Defining the RNA-protein interactions in the trypanosome preribosomal complex

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Eukaryotic 5S ribosomal RNA (rRNA) is synthesized in the nucleoplasm and transported to the nucleolus for ribosome assembly, facilitated by ribosomal protein L5.
• In trypanosomes, unique proteins P34 and P37 form a novel preribosomal complex with the conserved L5-5S rRNA complex in the nucleoplasm.
• Previous studies indicated that P34 stabilizes 5S rRNA by bridging interactions with L5, playing a key role in early ribosomal biogenesis.

Purpose of the Study:

• To delineate the specific domains and regions of L5, P34, and 5S rRNA involved in their interactions within the preribosomal complex.
• To understand how these interactions contribute to the stability and assembly of the trypanosome ribosome.

Main Methods:

• Protein domain analysis to identify critical regions for binding.
• RNA-binding assays to characterize protein-RNA interactions.
• Analysis of protein-protein interactions in the presence and absence of 5S rRNA.

Main Results:

• The L18 domain of L5 and the N-terminus/RNA recognition motif of P34 were identified as key 5S rRNA binding sites.
• Trypanosoma brucei L5 binds the β arm of 5S rRNA, while P34 binds loop A/stem V.
• 5S rRNA enhances the association between L5 and P34, with both the β arm and loop A/stem V contributing independently to this enhancement.
• Protein domains involved in protein-protein and protein-RNA interactions show overlap or proximity, suggesting conformational changes upon rRNA binding.

Conclusions:

• Specific domains within L5 and P34 interact with distinct regions of 5S rRNA, contributing to complex stability.
• 5S rRNA acts as a scaffold, promoting L5-P34 association through conformational modulation.
• These findings elucidate critical molecular mechanisms in the early stages of trypanosome ribosome biogenesis.