RNA is not a passive messenger
In the post genomic age, non-coding RNA sequences (those that do not encode proteins) have been found unexpectedly to be very important for regulation of gene expression and other cellular processes. Many of these are functional RNAs that can fold into stable three-dimensional structures and thereby perform work in the cell. A wide variety of riboswitches, which toggle gene expression on and off, have been identified, and catalytic RNAs, or ribozymes, are being discovered in a variety of contexts, including eukaryotic transcriptomes. It is therefore essential to develop a detailed understanding of these systems in order to understand the catalytic and regulatory functions of ribozymes and riboswitches. In our laboratory, we undertake structural and mechanistic analyses of functional RNAs and ribozymes to provide a picture of how these molecules can work within the cell.
The HDV ribozyme has a hybrid engine
The hepatitis delta virus (HDV) ribozyme is a small ribozyme originally identified in the genome of the HDV. HDV is a human pathogen that co-infects with the hepatitis B virus and thereby leads to liver disease. The ribozyme self-cleaves viral RNAs, synthesized as tandem repeats during rolling circle replication, into genome-sized pieces. This ribozyme has evolved to function within human cells and therefore has the potential to be used as a therapeutic and can be used as a molecular biological tool. Recently, functional HDV-like ribozymes have been identified within the human transcriptome, in plants, fungi and insects, and in the mosquito Anopheles gambiae where cleavage is activated in a developmentally-regulated fashion. Ribozyme self-cleavage thus represents a potential target for mosquito-borne pathogens, including Dengue virus, West Nile virus, and Malaria.
To create a snapshot of the ribozyme, we have trapped the molecule in a state prior to cleavage and solved its crystal structure. This gives us a picture of an RNA molecule poised to react. At the cleavage site, we observe an RNA nucleobase, cytosine 75 (C75), and a magnesium ion interacting with the cleavage site. This structure confirms a reaction mechanism in which C75 is initially protonated. We have shown that C75 possesses a dramatically shifted pKa and can therefore participate in proton transfer reactions at neutral pH. This property allows it to donate the proton to the 5’-hydroxyl leaving group, thereby activating it for catalysis. Surprisingly, we saw a magnesium ion interacting with both the 2’-hydroxyl attacking group and the cleavage site phosphate to help position the substrate for cleavage and to activate the nucleophile. In the use of a magnesium ion in the cleavage reaction, the HDV ribozyme functions similarly to the group I introns. This mechanism represents a paradigm shift because the ribozymes, such as the HDV ribozyme, were thought to be too small to bind and position metals for Lewis acid catalysis, a mechanism in which the metal ion interacts directly with the 2’-hydroxyl nucleophile. By both positioning a metal ion and by shifting the pKa of C75, the HDV ribozyme overcomes the intrinsic inertness of RNA. Thus, the HDV ribozyme is the first ribozyme that has been observed to use two distinct catalytic strategies to perform an RNA cleavage reaction.
These studies have provided new concepts in the understanding of RNA structure and RNA catalytic strategies that will aid in the understanding of other RNA catalysts. It is likely that the genomic sequencing enterprises will reveal new RNA catalysts that will either be widespread across evolution, or unique to individual organisms. Our work will aid in the process of identifying and characterizing novel ribozymes.