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.
Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec, Canada, H3G 1Y6.
Géraldine
G
Mathonnet
Marc R
MR
Fabian
Yuri V
YV
Svitkin
Armen
A
Parsyan
Laurent
L
Huck
Takayuki
T
Murata
Stefano
S
Biffo
William C
WC
Merrick
Edward
E
Darzynkiewicz
Ramesh S
RS
Pillai
Witold
W
Filipowicz
Thomas F
TF
Duchaine
Nahum
N
Sonenberg
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