RNA:RNA interaction in ternary complexes resolved by chemical probing

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Biology

Background:

• MicroRNA (miRNA) mediated gene regulation is crucial in cellular processes.
• Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a powerful tool for probing RNA structure and RNA:protein interactions.
• However, SHAPE has not been applied to study RNA:RNA interactions.

Purpose of the Study:

• To adapt and apply SHAPE for investigating RNA:RNA binding processes.
• To elucidate the structural mechanisms of microRNA-34a (miR-34a) binding to its mRNA target, silent information regulator 1 (mSIRT1).
• To examine the role of the Argonaute protein within the RNA-induced silencing complex (RISC) in this binding process.

Main Methods:

• Development and application of RNA:RNA binding by SHAPE (RABS) technique.
• Utilizing SHAPE probes to analyze RNA structural changes upon binding.
• Investigating miR-34a binding to mSIRT1 in the presence and absence of RISC.

Main Results:

• RABS successfully probed RNA:RNA interactions, including RNA:RNA-RBP complexes.
• Demonstrated that for miR-34a loaded into RISC, seed region binding to the mRNA target is required before compensatory region binding.
• Naked miR-34a can bind the compensatory region of the mRNA target without prior seed interaction.

Conclusions:

• RABS offers a novel method for structurally analyzing RNA:RNA binding events.
• The findings provide critical insights into the sequential binding mechanism of miRNAs to their mRNA targets within RISC.
• This technique complements existing methods like luciferase assays and has broad applicability to various nucleic acid-mediated binding processes.