CPEB3 Ribozymes Adopt an Unexpected Fold: From Monomer to Dimer and Back

Abstract

The International Year of Crystallography in 2014 was an event to celebrate the century of the discovery of X-ray crystallography. To this occasion Hao Wu from Harvard Medical School being interviewed said, “Structural analysis is like a detective story. There is no direct path from diffraction to structure”. However, once the structure is solved, the story continues and new peculiarity of the molecule under investigation can be discovered.
In 2006 the Szostak group discovered the Cytoplasmic Polyadenylation Element Binding Protein 3 (CPEB3) ribozyme in the human genome. This 67 nucleotide long RNA belongs to the large family of (hepatitis delta virus) HDV-like ribozymes. Representatives of this family are characterized by an intricate nested double pseudoknot fold with two parallel co-axial helical stacks. All HDV-like ribozymes have a cleavage site at the 5’-end of the ribozyme in common and show a very low sequence conservation among each other. One of the only six highly conserved nucleotides is the catalytically important cytosine. The CPEB3 ribozyme resides within the second intron of the cpeb3 gene coding for the CPEB3 protein that plays a crucial role in synaptic plasticity by translation regulation. Moreover, the CPEB3 ribozyme is highly conserved among all mammals implying its importance in protein level regulation. However, only very little is known about its structural and functional characteristics since its discovery.
The goal of this thesis was to solve the first crystal structures of the human and the chimpanzee CPEB3 ribozymes. These two sequences differ by only a single nucleotide but show a significant difference in cleavage rate of around ~1 order of magnitude. We are using X-ray crystallography in combination with Nuclear Magnetic Resonance (NMR) and enzymatic mapping to finally visualize the CPEB3 ribozyme structure and to confirm or deny its nested double pseudoknot fold.
To our surprise and oppositely to what is known from crystal structures of the genomic HDV ribozyme, both CPEB3 ribozymes crystallized as homodimers revealing a new open conformation. The high resolution of both structures allowed for comparison between human and chimpanzee ribozymes on atomic level and points out subtle structural variations that may explain the difference in their activity. However, homodimerization of both CPEB3 ribozymes identified by X-ray crystallography are in disagreement with supplementary Nuclear Magnetic Resonance (NMR) data, which clearly suggests monomeric states for both CPEB3 ribozymes. Size Exclusion Chromatography couped to Small Angle X-ray Scattering (SEC-SAXS) was additionally used to clarify which state, monomeric or dimeric, is present in solution. The identification of both species raised even more questions and illustrates an arm wrestling between monomers and dimers.
The existence of RNA oligomeric structures was for a long time taken just as an artefact frequently identified in laboratories. However, the CPEB3 ribozyme is not the first RNA found in a dimeric form. One of several examples found is the HDV ribozyme. In vitro studies conducted in 2012 showed that the HDV ribozyme can also form homodimers, which activate the Protein Kinase R (PKR). Thus, oligomeric structures adopted by RNA and solved by X-ray crystallography should not be automatically considered as artefacts of crystal packing, but can also imply functional relevance that should not be overlooked.