Poly(A)-binding protein modulates mRNA susceptibility to cap-dependent miRNA-mediated repression

Abstract

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally through binding specific sites within the 39 untranslated regions (UTRs) of their target mRNAs. Numerous investigations have documented repressive effects of miRNAs and identified factors required for their activity. However, the precise mechanisms by which miRNAs modulate gene expression are still obscure. Here, we have examined the effects of multiple miRNAs on diverse target transcripts containing artificial or naturally occurring 39 UTRs in human cell culture. In agreement with previous studies, we report that both the 59 m 7 G cap and 39 poly(A) tail are essential for maximum miRNA repression. These cis-acting elements also conferred miRNA susceptibility to target mRNAs translating under the control of viral-and eukaryotic mRNA-derived 59 UTR structures that enable cap-independent translation. Additionally, we evaluated a role for the poly(A)-binding protein (PABP) in miRNA function utilizing multiple approaches to modulate levels of active PABP in cells. PABP expression and activity inversely correlated with the strength of miRNA silencing, in part due to antagonism of target mRNA deadenylation. Together, these findings further define the cis-and trans-acting factors that modulate miRNA efficacy.

MicroRNA-mediated gene silencing: are we close to a unifying model?

“…However, we have not been able to rule out the involvement of rapid mRNA degradation in the observed silencing effect. These observations fi t well with previous fi ndings demonstrating the importance of m 7 G-cap recognition in mRNA processing and regulation, such as the work by Walters et al (47) who demonstrated the presence of a cap structure to be suffi cient to allow miRNA-mediated silencing to occur.…”

“…However, as previously highlighted, a degree of controversy exists in the literature over the interpretation of such results between contrasting studies; these are reviewed in Table 1 . Recent fi ndings by Walters et al (47) may allude to a more defi nitive explanation.…”

The complexity of miRNA-mediated repression

“…The extent of miRNA-mediated repression has been shown to be dependent on levels of the protein. 110,135 Paip2 is a negative regulator of PABP function, and acts by competing with eIF4G for interaction with PABP. 136,137 In accordance with the effects of PABP overexpression, increased levels of Paip2 were shown to enhance miRNA-mediated repression.…”

“…136,137 In accordance with the effects of PABP overexpression, increased levels of Paip2 were shown to enhance miRNA-mediated repression. 110 PABP also undergoes a number of posttranslational modifications, 138,139 which may in the future be shown to influence its effects in the miRNA repression pathway.…”

“…59 Not surprisingly therefore, the poly(A) tail is a contributing cis factor in miRNA-mediated repression and longer poly(A) tails have been shown to confer greater repressive effects on the mRNA. 63,109,110 Indeed, the role of the poly(A) tail was highlighted in a study describing the recapitulation of miRNA-mediated repression in RRL, where free poly(A) was shown to act in trans to stimulate miRNA-mediated repression in nuclease-treated RRL, possibly by modulating the interaction of PABP with eIF4G. 80 Importantly, PABP has been recently demonstrated to dissociate from the poly(A) tails of miRNA-targeted mRNAs in the absence of deadenylation and this effect was triggered by the recruitment of the Ccr4-NOT complex by RISC, 111,112 which could be an additional contributing factor to translational repression as PABP is known to stimulate translation by stabilising the mRNA closed-loop.…”