive molecular definition of ribonucleoprotein complexes (RNPs).
• The study advances understanding of how U1 regulates transcription termination and RNA processing.