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Updated: Mar 15, 2026
Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
Published on: May 3, 2014
Structural basis for single-stranded RNA recognition and cleavage by C3PO
Jing Zhang1, Hehua Liu1,2, Qingqing Yao2
1Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200438, China.
Abstract:
Translin and translin-associated factor-x are highly conserved in eukaroytes; they can form heteromeric complexes (known as C3POs) and participate in various nucleic acid metabolism pathways. In humans and Drosophila, C3POs cleave the fragmented siRNA passenger strands and facilitate the activation of RNA-induced silencing complex, the effector complex of RNA interference (RNAi). Here, we report three crystal structures of Nanoarchaeum equitans (Ne) C3PO. The apo-NeC3PO structure adopts an open form and unravels a potential substrates entryway for the first time. The NeC3PO:ssRNA and NeC3PO:ssDNA complexes fold like closed football with the substrates captured at the inner cavities. The NeC3PO:ssRNA structure represents the only catalytic form C3PO complex available to date; with mutagenesis and in vitro cleavage assays, the structure provides critical insights into the substrate binding and the two-cation-assisted catalytic mechanisms that are shared by eukaryotic C3POs. The work presented here further advances our understanding on the RNAi pathway.
Insights
Translin-associated factor-x (C3PO) complexes are crucial for RNA interference. We determined the structures of Nanoarchaeum equitans C3PO, revealing substrate binding and catalytic mechanisms essential for RNAi pathway function.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Translin and translin-associated factor-x form conserved C3PO complexes in eukaryotes.
- These complexes are involved in nucleic acid metabolism and RNA interference (RNAi).
- In humans and Drosophila, C3POs process siRNA passenger strands, activating the RNA-induced silencing complex.
Purpose of the Study:
- To elucidate the structural basis of C3PO function in Nanoarchaeum equitans.
- To understand substrate binding and catalytic mechanisms in C3POs.
Main Methods:
- X-ray crystallography to determine the structures of apo-NeC3PO, NeC3PO:ssRNA, and NeC3PO:ssDNA complexes.
- Site-directed mutagenesis.
- In vitro cleavage assays.
Main Results:
- The apo-NeC3PO structure revealed an open conformation with a potential substrate entryway.
- NeC3PO:ssRNA and NeC3PO:ssDNA complexes adopted a closed, football-like structure, capturing substrates internally.
- The catalytic NeC3PO:ssRNA structure provided insights into substrate binding and a conserved two-cation-assisted catalytic mechanism.
Conclusions:
- The determined structures offer the first view of a catalytic C3PO complex.
- This work advances the understanding of RNAi pathway mechanisms and conserved C3PO functions across eukaryotes.
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