Author

Jon Moulton
http://www.gene-tools.com     Email

Published on SDB CoRe: Apr 17 2012

Tools & Techniques: Molecular Techniques
Organism: Zebrafish, Xenopus, Chick, Mouse, Axolotl, Human, Medaka, Lamprey, Salamander
Stage of Development: Embryo

Object Description

The figure shows the steps in miRNA maturation where a Morpholino can inhibit the process (top) and how a Morpholino can protect the mRNA target of an miRNAa, preventing regulation of mRNA expression by the miRNA.  The Morpholinos are shown in green.

Morpholinos for Inhibiting microRNA

A microRNA (miRNA) matures in several steps and then becomes active; the steps in maturation can be inhibited by binding to a Morpholino oligo, as can the activity of the mature miRNA guide strand on RISC.  The pri-mRNA undergoes double-strand cleavage to form the hairpin pre-mRNA.  The pre-mRNA is double-strand cleaved to form the mature miRNA duplex.  The duplex loads onto RISC, where one strand is cleaved and dissociates while the other strand becomes the guide strand that binds to miRNA response elements (MRE) on mRNA.  If a Morpholino binds to the immature mRNA and displaces some of the RNA-RNA pairing, that displaced region is no longer a substrate for the nucleases that make the double-stranded cuts needed for miRNA maturation.  If a Morpholino is complementary to the guide strand, it can bind to that strand and prevent its interaction with MREs.

A Morpholino can also bind to an MRE, preventing miRNA guide strands from recognizing and binding to that MRE.  Because only about eight bases of the MRE must be complementary to the miRNAs seed sequence, there is great sequence variation between all the MREs recognized by a particular miRNA.  A Morpholino requires about 15 bases of contiguous complementarity to bind an RNA sequence tightly enough to have a stable effect on other processes.  Therefore, this “target protector” strategy can be used to inhibit the activity of an miRNA against a particular mRNA while leaving the miRNA’s action against other mRNAs unperturbed.

References

Kloosterman, W.P., Lagendijk, A.K., Ketting, R.F., Moulton, J.D., Plasterk, R.H.A. Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol., 2007, 5:e203.

Flynt, A.S., Li, N., Thatcher, E.J., Solnica-Krezel, L., Patton, J.G. Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate. Nature Genetics, 2007, 39:259-263.

Comment on this Object

You must be logged in to CoRe to comment. Please login or create an account.

Jon Moulton July 17, 2013 02:39 PM Citation for protecting an MRE: Choi WY, Giraldez AJ, Schier AF. Target Protectors Reveal Dampening and Balancing of Nodal Agonist and Antagonist by miR-430. Science. 2007 Oct 12;318(5848):271-4. Epub 2007 Aug 30. http://www.sciencemag.org/content/318/5848/271.long