A biochemical approach to identifying microRNA targets

Karginov, Fedor V.; Conaco, Cecilia; Xuan, Zhenyu; Schmidt, Bryan H.; Parker, Joel S.; Mandel, Gail; Hannon, Gregory J.

doi:10.1073/pnas.0709971104

Cited by 337 publications

(315 citation statements)

References 23 publications

(29 reference statements)

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“…We used a similar microarray-based analysis to identify mouse mRNAs downregulated by mature miR-124 or the miR-124-UU21 variant in undifferentiated P19 cells. The mRNAs downregulated by miR-124 were consistent with other reports, and included known targets of miR-124 [41] [47] [43] [81]. Comparison of mRNAs downregulated by miR-124 and miR-124-UU21 showed surprisingly limited overlap between mRNAs regulated by each miRNA.…”

Section: Discussionsupporting

confidence: 89%

“…Cao et al [44] observed defects in the basal lamina in response to ectopic miR-124 expression in chicken neural tube, and they linked these defects to reduced expression of integrin β1 and laminin proteins. In addition, since miRNAs can inhibit translation without reducing mRNA levels, it is likely that there are additional miR-124 target genes yet to be identified [81].…”

Section: Discussionmentioning

confidence: 99%

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MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

Chung

Deo

et al. 2008

Experimental Cell Research

290

211

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MicroRNAs (miRNAs) are small RNAs with diverse regulatory roles. The miR-124 miRNA is expressed in neurons in the developing and adult nervous system. Here we show that overexpression of miR-124 in differentiating mouse P19 cells promotes neurite outgrowth, while blocking miR-124 function delays neurite outgrowth and decreases acetylated α-tubulin. Altered neurite outgrowth also was observed in mouse primary cortical neurons when miR-124 expression was increased, or when miR-124 function was blocked. In uncommitted P19 cells, miR-124 expression led to disruption of actin filaments and stabilization of microtubules. Expression of miR-124 also decreased Cdc42 protein and affected the subcellular localization of Rac1, suggesting that miR-124 may act in part via alterations to members of the Rho GTPase family. Furthermore, constitutively active Cdc42 or Rac1 attenuated neurite outgrowth promoted by miR-124. To obtain a broader perspective, we identified mRNAs downregulated by miR-124 in P19 cells using microarrays. mRNAs for proteins involved in cytoskeletal regulation were enriched among mRNAs downregulated by miR-124. A miR-124 variant with an additional 5' base failed to promote neurite outgrowth and downregulated substantially different mRNAs. These results indicate that miR-124 contributes to the control of neurite outgrowth during neuronal differentiation, possibly by regulation of the cytoskeleton.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

Chung

Deo

et al. 2008

Experimental Cell Research

290

211

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“…4A and Table S8 lists all transcript data). Recent reports using Ago-2 to pull down miRNA targets in mammalian cells over-expressing a miRNA also showed limited overlap with targets downregulated at the mRNA level (20,23). To explore the basis for these differences we examined the two groups for distinct structural and molecular features, using all S2 mRNAs as a common control set.…”

Section: Comparison Of Targets Identified By Ago1 Ip and Ago1 Depletionmentioning

confidence: 99%

“…One such alternative involves identification of microRNA targets by virtue of their physical association with miRNAcontaining ribonucleoprotein complexes (19)(20)(21)(22)(23)(24). In ref.…”

mentioning

confidence: 99%

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

Hong

Hammell

Ambros

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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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).

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“…In such a experiment, we don't know which mRNAs are targets of which miRNA. To overcome this problem, the overexpression or inhibition of miRNA before the immunoprecipitation and comparison with control by microarray analysis will be useful (Karginov et al, 2007;Chi et al, 2009). cancer.…”

Section: Biogenesis Of Mirnas and Gene Regulation By Mirnasmentioning

confidence: 99%

MicroRNAs from biology to future pharmacotherapy: Regulation of cytochrome P450s and nuclear receptors

Nakajima

Yokoi

2011

Pharmacology & Therapeutics

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MicroRNAs (miRNAs) are a family of short, non-coding RNA whose final product is a 22-nucleotide functional RNA molecule. They regulate the expression of target genes by binding to complementary regions of transcripts to repress their translation or promote mRNA degradation. Since miRNAs regulate every aspect of cellular function, their dysregulation is associated with a variety of diseases including cancer, diabetes, and cardiovascular diseases.Therefore, miRNAs are now considered new therapeutic targets. However, the roles of miRNAs in the metabolism of xenobiotics and endobiotics have only recently been revealed.This review describes the current knowledge on the regulation of cytochrome P450s and nuclear receptors by miRNAs, the physiological and clinical significance. The miRNA expression is readily altered by chemicals, carcinogens, drugs, hormones, stress, or diseases, and the dysregulation of specific miRNAs might lead to changes in the drug metabolism potency or pharmacokinetics as well as pathophysiological changes. In the field of pharmacogenomics, the evaluation of miRNA-related polymorphisms would provide useful information for personalized medicine. Utilizing miRNAs opens a new era in the fields of drug metabolism and pharmacokinetics as well as toxicology.

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A biochemical approach to identifying microRNA targets

Cited by 337 publications

References 23 publications

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

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

MicroRNAs from biology to future pharmacotherapy: Regulation of cytochrome P450s and nuclear receptors

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