Formation of circular polyribosomes on eukaryotic mRNA without cap-structure and poly(A)-tail: a cryo electron...

Area of Science:

• Molecular Biology
• Structural Biology
• Gene Expression

Background:

• The prevailing model suggests mRNA circularization, crucial for efficient translation, relies on interactions between the cap-binding complex (eIF4F) and poly(A)-binding protein (PABP).
• This interaction is thought to bridge the 5' cap and 3' poly(A) tail, forming a closed-loop structure essential for multiple rounds of translation initiation.

Purpose of the Study:

• To investigate the structural basis of polyribosome formation and mRNA circularity using high-resolution cryo-electron tomography.
• To determine the role of mRNA cap structures and poly(A) tails in the formation of circular polyribosomes.

Main Methods:

• Wheat germ cell-free translation system to synthesize GFP-encoding mRNAs.
• Cryo-electron tomography (cryo-ET) for high-resolution imaging of polyribosomes.
• Sub-tomogram averaging and 3D reconstruction of individual polyribosomes.

Main Results:

• Reconstructed 3D polyribosome structures allowed visualization of the mRNA path, confirming circularization by connecting ribosome exit and entry sites.
• Approximately 50% of polyribosomes formed from capped, poly(A)-tailed mRNA were circular, aligning with the established circularization paradigm.
• Remarkably, similar proportions (around 40%) of circular polyribosomes were observed for mRNAs lacking poly(A) tails or cap structures, indicating circularization is largely independent of these elements.

Conclusions:

• The formation of circular polyribosomes is surprisingly independent of the presence of mRNA cap structures and poly(A) tails.
• This finding challenges the long-standing paradigm of mRNA circularization mechanisms, suggesting alternative pathways may contribute significantly to gene expression regulation.