Prediction of Human MicroRNA Targets

  • Bino John
  • Chris Sander
  • Debora S. Marks
Part of the Methods in Molecular Biology™ book series (MIMB, volume 342)


MicroRNAs (miRNAs) are small, nonprotein-coding RNAs that regulate gene expression. Although hundreds of human miRNA genes have been discovered, the functions of most of these are unknown. Computational predictions indicate that miRNAs, which account for at least 1% of human protein-coding genes, regulate protein production for thousands of or possibly all of human genes. We discuss the functions of mammalian miRNAs and the experimental and computational methods used to detect and predict human miRNA target genes. Anticipating their impact on genome-wide discovery of miRNA targets, we describe the various computational tools and web-based resources available to predict miRNA targets.

Key Words

MicroRNA microRNA targets gene regulation gene silencing translational repression computational prediction 


  1. 1.
    Lee, R. C., Feinbaum, R. L., and Ambros, V. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854.CrossRefPubMedGoogle Scholar
  2. 2.
    Pillai, R. S., Bhattacharyya, S. N., Artus, C. G., et al. (2005) Inhibition of translational initiation by let-7 MicroRNA in human cells. Science 309, 1573–1576.CrossRefPubMedGoogle Scholar
  3. 3.
    Yekta, S., Shih, I. H., and Bartel, D. P. (2004) MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594–596.CrossRefPubMedGoogle Scholar
  4. 4.
    Lim, L. P., Lau, N. C., Garrett-Engele, P., et al. (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773.CrossRefPubMedGoogle Scholar
  5. 5.
    Griffiths-Jones, S. (2004) The microRNA registry. Nucleic Acids Res. 32, D109–D111.CrossRefPubMedGoogle Scholar
  6. 6.
    Berezikov, E., Guryev, V., van de Belt, J., Wienholds, E., Plasterk, R. H., and Cuppen, E. (2005) Phylogenetic shadowing and computational identification of human microRNA genes. Cell 120, 21–24.CrossRefPubMedGoogle Scholar
  7. 7.
    Xie, X., Lu, J., Kulbokas, E. J., et al. (2005) Systematic discovery of regulatory motifs in human promoters and 3′ UTRs by comparison of several mammals. Nature 434, 338–345.CrossRefPubMedGoogle Scholar
  8. 8.
    Bentwich, I., Avniel, A., Karov, Y., et al. (2005) Identification of hundreds of conserved and nonconserved human microRNAs. Nat. Genet. 37, 766–770.CrossRefPubMedGoogle Scholar
  9. 9.
    Enright, A. J., John, B., Gaul, U., Tuschl, T., Sander, C., and Marks, D. S. (2003) MicroRNA targets in Drosophila. Genome Biol. 5, R1.CrossRefPubMedGoogle Scholar
  10. 10.
    John, B., Enright, A. J., Aravin, A., Tuschl, T., Sander, C., and Marks, D. S. (2004) Human microRNA targets. PLoS Biology 2, e363.CrossRefPubMedGoogle Scholar
  11. 11.
    Krek, A., Grun, D., Poy, M. N., et al. (2005) Combinatorial microRNA target predictions. Nat. Genet. 37, 495–500.CrossRefPubMedGoogle Scholar
  12. 12.
    Doench, J. G. and Sharp, P. A. (2004) Specificity of microRNA target selection in translational repression. Genes Dev. 18, 504–511.CrossRefPubMedGoogle Scholar
  13. 13.
    Kadonaga, J. T. (2004) Regulation of RNA polymerase II transcription by sequence-specific DNA binding factors. Cell 116, 247–257.CrossRefPubMedGoogle Scholar
  14. 14.
    Hobert, O. (2004) Common logic of transcription factor and microRNA action. Trends Biochem. Sci. 29, 462–468.CrossRefPubMedGoogle Scholar
  15. 15.
    Lagos-Quintana, M., Rauhut, R., Yalcin, A., Meyer, J., Lendeckel, W., and Tuschl, T. (2002) Identification of tissue-specific microRNAs from mouse. Curr. Biol. 12, 735–739.CrossRefPubMedGoogle Scholar
  16. 16.
    Krichevsky, A. M., King, K. S., Donahue, C. P., Khrapko, K., and Kosik, K. S. (2003) A microRNA array reveals extensive regulation of microRNAs during brain development. RNA 9, 1274–1281.CrossRefPubMedGoogle Scholar
  17. 17.
    Lagos-Quintana, M., Rauhut, R., Meyer, J., Borkhardt, A., and Tuschl, T. (2003) New microRNAs from mouse and human. RNA 9, 175–179.CrossRefPubMedGoogle Scholar
  18. 18.
