MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F.
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.
Mathonnet
Géraldine
G
Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec, Canada, H3G 1Y6.
Fabian
Marc R
MR
Svitkin
Yuri V
YV
Parsyan
Armen
A
Huck
Laurent
L
Murata
Takayuki
T
Biffo
Stefano
S
Merrick
William C
WC
Darzynkiewicz
Edward
E
Pillai
Ramesh S
RS
Filipowicz
Witold
W
Duchaine
Thomas F
TF
Sonenberg
Nahum
N
D000818:Animals; D002287:Carcinoma, Krebs 2; D002457:Cell Extracts; D004686:Encephalomyocarditis virus / Q000235:genetics; D039562:Eukaryotic Initiation Factor-4F / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D049410:Luciferases, Renilla / Q000235:genetics; D051379:Mice; D035683:MicroRNAs / Q000502:physiology*; D014176:Protein Biosynthesis / Q000502:physiology*; D012315:RNA Caps / Q000502:physiology*; D012270:Ribosomes / Q000378:metabolism
D002457:Cell Extracts; D039562:Eukaryotic Initiation Factor-4F; D035683:MicroRNAs; D012315:RNA Caps; D049410:Luciferases, Renilla