Reports

MicroRNA Inhibition of Translation Initiation in Vitro by Targeting the Cap-Binding Complex eIF4F

Géraldine Mathonnet,^1* Marc R. Fabian,^1* Yuri V. Svitkin,¹ Armen Parsyan,¹ Laurent Huck,¹ Takayuki Murata,¹ Stefano Biffo,^2,3 William C. Merrick,⁴ Edward Darzynkiewicz,⁵ Ramesh S. Pillai,⁶ Witold Filipowicz,⁶ Thomas F. Duchaine,¹ Nahum Sonenberg¹

MicroRNAs (miRNAs) play an important role in gene regulatory networks in animals. Yet, the mechanistic details of their function in translation inhibition or messenger RNA (mRNA) destabilization remain controversial. To directly examine the earliest events in this process, we have developed an in vitro translation system using mouse Krebs-2 ascites cell–free extract that exhibits an authentic miRNA response. We show here that translation initiation, specifically the 5' cap recognition process, is repressed by endogenous let-7 miRNAs within the first 15 minutes of mRNA exposure to the extract when no destabilization of the transcript is observed. Our results indicate that inhibition of translation initiation is the earliest molecular event effected by miRNAs. Other mechanisms, such as mRNA degradation, may subsequently consolidate mRNA silencing.

¹ Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec, Canada, H3G 1Y6.
² Department of Environmental and Life Sciences, University of Eastern Piedmont, 15100 Alessandria, Italy.
³ Laboratory of Molecular Histology, San Raffaele Scientific Institute, 20132 Milan, Italy.
⁴ Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106–4935, USA.
⁵ Department of Biophysics, Institute of Experimental Physics, Warsaw University, 02-089 Warsaw, Poland.
⁶ Friedrich Miescher Institute for Biomedical Research, Post Office Box 2543, 4002 Basel, Switzerland.

^* These authors contributed equally to this work.

To whom correspondence should be addressed. E-mail: nahum.sonenberg{at}mcgill.ca (N.S.); thomas.duchaine{at}mcgill.ca (T.F.D).

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Q. Yin, J. McBride, C. Fewell, M. Lacey, X. Wang, Z. Lin, J. Cameron, and E. K. Flemington (2008)
J. Virol. 82, 5295-5306
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X. Fu, M. Adamski, and E. M. Thompson (2008)
Mol. Biol. Evol. 25, 1067-1080
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MicroRNA-repressed mRNAs contain 40S but not 60S components.

B. Wang, A. Yanez, and C. D. Novina (2008)
PNAS 105, 5343-5348
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Target-specific requirements for enhancers of decapping in miRNA-mediated gene silencing.

A. Eulalio, J. Rehwinkel, M. Stricker, E. Huntzinger, S.-F. Yang, T. Doerks, S. Dorner, P. Bork, M. Boutros, and E. Izaurralde (2007)
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MicroRNAs repress translation of m7Gppp-capped target mRNAs in vitro by inhibiting initiation and promoting deadenylation.

N. Standart and R. J. Jackson (2007)
Genes & Dev. 21, 1975-1982
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