The type II poly(A)-binding protein PABP-2 is a downstream target of the "let-7" microRNA in the heterochronic pathway of "Caenorhabditis elegans" : mechanisms of microRNA-mediated gene silencing in "Caenorhabditis elegans"

microRNAs (miRNAs) are a large class of small, non-coding RNAs that post-transcriptionally regulate gene expression in animals, plants and protozoa. miRNAs are genomically encoded and transcribed by RNA polymerase II. Primary transcripts are sequentially processed by two RNase III enzymes via short, approximately 70 nucleotide long stem-loop containing precursor miRNAs into mature 21 to 23 nucleotide long miRNAs. Mature miRNAs are incorporated into the miRNA-induced silencing complex (miRISC), which, in its core, consists of an Argonaute and a GW182 family protein. miRNAs serve as guide molecules to direct miRISC to target mRNAs. Typically, miRNAs interact by base-pairing with partially complementary miRNA binding sites located in the 3’ untranslated regions of the targeted mRNA. Binding of miRISC ultimately prevents protein accumulation by mechanisms which are not well understood. miRNAs regulate diverse biological processes including development, proliferation, differentiation, apoptosis, host defense, and cancer. By estimation, miRNAs potentially regulate more than 60% of the human protein coding genome, leaving only few, if any, genetic pathway untouched.
The phylogenetically conserved miRNA lethal-7 (let-7) was first discovered as an essential developmental gene in the heterochronic pathway of the free-living nematode Caenorhabditis elegans. The genes of the heterochronic pathway direct the stage specific execution of cell fates during post-embryonic development of C. elegans.
We identified the type II poly(A)-binding protein PABP-2 in a suppressor screen for let-7 loss-of-function lethality. Mammalian PABP2 was initially identified as an enhancer of nuclear polyadenylation. In this work we show that depletion of PABP-2 not only rescues loss of let-7 function, but also causes let-7 gain-of-function phenotypes in wild-type animals. Surprisingly, efficient depletion of PABP-2 leaves global translation and mRNA levels largely unaffected, but causes premature accumulation of the LIN-29 transcription factor, the most downstream factor known in the heterochronic pathway. This is not due to an effect on let-7 biogenesis and let-7 activity, which are not affected by the level of PABP-2. However, we find that PABP�2 protein levels are developmentally regulated and decrease during larval development. Although PABP-2 is unlikely to be a direct target of let-7, decrease of PABP-2 in late larval development depends, at least in part, on let-7 activity.
The molecular mechanism of miRNA-mediated gene silencing has been subject to intense debate. Despite a plethora of often conflicting data, the emerging consensus is that repression of translation initiation and accelerated mRNA degradation are the prevailing mechanisms. However, it is not clear whether translational repression and mRNA degradation constitute two parallel mechanisms or whether translational repression and mRNA degradation are sequential events. Work done in our lab showed, that in C. elegans, miRNAs regulate their cognate target genes by repression of translation at the initiation stage, which often, but not always, coincides with reduced target mRNA levels. Furthermore, repression depended on the presence of AIN-1 and AIN-2, the C. elegans homologs of the GW182 protein family. AIN-1 and AIN-2 are highly divergent homologs of fly and vertebrate GW182 proteins. Moreover, AIN-1 and AIN-2 show only little similarity at the level of their protein sequences. In an extension of our previous work, we studied the individual contribution of AIN-1 and AIN-2 to miRNA mediated gene silencing by analyzing ain-1 and ain-2 single mutant animals. We find that translational repression, but not mRNA decay, relies on the presence of AIN-1. However, overexpression of AIN-2 rescues ain-1 specific developmental defects and restores wild-type translational repression. It is not clear why translational repression and mRNA degradation have a different requirement for overall GW182 protein levels. Thus far, our data proof that AIN-1 as well as AIN-2 act as bona fide GW182 proteins, mediating both translational repression and mRNA decay.