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A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA

Abstract

Structured RNAs embedded in the untranslated regions (UTRs) of messenger RNAs can regulate gene expression. In bacteria, control of a metabolite gene is mediated by the self-cleaving activity of a ribozyme embedded in its 5′ UTR1. This discovery has raised the question of whether gene-regulating ribozymes also exist in eukaryotic mRNAs. Here we show that highly active hammerhead ribozymes2,3 are present in the 3′ UTRs of rodent C-type lectin type II (Clec2) genes4,5,6,7. Using a hammerhead RNA motif search with relaxed delimitation of the non-conserved regions, we detected ribozyme sequences in which the invariant regions, in contrast to the previously identified continuous hammerheads8,9,10, occur as two fragments separated by hundreds of nucleotides. Notably, a fragment pair can assemble to form an active hammerhead ribozyme structure between the translation termination and the polyadenylation signals within the 3′ UTR. We demonstrate that this hammerhead structure can self-cleave both in vitro and in vivo, and is able to reduce protein expression in mouse cells. These results indicate that an unrecognized mechanism of post-transcriptional gene regulation involving association of discontinuous ribozyme sequences within an mRNA may be modulating the expression of several CLEC2 proteins that function in bone remodelling and the immune response of several mammals.

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Figure 1: Sequence arrangement and secondary structure model of the rodent Clec2d hammerhead ribozymes.
Figure 2: The discontinuous hammerhead ribozyme-containing 3′ UTRs (hcu) self-cleave in vitro.
Figure 3: The discontinuous mouse Clec2 hammerhead ribozymes embedded in the 3′ UTRs downregulate protein expression in vivo.
Figure 4: RT–PCR analysis of the in vivo expression products.

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Acknowledgements

We thank M. Hall and K. Chakrabarti for assistance with cell culture, members of Haussler and Ares laboratories for sharing tissue culture space, M. Robertson for the transcriptase and A. Zahler for discussion. This work was supported by the National Institutes of Health grant R01043393.

Author Contributions L.H.H. and M.M. did the sequence searches, designed the study, performed the experiments, analysed the data and wrote the manuscript.

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Correspondence to Monika Martick.

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Martick, M., Horan, L., Noller, H. et al. A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA. Nature 454, 899–902 (2008). https://doi.org/10.1038/nature07117

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