On 2016 Aug 30, Duke RNA Biology Journal Club commented:

This paper brought a new perspective to our discussion by focusing on the structural and biophysical characterization of RNA, in this case the mammalian ribozyme CPEB3, to gain insight into its biological function. Of particular interest to our group is understanding structure in the presence of a biologically relevant amount of magnesium, which remains a challenge in the field of RNA structural biology but is critical for understanding how RNA may function in the cell. In fact, magnesium acts as a crucial cofactor for many catalytic RNAs and is often required for folding of structured RNAs into their functionally competent state.

NMR is a powerful technique for studying biomolecular structure and dynamics at the atomic level, but much of the current NMR data describing RNA are reported in the absence of magnesium due to experimental limitations on signal sensitivity which worsens with high-conductivity samples.

This paper used previously established NMR methods such as Diffusion Ordered Spectroscopy (DOSY) and NOESY experiments to probe how CPEB3 ribozyme global conformation and local secondary structure are affected by magnesium. They were able to mitigate sensitivity issues by substituting magnesium with hexamminecobalt (III) Gonzalez RL Jr, 1999 as a probe of outer-sphere metal coordination to identify potential magnesium binding sites. While this technique is a useful starting point, concerns were raised that the authors used NOESY cross peaks of aromatic or sugar protons as the main evidence for direct magnesium binding since these resonances likely shift due to magnesium-induced conformational changes rather than site specific interaction. A better analysis would be to monitor nitrogen cross peaks such as N7 on guanines or adenines Fu DJ, 1992 which are insensitive to secondary structural rearrangements but sensitive to presence of nearby metal ions.

Nonetheless, this paper successfully characterized the impact of magnesium on secondary structure of a complicated RNA system comprised of a full-length nested double pseudoknot ribozyme which happens to be one of the few self-cleaving ribozymes identified in humans. We are excited for the development of new techniques directed toward studying specific RNA-metal interactions to better understand RNA structure as it exists in the cell.

On 2016 Aug 30, Duke RNA Biology Journal Club commented:

This paper brought a new perspective to our discussion by focusing on the structural and biophysical characterization of RNA, in this case the mammalian ribozyme CPEB3, to gain insight into its biological function. Of particular interest to our group is understanding structure in the presence of a biologically relevant amount of magnesium, which remains a challenge in the field of RNA structural biology but is critical for understanding how RNA may function in the cell. In fact, magnesium acts as a crucial cofactor for many catalytic RNAs and is often required for folding of structured RNAs into their functionally competent state.

NMR is a powerful technique for studying biomolecular structure and dynamics at the atomic level, but much of the current NMR data describing RNA are reported in the absence of magnesium due to experimental limitations on signal sensitivity which worsens with high-conductivity samples.

This paper used previously established NMR methods such as Diffusion Ordered Spectroscopy (DOSY) and NOESY experiments to probe how CPEB3 ribozyme global conformation and local secondary structure are affected by magnesium. They were able to mitigate sensitivity issues by substituting magnesium with hexamminecobalt (III) Gonzalez RL Jr, 1999 as a probe of outer-sphere metal coordination to identify potential magnesium binding sites. While this technique is a useful starting point, concerns were raised that the authors used NOESY cross peaks of aromatic or sugar protons as the main evidence for direct magnesium binding since these resonances likely shift due to magnesium-induced conformational changes rather than site specific interaction. A better analysis would be to monitor nitrogen cross peaks such as N7 on guanines or adenines Fu DJ, 1992 which are insensitive to secondary structural rearrangements but sensitive to presence of nearby metal ions.

Nonetheless, this paper successfully characterized the impact of magnesium on secondary structure of a complicated RNA system comprised of a full-length nested double pseudoknot ribozyme which happens to be one of the few self-cleaving ribozymes identified in humans. We are excited for the development of new techniques directed toward studying specific RNA-metal interactions to better understand RNA structure as it exists in the cell.