Final Report Summary - HUMANRIB (HumanRib: metal ion binding - stucture - function relations of the human CPEB3 ribozyme)
CPEB3 is one of the four human ribozymes known up to this date; the vast majority of ribozymes are found in lower organisms. The discovery of the human CPEB3 ribozyme, related to the Hepatitis Delta Virus one, was an enormous breakthrough, which gave rise to numerous scientific questions. We were able to answer many of them in the scope of this project, carried out in the group of Prof. Roland Sigel. Since many years, his group is experienced in obtaining structural and functional information on different families of RNAs, combining techniques such as smFRET, NMR, X-ray, CD and UV-VIS spectroscopy, etc.
In the scope of this project, we studied the structure-function and metal ion binding-function relationships of this 67 nucleobase long ribozyme, to understand its mechanism of action. The crucial role of metal ions in ribozymes is not limited to the screening of negative charge associated with the phosphate sugar backbone, but they are also directly involved in proper folding and catalysis. Despite their undoubtable importance, still only little information is available about the relationship between metal ion and ribozymes.
To achieve the challenging goal of this project, we obtained a high resolution NMR structures of several parts of this ribozyme and, using our NMR assignments and molecular dynamics studies, we are well advanced in solving the structure of the full-length CPEB3 ribozyme: This will be one of the largest and most complex RNA structures solved by NMR. NMR was also used to characterize specific metal binding sites, as well as structural and thermodynamic changes within the ribozyme upon addition of metal ions.
This study proved to be exceptionally challenging due to the signal overlap and the intrinsic dynamics of the 67 nucleotide long ribozyme. Assignment of the sequential walk region of the full length CPEB3 was based on a variety of different NMR experiments (e.g. [1H,1H]-NOESY, [1H,1H]-TOCSY, [1H,1H]-COSY, [1H,15N]-HMBC, [1H,15N] and [1H,13C]-HSQC, for which labelled samples are required) performed under different conditions. In addition, special samples were made, in which only one of the nucleobases was of natural abundance, while the others were fully deuterated in order to unambiguously assign the spectra.
Metal binding properties were studied in detail, by substituting Mg2+ with Mn2+ and [Co(NH3)6]3+, able to mimic inner-sphere and outer-sphere coordination properties of Mg2+, respectively.
We showed that the postcleavage CPEB3 ribozyme adopts its global fold in monovalent ions alone, but that Mg2+ binding to several sites is required to fully form the inner pseudoknot, active site and the compact native structure. The complete predicted tertiary structure is formed only in the presence of Mg2+ and includes the formation of an additional G•U wobble at the base of one of the pseudoknots. The coordinated magnesium ion is bound in an outer-sphere mode. Both findings are in line with the data on the HDV ribozyme.
The CPEB3 and HDV ribozymes are different from other small ribozymes investigated so far in that they crucially depend on the presence of divalent metal ions for catalysis, both in low and in high concentrations of monovalent ions. Also, their nested double pseudoknot fold is far more complicated than the secondary structures of the other small ribozymes. The data obtained in this project (i) prove that the CPEB3 ribozyme folds the same way as the related HDV and (ii) illustrate why the Mg2+ dependence does not only stem from the catalytic mechanism but is inherent to the particular structure of the CPEB3 and HDV ribozymes, by completely structuring their inner catalytic core.
This interdisciplinary project, which combined structural biology with coordination chemistry, resulted in an exciting and valuable piece of knowledge, crucial for understanding the complicated structure and pathway of metal mediated cleavage of one of the four known human ribozymes.
In the scope of this project, we studied the structure-function and metal ion binding-function relationships of this 67 nucleobase long ribozyme, to understand its mechanism of action. The crucial role of metal ions in ribozymes is not limited to the screening of negative charge associated with the phosphate sugar backbone, but they are also directly involved in proper folding and catalysis. Despite their undoubtable importance, still only little information is available about the relationship between metal ion and ribozymes.
To achieve the challenging goal of this project, we obtained a high resolution NMR structures of several parts of this ribozyme and, using our NMR assignments and molecular dynamics studies, we are well advanced in solving the structure of the full-length CPEB3 ribozyme: This will be one of the largest and most complex RNA structures solved by NMR. NMR was also used to characterize specific metal binding sites, as well as structural and thermodynamic changes within the ribozyme upon addition of metal ions.
This study proved to be exceptionally challenging due to the signal overlap and the intrinsic dynamics of the 67 nucleotide long ribozyme. Assignment of the sequential walk region of the full length CPEB3 was based on a variety of different NMR experiments (e.g. [1H,1H]-NOESY, [1H,1H]-TOCSY, [1H,1H]-COSY, [1H,15N]-HMBC, [1H,15N] and [1H,13C]-HSQC, for which labelled samples are required) performed under different conditions. In addition, special samples were made, in which only one of the nucleobases was of natural abundance, while the others were fully deuterated in order to unambiguously assign the spectra.
Metal binding properties were studied in detail, by substituting Mg2+ with Mn2+ and [Co(NH3)6]3+, able to mimic inner-sphere and outer-sphere coordination properties of Mg2+, respectively.
We showed that the postcleavage CPEB3 ribozyme adopts its global fold in monovalent ions alone, but that Mg2+ binding to several sites is required to fully form the inner pseudoknot, active site and the compact native structure. The complete predicted tertiary structure is formed only in the presence of Mg2+ and includes the formation of an additional G•U wobble at the base of one of the pseudoknots. The coordinated magnesium ion is bound in an outer-sphere mode. Both findings are in line with the data on the HDV ribozyme.
The CPEB3 and HDV ribozymes are different from other small ribozymes investigated so far in that they crucially depend on the presence of divalent metal ions for catalysis, both in low and in high concentrations of monovalent ions. Also, their nested double pseudoknot fold is far more complicated than the secondary structures of the other small ribozymes. The data obtained in this project (i) prove that the CPEB3 ribozyme folds the same way as the related HDV and (ii) illustrate why the Mg2+ dependence does not only stem from the catalytic mechanism but is inherent to the particular structure of the CPEB3 and HDV ribozymes, by completely structuring their inner catalytic core.
This interdisciplinary project, which combined structural biology with coordination chemistry, resulted in an exciting and valuable piece of knowledge, crucial for understanding the complicated structure and pathway of metal mediated cleavage of one of the four known human ribozymes.