Imp3 unfolds stem structures in pre-rRNA and U3 snoRNA to form a duplex essential for small subunit processing

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Eukaryotic ribosome biogenesis is a complex process involving U3 small nucleolar RNA (snoRNA) and pre-ribosomal RNA (pre-rRNA) interactions.
• Specific hybridization between U3 snoRNA and pre-rRNA is essential for cleavages that liberate the small ribosomal subunit precursor.
• The U3-18S duplex formation is hindered by conserved structures (U3 box A/A' stem-loop and 18S H1 helix) that must be unfolded.

Purpose of the Study:

• To elucidate the mechanism by which Imp3 and Imp4 proteins facilitate U3-18S duplex formation in eukaryotic ribosome biogenesis.
• To investigate the roles of Imp3 and Imp4 in unfolding conserved RNA structures required for U3 snoRNA-pre-rRNA hybridization.

Main Methods:

• Utilized chemical modification and ribonuclease activity assays to probe base and backbone accessibility of U3 snoRNA and pre-rRNA fragments.
• Employed in vitro assays with RNA fragments mimicking in vivo secondary structures.
• Investigated the effects of Imp3 and Imp4 proteins on RNA structure and hybridization.

Main Results:

• U3-18S hybridization requires only the Imp3 protein.
• Imp3 binding to RNA provides sufficient energy to unfold both the 18S H1 helix and the U3 box A/A' stem-loop structures.
• Imp3 binding increases accessibility at the U3-dependent A0 and A1 cleavage sites.
• Imp4 destabilizes the U3-18S duplex, facilitating U3 snoRNA release.

Conclusions:

• Imp3 is the primary protein responsible for initiating U3-18S hybridization by unfolding inhibitory RNA structures.
• The unfolding activity of Imp3 potentially signals pre-rRNA cleavage events.
• Imp4 plays a distinct role in releasing U3 snoRNA after hybridization, differentiating its function from Imp3.
• Protein-mediated unfolding acts as a regulatory switch, ensuring timely and accurate ribosome biogenesis by preventing premature interactions.