Short-range RNA-RNA crosslinking methods to determine rRNA structure and interactions

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• RNA structure and dynamics are crucial for biological function.
• Understanding RNA-RNA interactions provides insights into cellular processes.
• Photosensitive nucleotides enable crosslinking to probe RNA proximity.

Purpose of the Study:

• To describe detailed procedures for analyzing RNA-RNA crosslinks.
• To demonstrate the utility of crosslinking for monitoring RNA conformation.
• To establish methods for analyzing crosslinks in ribosomal RNA (rRNA) and associated nucleic acids.

Main Methods:

• Far-UV (< 300 nm) or near-UV (320-365 nm) irradiation of RNA containing 4-thiouridine.
• Production of zero-length RNA-RNA crosslinks.
• Gel electrophoresis for separation and quantification of crosslinked products.
• Ultracentrifugation for RNA recovery from gels.
• Primer extension and RNA sequencing for site analysis.

Main Results:

• Established procedures for generating and analyzing RNA-RNA crosslinks.
• Demonstrated the application to 16S rRNA, mRNA, and tRNA.
• Successfully purified crosslinked RNA molecules.
• Enabled precise mapping of crosslinking sites.

Conclusions:

• UV-induced RNA-RNA crosslinking is a valuable technique for studying RNA structure and interactions.
• The described methods are adaptable to various RNA molecules.
• This approach provides a powerful tool for investigating RNA conformation and proximity in biological systems.