Article thumbnail

Experimental strategies for microRNA target identification

By Daniel W. Thomson, Cameron P. Bracken and Gregory J. Goodall


MicroRNAs (miRNAs) are important regulators of eukaryotic gene expression in most biological processes. They act by guiding the RNAi-induced silencing complex (RISC) to partially complementary sequences in target mRNAs to suppress gene expression by a combination of translation inhibition and mRNA decay. The commonly accepted mechanism of miRNA targeting in animals involves an interaction between the 5′-end of the miRNA called the ‘seed region’ and the 3′ untranslated region (3′-UTR) of the mRNA. Many target prediction algorithms are based around such a model, though increasing evidence demonstrates that targeting can also be mediated through sites other than the 3′-UTR and that seed region base pairing is not always required. The power and validity of such in silico data can be therefore hindered by the simplified rules used to represent targeting interactions. Experimentation is essential to identify genuine miRNA targets, however many experimental modalities exist and their limitations need to be understood. This review summarizes and critiques the existing experimental techniques for miRNA target identification

Topics: Survey and Summary
Publisher: Oxford University Press
OAI identifier:
Provided by: PubMed Central

Suggested articles


  1. (2007). A biochemical approach to identifying microRNA targets.
  2. (2008). A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition.
  3. (2006). A guide through present computational approaches for the identification of mammalian microRNA targets.
  4. (2008). A link between RNA metabolism and silencing affecting Arabidopsis development.
  5. (2008). A novel biochemical method to identify target genes of individual microRNAs: identification of a new Caenorhabditis elegans let-7 target.
  6. (2006). A novel method to detect functional microRNA targets.
  7. (2009). A three-dimensional view of the molecular machinery of RNA interference.
  8. (2010). Affinity purification of microRNA-133a with the cardiac transcription factor,
  9. (2008). Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver.
  10. (2009). Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps.
  11. (2004). Argonaute2 is the catalytic engine of mammalian RNAi.
  12. (2002). Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA.
  13. (2009). CleaveLand: a pipeline for using degradome data to find cleaved small RNA targets.
  14. (2009). Coherent but overlapping expression of microRNAs and their targets during vertebrate development.
  15. (2005). Combinatorial microRNA target predictions.
  16. (2010). Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans.
  17. (2009). Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA.
  18. (2005). Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.
  19. (2009). Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome.
  20. (2004). Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways.
  21. (2010). Diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon microRNAs, Drosha, and additional nucleases.
  22. (2008). Ensembl
  23. (2004). Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses.
  24. (2010). Expanding the microRNA targeting code: functional sites with centered pairing.
  25. (2008). Experimental validation of miRNA targets.
  26. (2011). Global analysis of the mammalian RNA degradome reveals widespread miRNA-dependent and miRNA-independent endonucleolytic cleavage. Nucleic Acids Res.,
  27. (2008). Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends.
  28. (2004). Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.
  29. (2003). Identification of drosophila microRNA targets.
  30. (2007). Identification of human microRNA targets from isolated argonaute protein complexes.
  31. (2006). Identification of miRNA targets with stable isotope labeling by amino acids in cell culture.
  32. (2010). Identifying targets of miR-143 using a SILAC-based proteomic approach.
  33. (2008). Interplay between microRNAs and RNA-binding proteins determines developmental processes.
  34. (2009). Investigating translational repression by microRNAs in Arabidopsis.
  35. (2007). Isolation of microRNA targets by miRNP immunopurification.
  36. (2007). Isolation of microRNA targets using biotinylated synthetic microRNAs.
  37. (2008). LNA-mediated microRNA silencing in non-human primates.
  38. (2009). Lost in translation: an assessment and perspective for computational microRNA target identification.
  39. (2010). Mammalian microRNAs predominantly act to decrease target mRNA levels.
  40. (2005). Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.
  41. (2007). MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells.
  42. (2007). MicroRNA targeting specificity
  43. (2008). MicroRNA-10a binds the 5’UTR of ribosomal protein mRNAs and enhances their translation.
  44. (2007). MicroRNA-21 targets the tumor suppressor gene tropomyosin 1 (TPM1).
  45. (2008). MicroRNA-373 induces expression of genes with complementary promoter sequences.
  46. (2004). MicroRNA-directed cleavage of HOXB8 mRNA.
  47. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function.
  48. (2009). miR-24 Inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to ‘‘seedless’’ 30UTR microRNA recognition elements.
  49. (2009). MiRNA-29a regulates the expression of numerous proteins and reduces the invasiveness and proliferation of human carcinoma cell lines.
  50. (2007). miRNAs play a tune.
  51. (2010). MiRonTop: mining microRNAs targets across large scale gene expression studies.
  52. (2007). Molecular basis for target RNA recognition and cleavage by human RISC.
  53. (2006). mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.
  54. (2010). Multiple microRNAs modulate p21Cip1/Waf1 expression by directly targeting its 3’ untranslated region.
  55. (2006). Perfect seed pairing is not a generally reliable predictor for miRNA-target interactions.
  56. (2005). Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs.
  57. (2009). Predicting microRNA targets and functions: traps for the unwary.
  58. (2005). Prediction and validation of microRNAs and their targets.
  59. (2003). Prediction of mammalian microRNA targets.
  60. (2005). Principles of microRNA-target recognition.
  61. (2005). Silencing of microRNAs in vivo with ‘antagomirs’.
  62. (2004). Specificity of microRNA target selection in translational repression.
  63. (2007). Switching from repression to activation: microRNAs can up-regulate translation.
  64. (2008). Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance.
  65. (2009). Systematic validation of predicted microRNAs for cyclin D1.
  66. (2007). Target mRNAs are repressed as efficiently by microRNA-binding sites
  67. (2010). Targeting of mRNAs by multiple miRNAs: the next step.
  68. (2004). The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3’UTR.
  69. (2006). The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells.
  70. (2008). The genome browser at UCSC for locating genes, and much more!
  71. (2008). The impact of microRNAs on protein output.
  72. (2008). The mechanism of micro-RNA-mediated translation repression is determined by the promoter of the target gene.
  73. (2003). The microRNAs of Caenorhabditis elegans.
  74. (2008). The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2.
  75. (2010). The widespread regulation of microRNA biogenesis, function and decay.
  76. (2010). Transcriptome and targetome analysis in MIR155 expressing cells using RNA-seq.
  77. (2010). Transcriptome-wide identification of microRNA targets in rice.
  78. (2010). Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.
  79. (2009). Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs.
  80. (2009). Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells.
  81. (2008). Widespread changes in protein synthesis induced by microRNAs.

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.