Recapitulation of short RNA-directed translational gene silencing in vitro.
microRNAs (miRNAs) are a large class of endogenous short RNAs that repress gene expression. Many miRNAs are conserved throughout evolution, and dysregulation of miRNA pathways has been correlated with an increasing number of human diseases. In animals, miRNAs typically bind to the 3' untranslated region (3'UTR) of target mRNAs with imperfect sequence complementarity and repress translation. Despite their importance in regulating biological processes in numerous organisms, the mechanisms of miRNA function are largely unknown. Here, we report in vitro reactions for miRNA-directed translational gene silencing. These reactions faithfully recapitulate known in vivo hallmarks of mammalian miRNA function, including a requirement for a 5' phosphate and perfect complementarity to the mRNA target in the 5' seed region. Translational gene silencing by miRNAs in vitro requires target mRNAs to possess a 7-methyl G cap and a polyA tail, whereas increasing polyA tail length alone can increase miRNA silencing activity.
Wang
Bingbing
B
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Love
Tara M
TM
Call
Matthew E
ME
Doench
John G
JG
Novina
Carl D
CD
D000818:Animals; D001483:Base Sequence; D020868:Gene Silencing; D017930:Genes, Reporter; D006801:Humans; D066298:In Vitro Techniques; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D014176:Protein Biosynthesis; D011817:Rabbits; D019718:Receptors, CXCR4 / Q000235:genetics; D012156:Reticulocytes / Q000378:metabolism
D035683:MicroRNAs; D019718:Receptors, CXCR4