Medicinal Chemistry & Chemical Biology, Short talk
MC-016

Human and Chimp CPEB3 ribozymes: unexpected fold of the HDV-like ribozymes 

A. I. Przytula-Mally1, S. Engilberge2, S. Johannsen1, E. Freisinger1, V. Olieric2, R. K. Sigel1*
1University of Zurich, Department of Chemistry, Winterthurerstrasse 190, 8057 Zurich, 2Paul Scherrer Institute, Forschungsstrasse 111, 5232 Villigen PSI

Cytoplasmic polyadenylation element binding (CPEB) proteins are involved in many vital processes including cell division, synaptic plasticity, learning and memory. A highly conserved, short mammalian ribozyme was found within an intron of the CPEB3 protein. This CPEB3 ribozyme is the third ribozyme confirmed in humans, besides the ribosome and the spliceosome, and belongs to the broad family of the hepatitis delta virus (HDV)-like ribozyme. All members of the HDV-like family have several features in common: (i) a nested double-pseudoknot structure, (ii) 5’-end self-cleavage activity, and (iii) a conserved cytosine in the catalytic core. However, their self-cleavage rates are highly divergent and it is assumed that the rates directly depend on the overall stabilization of the catalytic core. Since the first crystal structure of the cleaved HDV ribozyme in 1998 followed by structures of uncleaved, mutant-inhibited and ion-complexed forms, no three-dimensional structure of any other ribozyme of this family was published.

Here we present the first crystal structures of the cleaved human and chimp CPEB3 ribozyme in complex with the U1A spliceosomal protein. Theses sequences differ by only a single nucleotide but show a difference in cleavage rate by around  ~1 order of magnitude. Our crystallographic data disclosed two highly similar structures with the proposed sophisticated double-pseudoknot fold. However, only the four helical regions P1, P2, P3 and P4 are present, whereas P1.1 consisting of only one Watson-Crick base pair and a U-U wobble is not formed. Instead, we observed an alternative interaction in which two copies of RNA base pair within the L3 region with each other. The dimer formation was also consistent with SEC-SAXS experiments and suggesting the highly dynamic behavior of the catalytic core. This is well in accordance with our NMR data of the cleaved wild-type human and chimp ribozyme in which P1.1 formation occurs only in the presence of at least 5 mM Mg2+ ions.