Cooperative target mRNA destabilization and translation inhibition by miR-58 microRNA family in C. elegans

A combined selected reaction monitoring (SRM) and SILAC approach reveals additive target protein regulation of miR-58 family members. (A) Cumulative distributions of log₂ fold expression changes of the 63 TargetScan-predicted miR-58 targets in the mir-58.1, mir-80; mir-58.1, and mir-80; mir-58.1; mir-81-82 mutants relative to the wild type (WT) indicate increased protein abundance with each miR-58 family mutation introduced. Quantified TargetScan-predicted targets identified in SRM, fractionated SILAC, and unfractionated SILAC were compared to a group of 518 nontargets identified in unfractionated SILAC measurements for which we also had transcript abundance data. P-values were calculated using Kolmogorov–Smirnov (KS) test comparing the fold change distributions (log₂) of targets and nontargets. (B) Mean log₂ fold changes of protein abundances of miRNA targets and nontargets in the different miR-58 family mutants relative to the WT indicate a cumulative effect of miRNA mutations. (C) Differential expression of 55 TargetScan-predicted miR-58 family targets quantified by SRM and unfractionated and fractionated SILAC in mir-58.1, mir-80; mir-58.1, and mir-80; mir-58.1; mir-81-82 relative to the WT. The protein ID indicates the specific protein quantified in different mutants relative to the WT. Proteins are sorted according to increasing up-regulation in the mir-80; mir-58.1; mir-81-82 quadruple mutant. It is apparent that each additional miRNA mutations further increases the expression of TargetScan-predicted targets compared with random controls. (D) Cumulative distributions of log₂ protein-level fold changes of 58 noncanonical targets identified by MIRZA (score cutoff > 10) quantified by unfractionated and fractionated SILAC and of 518 nontargets described above in different miR-58 family mutant backgrounds. P-values were calculated using KS test comparing the fold change distributions (log₂) of targets and nontargets. (E) Cumulative distributions of log₂ protein-level fold changes of ALG-1–bound miR-58 family targets and of 518 nontargets described above in different miR-58 family mutant backgrounds compared with the WT. Requiring that a target reported by Grosswendt et al. (2014) is supported by at least five reads yielded 43 miR-58 family targets; of these, we obtained SILAC data for 16 targets. P-values were calculated using KS test comparing the fold change distributions (log₂) of targets and nontargets.