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 re...

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Bibliographic Details
Published in:Molecular cell 2006-05, Vol.22 (4), p.553-560
Main Authors: Wang, Bingbing, Love, Tara M., Call, Matthew E., Doench, John G., Novina, Carl D.
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Gene Silencing
Genes, Reporter
Humans
In Vitro Techniques
MicroRNAs - chemistry
MicroRNAs - genetics
MicroRNAs - metabolism
Models, Biological
Mutation
Protein Biosynthesis
Rabbits
Receptors, CXCR4 - genetics
Reticulocytes - metabolism
RNA
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Summary: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.
ISSN:1097-2765
1097-4164
DOI:10.1016/j.molcel.2006.03.034