Recapitulation of Short RNA-Directed Translational Gene Silencing In Vitro

Abstract

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.

MicroRNAs and genomic instability

“…Due to the limited requirement for sequence complementarity between a miRNA and a target transcript, any one miRNA has the potential to regulate the expression of many different genes. There is also supporting evidence for the multiple binding of miRNAs (whether the same or different) to unique sites within a single transcript and that the cumulative effect of these binding events may be required for maximal repression of translation [41].…”

Principles and effects of microRNA-mediated post-transcriptional gene regulation

“…However, mRNAs do not always respond with significantly decreased stability when targeted by miRNAs. (Humphreys et al, 2005;Pillai et al, 2005;Bhattacharyya et al, 2006;Petersen et al, 2006;Wang et al, 2006). miRNA targeted, translationally repressed transcripts can be transported to PBs without destruction suggesting that PB may function as a reservoir where quiescent mRNAs stored, possibly allowing their reuse for translation (Pillai et al, 2005;Bhattacharyya et al, 2006).…”

The role of microRNA in human leukemia: a review

“…It has been shown that reporter genes with the m 7 G-capped mRNA are repressed by miRNAs but not those with internal ribosome entry site sequences, suggesting some effect of miRNAs on initiation. 13 However, some miRNAs cause repression of mRNA with internal ribosome entry site sequences. 14 Also, polysomes cosediment with miRNAs and their target mRNAs, suggesting an influence on the elongation process of translation.…”