In vitro Drosophila study supports theory that microRNAs inhibit initiation of protein translation
By Melissa Lee Phillips | May 17, 2007
MicroRNAs inhibit protein synthesis in Drosophila by preventing the assembly of translation machinery, according to a study in this week's Nature. The researchers used a novel in vitro system to show that microRNAs can inhibit the initiation of protein synthesis.
"They have a nice system for repression, it seems, in an in vitro extract, and that's been what everybody's wanted," said Rachel Green of Johns Hopkins University in Baltimore, Md., who was not involved in the work.
MicroRNAs regulate gene expression in plants and animals by repressing protein translation or degrading messenger RNA (mRNA) molecules, but their precise mechanism of action has been controversial, said senior author Matthias Hentze of the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany.
Studies in Caenorhabditis elegans and human cells have shown that polysomes -- ribosomal complexes involved in translating mRNA into protein -- appear to be present on mRNAs repressed by microRNAs, suggesting that translation is inhibited sometime after initiation, Hentze said. On the other hand, some studies have shown that altering the mRNA site where translation begins can prevent microRNA-mediated repression, suggesting that microRNAs block the beginning of translation.
Hentze and Rolf Thermann, also of EMBL, developed an in vitro system to test how the microRNA miR2 inhibits protein translation from its Drosophila melanogaster mRNA target. They inserted several copies of the miR2 binding site into a genetic construct based on the mRNA target and created a control construct that lacked the miR2 binding sites. The authors then transfected both constructs into D. melanogaster cells. As expected, the mRNA with the miR2 binding sites was translated about 3.5 times less efficiently than was the control mRNA. The researchers found that the two mRNA molecules were equally stable, suggesting that miR2 does not regulate protein translation by degrading the mRNA.
They next found that the assembly of polysomes is severely inhibited on mRNA with miR2 binding sites. Instead of ribosomal complexes, large microRNA complexes assemble on the mRNA molecule. According to the authors, these complexes -- which they call "pseudo-polysomes" -- may have been mistaken for polysomes in previous studies, prompting the belief that microRNA-mediated repression happens sometime after polysome assembly and the initiation of translation.
However, those past studies had strong data to suggest that repressed mRNAs associate with true polysomes, according to Green. "I don't think [this study] explains away" those data, she told The Scientist.
The study also does not rule out the possibility that multiple mechanisms may be involved in microRNA-mediated repression in different situations, Hentze said.
It's not yet clear if these pseudo-polysomes are required for repression or if they simply accompany microRNA-mediated repression, Hentze said. "That is, in my view, an open question," he told The Scientist.
"Recapitulating microRNA-based regulation in vitro would be a huge step forward for the field," according to Phillip Zamore of the University of Massachusetts Medical School in Worcester, who was not involved in the study. "But a lot of work remains to be done to show that any [in vitro system] really can resolve the controversy," Zamore told The Scientist, "especially since the controversy in the literature over how microRNAs work is based on experiments in living cells."
In the same issue of Nature, researchers led by Thimmaiah Chendrimada of the Wistar Institute in Philadelphia, Pa., reveal an evolutionarily conserved mechanism of microRNA-mediated silencing. They found that a factor called eIF6, which prevents ribosome assembly, regulates microRNA-mediated repression in both human cells and C. elegans - further evidence that microRNAs affect the beginning of translation.
Melissa Lee Phillips
Links within this article
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R. Thermann, M.W. Hentze, "Drosophila miR2 induces pseudo-polysomes and
inhibits translation initiation," Nature, published online May 16, 2007.
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D.P. Bartel, "MicroRNAs: genomics, biogenesis, mechanism, and function," Cell, January 23, 2004.
P.H. Olsen, V. Ambros, "The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation," Developmental Biology, December 15, 1999.
C.P. Petersen et al., "Short RNAs repress translation after initiation in mammalian cells," Molecular Cell, February 17, 2006.
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