    Dostie, J., Mourelatos, Z., Yang, M., Sharma, A., and Dreyfuss, G. (2003) Numerous microRNPs in neuronal cells containing novel microRNAs. RNA 9, 180–186.CrossRefPubMedGoogle Scholar
  19. 19.
    Sempere, L. F., Freemantle, S., Pitha-Rowe, I., Moss, E., Dmitrovsky, E., and Ambros, V. (2004) Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol. 5, R13.CrossRefPubMedGoogle Scholar
  20. 20.
    Miska, E. A., Alvarez-Saavedra, E., Townsend, M., et al. (2004) Microarray analysis of microRNA expression in the developing mammalian brain. Genome Biol. 5, R68.CrossRefPubMedGoogle Scholar
  21. 21.
    Babak, T., Zhang, W., Morris, Q., Blencowe, B. J., and Hughes, T. R. (2004) Probing microRNAs with microarrays: tissue specificity and functional inference. RNA 10, 1813–1819.CrossRefPubMedGoogle Scholar
  22. 22.
    Liu, C. G., Calin, G. A., Meloon, B., et al. (2004) An oligonucleotide microchip for genomewide microRNA profiling in human and mouse tissues. Proc. Natl. Acad. Sci. USA 101, 9740–9744.CrossRefPubMedGoogle Scholar
  23. 23.
    Thomson, J. M., Parker, J., Perou, C. M., and Hammond, S. M. (2004) A custom microarray platform for analysis of microRNA gene expression. Nat. Methods 1, 47–53.CrossRefPubMedGoogle Scholar
  24. 24.
    Baskerville, S. and Bartel, D. P. (2005) Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA 11, 241–247.CrossRefPubMedGoogle Scholar
  25. 25.
    Houbaviy, H. B., Murray, M. F., and Sharp, P. A. (2003) Embryonic stem cell-specific MicroRNAs. Dev. Cell 5, 351–358.CrossRefPubMedGoogle Scholar
  26. 26.
    Suh, M. R., Lee, Y., Kim, J. Y., et al. (2004) Human embryonic stem cells express a unique set of microRNAs. Dev. Biol. 270, 488–498.CrossRefPubMedGoogle Scholar
  27. 27.
    Calin, G. A., Liu, C. G., Sevignani, C., et al. (2004) MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc. Natl. Acad. Sci. USA 101, 11,755–11,760.CrossRefPubMedGoogle Scholar
  28. 28.
    Takamizawa, J., Konishi, H., Yanagisawa, K., et al. (2004) Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res. 64, 3753–3756.CrossRefPubMedGoogle Scholar
  29. 29.
    Poy, M. N., Eliasson, L., Krutzfeldt, J., et al. (2004) A pancreatic islet-specific microRNA regulates insulin secretion. Nature 432, 226–230.CrossRefPubMedGoogle Scholar
  30. 30.
    Pfeffer, S., Zavolan, M., Grasser, F. A., et al. (2004) Identification of virus-encoded microRNAs. Science 304, 734–736.CrossRefPubMedGoogle Scholar
  31. 31.
    Omoto, S., Ito, M., Tsutsumi, Y., et al. (2004) HIV-1 nef suppression by virally encoded microRNA. Retrovirology 1, 44.CrossRefPubMedGoogle Scholar
  32. 32.
    Pfeffer, S., Sewer, A., Lagos-Quintana, M., et al. (2005) Identification of microRNAs of the herpesvirus family. Nat. Methods 2, 269–276.CrossRefPubMedGoogle Scholar
  33. 33.
    Cai, X., Lu, S., Zhang, Z., Gonzalez, C. M., Damania, B., and Cullen, B. R. (2005) Kaposi’s sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells. Proc. Natl. Acad. Sci. USA 102, 5570–5575.CrossRefPubMedGoogle Scholar
  34. 34.
    Brennecke, J., Stark, A., Russell, R. B., and Cohen, S. M. (2005) Principles of MicroRNATarget Recognition. PLoS. Biol. 3, e85.CrossRefPubMedGoogle Scholar
  35. 35.
    Vella, M. C., Reinert, K., and Slack, F. J. (2004) Architecture of a validated microRNA::target interaction. Chem. Biol. 11, 1619–1623.CrossRefPubMedGoogle Scholar
  36. 36.
    Vella, M. C., Choi, E. Y., Lin, S. Y., Reinert, K., and Slack, F. J. (2004) The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3′UTR. Genes Dev. 18, 132–137.CrossRefPubMedGoogle Scholar
  37. 37.
