Immunopurification of Ago1 miRNPs selects for a distinct class of microRNA targets

Abstract

microRNAs comprise a few percent of animal genes and have been recognized as important regulators of a diverse range of biological processes. Understanding the biological functions of miRNAs requires effective means to identify their targets. Combined efforts from computational prediction, miRNA over-expression or depletion, and biochemical purification have identified thousands of potential miRNA-target pairs in cells and organisms. Complementarity to the miRNA seed sequence appears to be a common principle in target recognition. Other features, including miRNAtarget duplex stability, binding site accessibility, and local UTR structure might affect target recognition. Yet computational approaches using such contextual features have yielded largely nonoverlapping results and experimental assessment of their impact has been limited. Here, we compare two large sets of miRNA targets: targets identified using an improved Ago1 immunopurification method and targets identified among transcripts upregulated after Ago1 depletion. We found surprisingly limited overlap between these sets. The two sets showed enrichment for target sites with different molecular, structural and functional properties. Intriguingly, we found a strong correlation between UTR length and other contextual features that distinguish the two groups. This finding was extended to all predicted microRNA targets. Distinct repression mechanisms could have evolved to regulate targets with different contextual features. This study reveals a complex relationship among different features in miRNAtarget recognition and poses a new challenge for computational prediction.Argonaute ͉ gene regulation ͉ RISC complex A nimal genomes contain hundreds of microRNA genes (miRBase 13.0). Recent estimates suggest that miRNAs comprise Ϸ1% of genes in Drosophila and Caenorhabditis elegans and 2-3% of genes in mouse and human. To date, functional analysis in vivo has revealed biological roles for only a small fraction of these (1-3). One issue limiting progress in understanding the miRNA functions is identification of the target mRNAs that they regulate. Computational target identification is primarily based on sequence complementarity to the miRNA (reviewed in ref. 2), but many computational strategies also make use of sequence context to predict miRNA targets. Comparisons of different methods show limited overlap among the predicted targets, although those that place more emphasis on pairing to the seed sequence at the 5Ј end of the miRNA tend to produce similar results. Most of these methods identify many possible targets for each miRNA, often hundreds (e.g., refs. 4-9).

MicroRNA transfection and AGO-bound CLIP-seq data sets reveal distinct determinants of miRNA action

“…1A). This is consistent with the finding of Hong et al (2009) of no enrichment for GU pairing or mismatches in RIP-chip data.…”

“…Two recent studies (Hausser et al 2009;Hong et al 2009) have compared miRNA target features determined from such expression analyses with the results of Argonaute immunopurification (RIP-chip) experiments, and noted differences in the target features. For example, in contrast to earlier studies which have found that 39 UTR length is increased in miRNA targets, these studies found that a distinguishing feature of miRNP binding was short 39 UTR length.…”

General and MicroRNA-Mediated mRNA Degradation Occurs on Ribosome Complexes in Drosophila Cells

“…We found 1,115 transcripts upregulated in AGO1 knockdown cells (see Table S6 in the supplemental material). In order to verify some of the targets, we compared the transcripts to the data sets available from Hong et al (76), and 256 transcripts had been de- scribed to be AGO1 targets. Next, we were interested in whether we had also detected these 1,115 transcripts in our decapped RNAseq libraries of XRN1 knockdown cells (Fig.…”

RNA regulons and the RNA-protein interaction network

“…Thanks to its simplicity and robustness, this method has since been employed to determine the RNA targets of more than one hundred RBPs in mammalian cells, fl ies, worms, trypanosomes, and in yeast [a comprehensive list of RBP experiments is given in (28) ]. It was also the fi rst technique that was applied for the experimental exploration of miRNA targets through immunopurifi cation of Ago proteins and other miRISC proteins from human cells (29 -36) , fl ies (37,38) , and worms (39,40) . Of note, RIP allows concomitant identifi cation of the protein components of RNPs and other associated regulatory proteins by mass-spectrometry (MS) (41) .…”