Hairpin ribozyme

Biological function

The hairpin ribozyme is an RNA motif that catalyzes RNA processing reactions essential for replication of the satellite RNA molecules in which it is embedded. These reactions are self-processing, i.e. a molecule rearranging its own structure. Both cleavage and end joining reactions are mediated by the ribozyme motif, leading to a mixture of interconvertible linear and circular satellite RNA molecules. These reactions are important for processing the large multimeric RNA molecules that are generated by rolling circle replication. At the end of the replication cycle, these large intermediates of satellite RNA replication are processed down to unit length molecules (circular or linear) before they can be packaged by viruses and carried to other cells for further rounds of replication.

Natural versions of the hairpin ribozyme

In the 1980s, the hairpin ribozyme was identified in 3 naturally occurring and well-characterized sequences:

• satellite RNA of tobacco ringspot virus (sTRSV)
• satellite RNA of chicory yellow mottle virus (sCYMV)
• satellite RNA of arabis mosaic virus (sARMV)

Later work in 2021 revealed almost 1000 hairpin ribozyme sequences in largely unknown organisms found in metatranscriptome data. These newer sequences were hypothesized to occur in organisms that, like those containing the three previously found hairpin ribozymes, use single-stranded, circular RNA genomes. The circularity of the genomes was supported experimentally, but the further nature of the organisms is not yet well studied.

Artificial versions of the hairpin ribozyme

Smaller artificial versions of the hairpin ribozyme have been developed to enable a more detailed experimental analysis of the molecule.{{Cite journal

Reaction chemistry

In common with several other ribozymes and protein ribonucleases, the cleavage reaction of the hairpin ribozyme generates RNA fragments with termini consisting of a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The ligation reaction appears to be a simple reversal of cleavage, i.e. covalent joining of RNA fragments ending with a 2',3'-cyclic phosphate and a 5'-hydroxyl group to generate the ordinary 3'-5' phosphodiester linkage used in both RNA and DNA.

Studies of this reaction in multiple ribozymes have served to establish that the reaction chemistry (catalytic mechanism) is an endogenous property of the RNA molecule itself and is not mediated by metal ions, as is true for some protein enzymes and some other ribozymes.{{Cite journal

Structure

The minimal hairpin ribozyme-substrate complex folds into a secondary structure that includes two domains, each consisting of two short base paired helices separated by an internal loop. Domain A (helix 1 – loop A – helix 2) contains the substrate and the primary substrate-recognition region of the ribozyme. Domain B (helix 3 – loop B – helix 4) is larger and contains the primary catalytic determinants of the ribozyme. The two domains are covalently joined via a phosphodiester linkage that connects helix 2 to helix 3. These domains must interact with one another for catalysis to occur.{{Cite journal | doi-access = free

When the minimal ribozyme-substrate complex is allowed to fold under conditions of low ionic strength, the two domains stack one atop the other, forming an inactive, extended structure that resembles a hairpin.{{Cite journal

The structure and activity of the hairpin ribozyme has been explored using a wide range of complementary experimental methods, including nucleotide replacement, functional group substitution, combinatorial selection, fluorescence spectroscopy, covalent crosslinking, NMR analysis and x-ray crystallography. These studies have been facilitated by the ability of the functional complex to self-assemble from segments made by solid phase chemical RNA synthesis, permitting the incorporation of a wide variety of modified nucleotides that are not naturally found in RNA. Together, the results of these experiments present a highly congruent picture of the catalytic cycle, i.e. how the hairpin ribozyme binds its substrate, folds into a specific three-dimensional structure, catalyzes the reaction, and releases the product(s) of the reaction.{{Cite journal

Targeted RNA cleavage and antiviral activity

Hairpin ribozymes have been modified in such a way that they can be used to target cleavage of other RNA molecules. This is possible because much of the substrate specificity of the hairpin ribozyme results from simple Watson-Crick base pairing within helices 1 and 2.{{Cite journal

One area of particular interest has been the development of hairpin ribozymes for potential therapeutic use, for example by preventing the replication of pathogenic viruses. Antiviral hairpin ribozymes have been generated and expressed within mammalian cells, and cells expressing different engineered ribozymes have been shown to be resistant to infection by HIV-1,{{Cite journal

References

References

1. Symons, RH. (1997). "Plant pathogenic RNAs and RNA catalysis". Nucleic Acids Res.
2. Feldstein, PA. (October 15, 1989). "Two sequences participating in the autolytic processing of satellite tobacco ringspot virus complementary RNA.". Gene.
3. Hampel, A. (June 13, 1989). "RNA catalytic properties of the minimum (-)sTRSV sequence.". Biochemistry.
4. Rubino, L. (September 1990). "Nucleotide sequence and structural analysis of two satellite RNAs associated with chicory yellow mottle virus.". The Journal of General Virology.
5. Kaper, JM. (July 15, 1988). "Nucleotide sequence predicts circularity and self-cleavage of 300-ribonucleotide satellite of arabis mosaic virus.". Biochemical and Biophysical Research Communications.
6. (June 2021). "Identification of over 200-fold more hairpin ribozymes than previously known in diverse circular RNAs". Nucleic Acids Res.
7. (October 1, 1997). "Metal Ions Play a Passive Role in the Hairpin Ribozyme Catalysed Reaction". Nucleic Acids Research.
8. (April 12, 2001). "Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis". Nature.
9. (November 15, 2002). "Transition state stabilization by a catalytic RNA". Science.
10. (June 10, 2005). "Inhibition of HIV-1 replication by RNA targeted against the LTR region.". AIDS.