Dosage and Temporal Thresholds in microRNA Proteomics*

Abstract

MicroRNAs (miRNAs) modulate protein and mRNA expression through translational repression and/or mRNA decay. In this study, we combined SILAC-based proteomics and RNAseq to identify primary targets based on measurements of protein and mRNA repression and analysis of transcript 3UTR sequences. The primary target set was used to compare different prediction algorithms, revealing higher stringency of selection by Targetscan and PITA compared with miRanda, at the expense of higher false negatives. A key finding was that significant and unexpected variations occurred in the kinetics of repression as well as the sensitivity to exogeneous miRNA concentration. Bimodal thresholds were observed, which distinguished responses to low (10 nM) versus high (50 -100 nM) miRNA, as well as the onset of repression at early (12-18 h) versus late (36 -48 h) times. Similar behavior was seen at the transcript level with respect to kinetics of repression. The differential thresholds were most strongly correlated with ⌬⌬G, the net free energy of miRNA-target interactions, which mainly reflected inverse correlations with ⌬G open , the free energy of forming 3UTR secondary structures, at or nearby the miRNA seed matching sites. Thus, our working model is that protein binding or other competitive mechanisms variably interfere with the accessibility of miRISC to the transcript binding site. In addition, biphasic responses were observed in a subset of proteins that were partially down-regulated at early times, and further down-regulated at later times. Taken together, our findings provide evidence for varying modes of miRNA target repression, which lead to different thresholds of target responses with respect to kinetics and concentration, and predict that certain transcripts will show graded responses in sensitivity and fold-change under cellular conditions that lead to varying steady state miRNA levels. Molecular & Cellular Proteomics 14: 10.1074/mcp.M114.043851, 289-302, 2015. microRNAs (miRNAs)1 are small noncoding RNAs that modulate cellular protein levels, impacting a wide range of processes ranging from cellular development and differentiation to cancer, metabolic disorder, and other human diseases (1-5). Mature miRNAs interact with Argonaute (AGO) family proteins to form miRNA-induced silencing complexes (miRISCs) (6, 7), which in turn attenuate protein expression through translational repression and/or mRNA decay mechanisms. In animal cells, miRISC interacts with mRNA-bound poly(A) binding protein C (PABPC) through a complex between AGO and the GW182 trinucleotide-repeat-containing protein. This interaction recruits deadenylation, 5Ј-decapping, and mRNA degrading enzymes, and also blocks eukaryotic translation initiation factor G (eIFG) binding to confer translational repression (8 -11). Although the degree of protein or mRNA repression by miRNAs is often moderate (1.5-to 4-fold), fine-tuning at this level can be enough to shift cell and organismal phenotypes, or to set thresholds for gene and protein expression changes which control...

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