Abstract
MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5′-fragments were 3′-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5′-3′ degradation or other molecular fates. Some 3′-5′ decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation.
Highlights
MicroRNAs are noncoding RNAs that have been shown to posttranscriptionally regulate gene expression and protein synthesis, in mammalian cells, by partially base-pairing to complementary sequences in the 3′-untranslated regions (3′UTRs) of their target mRNAs1–3
A cleaved 5′-mRNA fragment with its 3′terminal sequence complementarily matched with the probe sequence of an SLA-RT primer was and most efficiently reverse-transcribed, and its relative abundance was subsequently determined by both end-point PCR and quantitative RT-PCR (qRT-PCR)
The accumulation of a cleaved 5′-mRNA fragment at a specific cleavage site within and near the predicted let-7:TUSC2 target pairing sequences was represented by the relative intensity of each specific SLA–RT-PCR amplicon resolved on an agarose gel (Fig. 1a-I, upper panel) and by the relative fragment abundance (RFA) on a qRT-PCR histogram (Fig. 1a-I, lower panel)
Summary
Multiple models have been proposed for the molecular mechanics of miRNA-mediated posttranscriptional repression of target gene expression either by inhibition of mRNA translation, with no apparent impact on mRNA stability, or by initiation of mRNA decay[1,2,7,8,30,31]. Based on the unique and dominant locations of the cleaved fragments accumulated in the seed region and the 3′supplementary pairing region, it is possible that the miRISC stationed on miRNA:mRNA target sites could provide a barricade to the exonuclease-mediated 3′-5′RNA decay machinery and facilitate the recruitment of specific TUTases or uridyl polymerases to the miRISC for adding oligouridines to 3′-termini of the cleaved 5′mRNA fragments. This miRISC-imposed blockage to RNA 3′-5′decay may imply a cooperative role of noncatalytic miRISCs in the gene silencing pathway. Further studies are needed to understand the precise mechanism of miRNA action in association with specific co-factors in miRISC on posttranscriptional regulation of target gene expression in a complex mammalian cellular system
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