The type II poly(A)-binding protein PABP-2 genetically interacts with the let-7 miRNA and elicits heterochronic phenotypes in Caenorhabditis elegans.

The type II poly(A)-binding protein PABP2/PABPN1 functions in general mRNA metabolism by promoting poly(A) tail formation in mammals and flies. It also participates in poly(A) tail shortening of specific mRNAs in flies, and snoRNA biogenesis in yeast. We have identified Caenorhabditis elegans pabp-2 as a genetic interaction partner of the let-7 miRNA, a widely conserved regulator of animal stem cell fates. Depletion of PABP-2 by RNAi suppresses loss of let-7 activity, and, in let-7 wild-type animals, leads to precocious differentiation of seam cells. This is not due to an effect on let-7 biogenesis and activity, which remain unaltered. Rather, PABP-2 levels are developmentally regulated in a let-7-dependent manner. Moreover, using RNAi PABP-2 can be depleted by >80% without significantly impairing larval viability, mRNA levels or global translation. Thus, it unexpectedly appears that the bulk of PABP-2 is dispensable for general mRNA metabolism in the larva and may instead have more restricted, developmental functions. This observation may be relevant to our understanding of why the phenotypes associated with human PABP2 mutation in oculopharyngeal muscular dystrophy (OPMD) seem to selectively affect only muscle cells.

A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans.

Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and luciferase assays, that validation by targeted proteomics substantially enriched for biologically relevant let-7 interactors. For example, we found that the zinc finger protein ZTF-7 was a bona fide let-7 miRNA target. We also validated predicted miR-58 targets, demonstrating that this approach is adaptable to other miRNAs. We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational methods or in large-scale experiments, and that the described strategy should be readily adaptable to other organisms.

Translational control of endogenous microRNA target genes in C. elegans.

lin-4 and let-7 are the founding members of the large microRNA (miRNA) family of regulatory RNAs and were originally identified as components of a C. elegans developmental pathway that controls temporal cell fates. Consistent with their pioneering role, lin-4 and let-7 were studied widely as "model miRNAs" in efforts to reveal the mode of action of miRNAs. Early work on lin-4 thus established a paradigm that miRNAs inhibit translation of their target mRNAs at a step downstream from initiation, without affecting mRNA stability. Although some studies on mammalian miRNAs in cell culture reached similar conclusions, most of those studies indicated that miRNAs repressed translation initiation and frequently also promoted target mRNA degradation. We will discuss here what is known about modes of miRNA target gene repression in C. elegans, highlighting recent work that demonstrates that both mRNA degradation and repression of translation initiation are mechanisms employed in vivo by let-7 and, unexpectedly, lin-4 to silence their endogenous targets. We will also discuss the roles of the GW182 homologous AIN-1 and AIN-2 proteins in this process.