Molecular basis for RNA kink-turn recognition by the h15.5K small RNP protein

Area of Science:

• Molecular Biology
• RNA Biology
• Biochemistry

Background:

• Box C/D small nucleolar RNAs (snoRNAs) are crucial for rRNA modification.
• The 15.5K protein is essential for nucleating snoRNP assembly by binding snoRNAs.
• Eukaryotic snoRNAs often possess two potential 15.5K binding sites, but only one is typically used in vivo.

Purpose of the Study:

• To investigate the molecular basis for the selective binding of the 15.5K protein to one of two potential sites on snoRNAs.
• To determine the structural requirements for preferential 15.5K protein interaction with snoRNAs.
• To elucidate the energetic contributions of specific RNA functional groups to the binding interaction.

Main Methods:

• In vitro mobility shift assays were employed to study RNA-protein interactions.
• Nucleotide analog interference mapping was used to analyze functional group contributions to binding.
• Xenopus U25 snoRNA and a circularly permuted variant were utilized in the experiments.

Main Results:

• Preferential binding of the 15.5K protein is not dependent on the proximity of RNA ends.
• A structural context beyond the canonical kink-turn motif is required for selective binding.
• Both backbone atoms and base functionalities within the kink-turn are critical for 15.5K interaction.
• An intramolecular RNA-RNA contact may stabilize the complex over a putative A-minor interaction.

Conclusions:

• The selection of a specific 15.5K binding site on snoRNAs is dictated by structural context beyond the kink-turn motif.
• Specific RNA functional groups and backbone atoms are essential for high-affinity binding.
• The findings provide insights into the precise mechanism of snoRNP assembly initiation.