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An Allosteric Self-Splicing Ribozyme Triggered by a Bacterial Second Messenger

Elaine R. Lee, Jenny L. Baker, Zasha Weinberg, Narasimhan Sudarsan, Ronald R. Breaker

Science Vol. 329. no. 5993, pp. 845 – 848, 13 August 2010.
DOI: 10.1126/science.1190713

This week’s methodical summary and analysis by Alex Subtelny:

From the lab that discovered riboswitches comes this paper, which describes a bacterial riboswitch that allosterically controls the self-splicing of a ribozyme located immediately downstream. This unusual tandem arrangement was discovered upstream of a putative C. difficile virulence gene (CD3246) during a computational search for new riboswitches, including those for cyclic di-guanosyl 5’-monophosphate (c-di-GMP), an important bacterial second messenger that regulates the transition between motile and biofilm states. Interestingly, the riboswitch in question was located far (~600 nucleotides) upstream of its associated ORF and appeared to lack the typical expression structures associated with riboswitches. Instead, the intervening sequence between the riboswitch and the ORF contained what looked like a group I ribozyme. This raised two intriguing possibilities: i) that the c-di-GMP aptamer allosterically regulates self-splicing of the ribozyme, and ii) that unlike most group I ribozymes, which are part of selfish genetic elements, this one might perform a beneficial function for its host.

The authors first demonstrate that the putative riboswitch aptamer indeed binds c-di-GMP with high affinity and specificity. Then, they dissect the mechanism of the tandem riboswitch-ribozyme through a beautiful series of in vitro experiments with mutants that disrupt or restore key secondary structure elements. Binding of c-di-GMP to the aptamer stabilizes a base-pairing architecture that favors splicing of the region upstream of the ribozyme (the 5’ exon) to the region downstream (the 3’ exon), which contains the ORF for the virulence gene. In the absence of the ligand, a different base-pairing structure is favored, leading to the formation of an alternative excision product consisting of a fragment of the 3’ exon. The authors support their splicing assays with kinetic experiments showing that c-di-GMP causes a ~12-fold increase in the rate of 5’-3’ spliced product formation and a modest decrease in the rate of formation of the alternative 3’ excision product. Finally, the authors present an elegantly convincing model to explain how alternative processing of the mRNA might affect the expression of the virulence gene. 5’-3’ splicing, which is favored in the presence of c-di-GMP, generates a ribosome binding site situated an optimal distance from the start codon, which in the precursor mRNA is concealed by being part of a stem-loop. In contrast, the alternative 3’ excision product lacks a ribosome binding site (since only five nucleotides are left upstream of the start codon), preventing translation of the downstream ORF. Thus, according to this model, the mRNA for the virulence gene is competent for translation only in the presence of c-di-GMP.

While the authors do an excellent job of showing that c-di-GMP regulates alternative ribozyme self-splicing in vitro and present a highly plausible model for how this might regulate virulence gene expression, they stop there. They provide little evidence to support the in vivo relevance of the riboswitch-regulated ribozyme, and, in particular, to show that it performs a beneficial function for the host. In one of their supplemental figures, the authors show that the major RT-PCR product for CD3246 (using primers corresponding to the aptamer and the interior of the ORF) is 5’-3’ spliced, and that the extent of splicing increases with culture age, which is associated with an increased concentration of c-di-GMP. However, they do not show that 5’-3’ splicing results in increased protein output. This could conceivably be accomplished by placing the riboswitch-ribozyme (or mutants thereof) upstream of a reporter gene, introducing this fusion into their C. difficile strain or another bacterial species, and measuring levels of the reporter normalized to another, control reporter. Moreover, the authors do not address in the paper the (rather unlikely but) possible existence of alternative transcriptional start sites within the body of the riboswitch-ribozyme that, if highly used, might call into question the relevance of their model for the translational regulation of CD3246 expression. In addition, we are left with several other key questions: what is the function of CD3246? And why is it important for its expression to be regulated by c-di-GMP? Insight into these questions, as well as those discussed earlier, would strengthen the authors’ hypothesis that group I ribozymes can be co-opted into performing beneficial functions for their hosts.