    Stark, A., Brennecke, J., Russell, R. B., and Cohen, S. M. (2003) Identification of Drosophila microRNA targets. PLoS. Biol. 3, e60.CrossRefGoogle Scholar
  38. 38.
    Lewis, B. P., Shih, I., Jones-Rhoades, M. W., Bartel, D. P., and Burge, C. B. (2003) Prediction of mammalian microRNA targets. Cell 115, 787–798.CrossRefPubMedGoogle Scholar
  39. 39.
    Poy, M. N., Eliasson, L., Krutzfeldt, J., et al. (2004) A pancreatic islet-specific microRNA regulates insulin secretion. Nature 432, 226–230.CrossRefPubMedGoogle Scholar
  40. 40.
    Krek, A., Grün, D., Poy, P., et al. (2005) Combinatorial microRNA target predictions. Nat. Genet. 37, 226–230.CrossRefGoogle Scholar
  41. 41.
    Kiriakidou, M., Nelson, P. T., Kouranov, A., et al. (2004) A combined computationalexperimental approach predicts human microRNA targets. Genes Dev. 18, 1165–1178.CrossRefPubMedGoogle Scholar
  42. 42.
    Robins, H., Li, Y., and Padgett, R. W. (2005) Incorporating structure to predict microRNA targets. Proc. Natl. Acad. Sci. USA 102, 4006–4009.CrossRefPubMedGoogle Scholar
  43. 43.
    Chen, C. Z., Li, L., Lodish, H. F., and Bartel, D. P. (2004) MicroRNAs modulate hematopoietic lineage differentiation. Science 303, 83–86.CrossRefPubMedGoogle Scholar
  44. 44.
    Mansfield, J. H., Harfe, B. D., Nissen, R., et al. (2004) MicroRNA-responsive’ sensor’ transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nat. Genet. 36, 1079–1083.CrossRefPubMedGoogle Scholar
  45. 45.
    Zhao, Y., Samal, E., and Srivastava, D. (2005) Serum response factor regulates a musclespecific microRNA that targets Hand2 during cardiogenesis. Nature 436, 214–220.CrossRefPubMedGoogle Scholar
  46. 46.
    Yu, Z., Raabe, T., and Hecht, N. B. (2005) MicroRNA122a reduces expression of the post-transcriptionally regulated germ cell transition protein 2 (Tnp2) messenger RNA (mRNA) by mRNA cleavage. Biol. Reprod. 73, 427–433.CrossRefPubMedGoogle Scholar
  47. 47.
    Chang, J., Nicolas, E., Marks, D., et al. (2004) miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may down-regulate the high affinity cationic amino acid transporter CAT-1. RNA Biology 1, 106–113.PubMedGoogle Scholar
  48. 48.
    Johnson, S. M., Grosshans, H., Shingara, J., et al. (2005) RAS is regulated by the let-7 microRNA family. Cell 120, 635–647.CrossRefPubMedGoogle Scholar
  49. 49.
    O’Donnell, K. A., Wentzel, E. A., Zeller, K. I., Dang, C. V., and Mendell, J. T. (2005) c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435, 839–843.CrossRefGoogle Scholar
  50. 50.
    Matsumura, I., Tanaka, H., and Kanakura, Y. (2003) E2F1 and c-Myc in cell growth and death. Cell Cycle 2, 333–338.CrossRefPubMedGoogle Scholar
  51. 51.
    He, L., Thomson, J. M., Hemann, M. T., et al. (2005) A microRNA polycistron as a potential human oncogene. Nature 435, 828–833.CrossRefPubMedGoogle Scholar
  52. 52.
    Esau, C., Kang, X., Peralta, E., et al. (2004) MicroRNA-143 regulates adipocyte differentiation. J. Biol. Chem. 279, 52,361–52,365.CrossRefPubMedGoogle Scholar
  53. 53.
    Lecellier, C. H., Dunoyer, P., Arar, K., et al. (2005) A cellular microRNA mediates antiviral defense in human cells. Science 308, 557–560.CrossRefPubMedGoogle Scholar
  54. 54.
    Bullrich, F. and Croce, C. M. (2001) Chronic Lymphoid Leukemia, Dekker, New York, pp. 6640–6648.Google Scholar
  55. 55.
    Calin, G. A., Dumitru, C. D., Shimizu, M., et al. (2002) Frequent deletions and down-regulation of microRNA genes miR-15 and miR-16 at 13q14 in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA 99, 15,524–15,529.CrossRefPubMedGoogle Scholar
  56. 56.
