Drosophila miR2 primarily targets the m7GpppN cap structure for translational repression.
Understanding the molecular mechanism(s) of how miRNAs repress mRNA translation is a fundamental challenge in RNA biology. Here we use a validated cell-free system from Drosophila embryos to investigate how miR2 inhibits translation initiation. By screening a library of chemical m7GpppN cap structure analogs, we identified defined modifications of the triphosphate backbone that augment miRNA-mediated inhibition of translation initiation but are "neutral" toward general cap-dependent translation. Interestingly, these caps also augment inhibition by 4E-BP. Kinetic dissection of translational repression and miR2-induced deadenylation shows that both processes proceed largely independently, with establishment of the repressed state involving a slow step. Our data demonstrate a primary role for the m7GpppN cap structure in miRNA-mediated translational inhibition, implicate structural determinants outside the core eIF4E-binding region in this process, and suggest that miRNAs may target cap-dependent translation through a mechanism related to the 4E-BP class of translational regulators.
European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg 69117, Germany.
Agnieszka
A
Zdanowicz
Rolf
R
Thermann
Joanna
J
Kowalska
Jacek
J
Jemielity
Kent
K
Duncan
Thomas
T
Preiss
Edward
E
Darzynkiewicz
Matthias W
MW
Hentze
D000818:Animals; D001665:Binding Sites; D002474:Cell-Free System; D002478:Cells, Cultured; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D020868:Gene Silencing; D007700:Kinetics; D035683:MicroRNAs / Q000378:metabolism*; D014176:Protein Biosynthesis; D011755:Pyrophosphatases / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism*; D020871:RNA Stability; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D029721:Drosophila Proteins; D035683:MicroRNAs; D012315:RNA Caps; D034743:RNA-Induced Silencing Complex; C105238:cup protein, Drosophila; D011755:Pyrophosphatases; C021798:5'-(N(7)-methyl 5'-triphosphoguanosine)-(mRNA) diphosphatase