Mapping contacts between gRNA and mRNA in trypanosome RNA editing

Area of Science:

• Molecular Biology
• RNA Biology
• Biochemistry

Background:

• Guide RNAs (gRNAs) possess 5' anchor and 3' uridylate tail sequences.
• The 5' anchor is crucial for in vitro editing and mRNA binding.
• The precise role of gRNAs in directing RNA editing remains largely unknown.

Purpose of the Study:

• To investigate the structural interactions between gRNAs and pre-edited mRNA.
• To elucidate the function of the 5' anchor and 3' uridylate tail in RNA editing.

Main Methods:

• Utilized photo-reactive crosslinking agent azidophenacyl (APA) attached to gRNA termini.
• Mapped crosslinked conjugates to determine gRNA-mRNA structural relationships.
• Analyzed crosslinks at 5' and 3' ends of gRNAs for three gRNA/mRNA pairs.

Main Results:

• 5'-terminal APA crosslinks confirmed the anchor's role in positioning molecules.
• 3'-terminal crosslinks revealed the U-tail interacts with nucleotides upstream of the first edited site.
• The U-tail preferentially binds to purine-rich regions near initial editing sites.

Conclusions:

• The gRNA's 5' anchor correctly positions interacting molecules during RNA editing.
• The 3' uridylate tail interacts with upstream mRNA regions, potentially facilitating secondary structure melting.
• Combined action of the anchor and U-tail may enhance editing complex accessibility to target sites.