    Michael, M. Z., O’Connor, S. M., van Holst Pellekaan, N. G., Young, G. P., and James, R. J. (2003) Reduced accumulation of specific microRNAs in colorectal neoplasia. Mol. Cancer Res. 1, 882–891.PubMedGoogle Scholar
  57. 57.
    Metzler, M., Wilda, M., Busch, K., Viehmann, S., and Borkhardt, A. (2004) High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma. Genes Chromosomes Cancer 39, 167–169.CrossRefPubMedGoogle Scholar
  58. 58.
    Calin, G. A., Sevignani, C., Dumitru, C. D., et al. (2004) Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc. Natl. Acad. Sci. USA 101, 2999–3004.CrossRefPubMedGoogle Scholar
  59. 59.
    Eis, P. S., Tam, W., Sun, L., et al. (2005) Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc. Natl. Acad. Sci. USA 102, 3627–3632.CrossRefPubMedGoogle Scholar
  60. 60.
    Lu, J., Getz, G., Miska, E. A., et al. (2005) MicroRNA expression profiles classify human cancers. Nature 435, 834–838.CrossRefPubMedGoogle Scholar
  61. 61.
    Ciafre, S. A., Galardi, S., Mangiola, A., et al. (2005) Extensive modulation of a set of microRNAs in primary glioblastoma. Biochem. Biophys. Res. Commun. 334, 1351–1358.CrossRefPubMedGoogle Scholar
  62. 62.
    Caudy, A. A., Myers, M., Hannon, G. J., and Hammond, S. M. (2002) Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev. 16, 2491–2496.CrossRefPubMedGoogle Scholar
  63. 63.
    Jin, P., Zarnescu, D. C., Ceman, S., et al. (2004) Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway. Nat. Neurosci. 7, 113–117.CrossRefPubMedGoogle Scholar
  64. 64.
    Jin, P., Alisch, R. S., and Warren, S. T. (2004) RNA and microRNAs in fragile X mental retardation. Nat. Cell Biol. 6, 1048–1053.CrossRefPubMedGoogle Scholar
  65. 65.
    Veneri, M., Zalfa, F., and Bagni, C. (2004) FMRP and its target RNAs: fishing for the specificity. Neuroreport 15, 2447–2450.CrossRefPubMedGoogle Scholar
  66. 66.
    Dong, J. T., Boyd, J. C., and Frierson, H. F. Jr. (2001) Loss of heterozygosity at 13q14 and 13q21 in high grade, high stage prostate cancer. Prostate 49, 166–171.CrossRefPubMedGoogle Scholar
  67. 67.
    Lee, Y. S., Kim, H. K., Chung, S., Kim, K. S., and Dutta A. (2005) Depletion of human micro-RNA miR-125b reveals that it is critical for the proliferation of differentiated cells but not for the down-regulation of putative targets during differentiation. J. Biol. Chem. 280, 16,635–16,641.CrossRefPubMedGoogle Scholar
  68. 68.
    Chan, J. A., Krichevsky, A. M., and Kosik, K. S. (2005) MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 65, 6029–6033.CrossRefPubMedGoogle Scholar
  69. 69.
    Cheng, A. M., Byrom, M. W., Shelton, J., and Ford, L. P. (2005) Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res. 33, 1290–1297.CrossRefPubMedGoogle Scholar
  70. 70.
    Lai, E. C. (2004) Predicting and validating microRNA targets. Genome Biol. 5, 115.CrossRefPubMedGoogle Scholar
  71. 71.
    Lee, Y. S., Kim, H. K., Chung, S., Kim, K. S., and Dutta, A. (2005) Depletion of human microRNA miR-125b reveals that it is critical for the proliferation of differentiated cells but not for the down-regulation of putative targets during differentiation. J. Biol. Chem. 280, 16,635–16,641.CrossRefPubMedGoogle Scholar
  72. 72.
    Meister, G., Landthaler, M., Dorsett, Y., and Tuschl, T. (2004) Sequence-specific inhibition of microRNA-and siRNA-induced RNA silencing. RNA 10, 544–550.CrossRefPubMedGoogle Scholar
  73. 73.
    Braasch, D. A. and Corey, D. R. (2001) Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chem. Biol. 8, 1–7.CrossRefPubMedGoogle Scholar
  74. 74.
    Valoczi, A., Hornyik, C., Varga, N., Burgyan, J., Kauppinen, S., and Havelda, Z. (2004) Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res. 32, e175.CrossRefPubMedGoogle Scholar
  75. 75.
    Summerton, J. and Weller, D. (1997) Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 7, 187–195.PubMedGoogle Scholar
  76. 76.
    Wheeler, D. L., Barrett, T., Benson, D. A., et al. (2005) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 33, D39–D45.CrossRefPubMedGoogle Scholar
  77. 77.
    Birney, E., Andrews, D., Bevan, P., et al. (2004) Ensembl 2004. Nucleic Acids Res. 32 (Database issue), D468–D470.CrossRefPubMedGoogle Scholar
  78. 78.
    Saxena, S., Jonsson, Z. O., and Dutta, A. (2003) Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells. J. Biol. Chem. 278, 44,312–44,319.CrossRefPubMedGoogle Scholar
  79. 79.
    Lewis, B. P., Shih, I. H., Jones-Rhoades, M. W., Bartel, D. P., and Burge, C. B. (2003) Prediction of mammalian microRNA targets. Cell 115, 787–798.CrossRefPubMedGoogle Scholar
  80. 80.
    Rehmsmeier, M., Steffen, P., Hochsmann, M., and Giegerich, R. (2004) Fast and effective prediction of microRNA/target duplexes. RNA 10, 1507–1517.CrossRefPubMedGoogle Scholar
  81. 81.
    Rajewsky, N. and Socci, N. D. (2004) Computational identification of microRNA targets. Dev. Biol. 267, 529–535.CrossRefPubMedGoogle Scholar
  82. 82.
    Grosshans, H., Johnson, T., Reinert, K. L., Gerstein, M., and Slack, F. J. (2005) The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans. Dev. Cell 8, 321–330.CrossRefPubMedGoogle Scholar
  83. 83.
    Smalheiser, N. R. and Torvik, V. I. (2004) A population-based statistical approach identifies parameters characteristic of human microRNA-mRNA interactions. BMC Bioinformatics 5, 139.CrossRefPubMedGoogle Scholar
  84. 84.
    Lewis, B. P., Burge, C. B., and Bartel, D. P. (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20.CrossRefPubMedGoogle Scholar
  85. 85.
    Rusinov, V., Baev, V., Minkov, I. N., and Tabler, M. (2005) MicroInspector: a web tool for detection of miRNA binding sites in an RNA sequence. Nucleic Acids Res. 33, W696–W700.CrossRefPubMedGoogle Scholar
  86. 86.
    Leaman, D., Chen, P. Y., Fak, J., et al. (2005) Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development. Cell 121, 1097–1108.CrossRefPubMedGoogle Scholar
  87. 87.
    Chen, P. Y., Manninga, H., Slanchev, K., et al. (2005) The developmental miRNA profiles of zebrafish as determined by small RNA cloning. Genes Dev. 19, 1288–1293.CrossRefPubMedGoogle Scholar
  88. 88.
    Griffiths-Jones, S., Bateman, A., Marshall, M., Khanna, A., and Eddy, S. R. (2003) Rfam: an RNA family database. Nucleic Acids Res. 31, 439–441.CrossRefPubMedGoogle Scholar
  89. 89.
    Weber, M. J. (2005) New human and mouse microRNA genes found by homology search. FEBS J. 272, 59–73.CrossRefPubMedGoogle Scholar
  90. 90.
    Altschul, S. F., Madden, T. L., Schaffer, A. A., et al. (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402.CrossRefPubMedGoogle Scholar
  91. 91.
    Bray, N. and Pachter, L. (2004) MAVID: constrained ancestral alignment of multiple sequences. Genome Res. 14, 693–699.CrossRefPubMedGoogle Scholar
  92. 92.
    Bray, N. and Pachter, L. (2003) MAVID multiple alignment server. Nucleic Acids Res. 31, 3525–3526.CrossRefPubMedGoogle Scholar
  93. 93.
    Kent, W. J., Sugnet, C. W., Furey, T. S., et al. (2002) The human genome browser at UCSC. Genome Res. 12, 996–1006.PubMedGoogle Scholar
  94. 94.
    Wuchty, S., Fontana, W., Hofacker, I. L., and Schuster, P. (1999) Complete suboptimal folding of RNA and the stability of secondary structures. Biopolymers 49, 145–165.CrossRefPubMedGoogle Scholar
  95. 95.
    Burgler, C. and Macdonald, P. M. (2005) Prediction and verification of microRNA targets by MovingTargets, a highly adaptable prediction method. BMC Genomics 6, 88.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Bino John
    • 1
  • Chris Sander
    • 1
  • Debora S. Marks
    • 2
  1. 1.Computational Biology CenterMemorial Sloan-Kettering Cancer CenterNew York
  2. 2.Department of Systems BiologyHarvard Medical SchoolBoston

Personalised recommendations