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Cited by 394MicroRNA in disease regulationRNA Interference and Gene DeliveryPlant Molecular Biology Research

High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries

Open access
Mullokandov, Gavriel, Baccarini, Alessia, Ruzo, Albert, Jayaprakash, Anitha D, Tung, Navpreet, Israelow, Benjamin, Evans, Matthew J, Sachidanandam, Ravi +1 more·2012·Nature Methods
DOI 10.1038/nmeth.2078PMC PMC3518396
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Abstract

We introduce two large-scale resources for functional analysis of microRNA (miRNA): a decoy library for inhibiting miRNA function and a sensor library for monitoring microRNA activity. To take advantage of the sensor library, we developed a high-throughput assay called Sensor-seq to simultaneously quantify the activity of hundreds of miRNAs. Using this approach, we show that only the most abundant miRNAs in a cell mediate target suppression. Over 60% of detected miRNAs had no discernible activity, which indicated that the functional 'miRNome' of a cell is considerably smaller than currently inferred from profiling studies. Moreover, some highly expressed miRNAs exhibited relatively weak activity, which in some cases correlated with a high target-to-miRNA ratio or increased nuclear localization of the miRNA. Finally, we show that the miRNA decoy library can be used for pooled loss-of-function studies. These tools are valuable resources for studying miRNA biology and for miRNA-based therapeutics.

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  • This number of new microRNAs seems impossibly high and likely mostly false positives and/or nonfunctional transcripts.

    “This number of new microRNAs seems impossibly high and likely mostly false positives and/or nonfunctional transcripts (Mullokandov et al. 2012).”

    From Toward the human cellular microRNAome (2017) · cites this paper as Mullokandov et al. 2012

    The Discussion (page unavailable in our copy) reports that almost 60% of miRNAs detected in the cells studied had no discernible suppressive activity, supporting concern that many detected miRNAs may have little biological activity. It does not assess the number of *new* miRNAs or conclude that most are false-positive identifications. The Results’ reference to possible false positives concerns a small set of sensor results, not the miRNA transcripts themselves. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • miRNA inhibition using decoy or sponge-like constructs has potential therapeutic benefit.

    “It has already been shown that miRNA inhibition using decoy or sponge-like constructs have a potential therapeutic benefit.”

    From Potential Therapeutic Effects of Exosomes Packed With a miR-21-Sponge Construct in a Rat Model of Glioblastoma (2019) · cites this paper as [30]

  • Sponge constructs are designed to bind complementary miRNA(s) or their seed sequences, and hence block the binding of the miRNA to its biological targets.

    “The sponge construct are designed to bind its complementary miRNA(s) or their seed sequences, and hence block the binding of the miRNA to its biological targets (29–31).”

    From Potential Therapeutic Effects of Exosomes Packed With a miR-21-Sponge Construct in a Rat Model of Glioblastoma (2019) · cites this paper as [30]

  • Using decoy or sponge-like constructs for miRNA inhibition has been shown to bind complementary miRNA(s) or their seed sequences and block their effects.

    “Using decoy or sponge-like constructs for miRNA inhibition have been already shown before with good effect in binding to its complementary miRNA(s) or their seed sequences and blocking their effects (29–31, 56, 57).”

    From Potential Therapeutic Effects of Exosomes Packed With a miR-21-Sponge Construct in a Rat Model of Glioblastoma (2019) · cites this paper as [30]

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References 47 · Cited by 394

Research works printed but not linked: 41

  1. [2]He L Hannon GJ MicroRNAs: small RNAs with a big role in gene regulation Nat Rev Genet 5 522 531 2004 15211354
  2. [3]Bartel DP MicroRNAs: target recognition and regulatory functions Cell 136 215 233 2009 19167326 PMC3794896
  3. [4]Brown BD Naldini L Exploiting and antagonizing microRNA regulation for therapeutic and experimental applications Nat Rev Genet 10 578 585 2009 19609263
  4. [5]Mansfield JH MicroRNA-responsive ‘sensor’ transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression Nat Genet 36 1079 1083 2004 15361871
  5. [6]Gottwein E Cai X Cullen BR A novel assay for viral microRNA function identifies a single nucleotide polymorphism that affects Drosha processing J Virol 80 5321 5326 2006 16699012 PMC1472151
  6. [7]Brown BD Venneri MA Zingale A Sergi Sergi L Naldini L Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer Nat Med 12 585 591 2006 16633348
  7. [8]Kelly EJ Hadac EM Greiner S Russell SJ Engineering microRNA responsiveness to decrease virus pathogenicity Nat Med 14 1278 1283 2008 18953352
  8. [9]Barnes D Kunitomi M Vignuzzi M Saksela K Andino R Harnessing endogenous miRNAs to control virus tissue tropism as a strategy for developing attenuated virus vaccines Cell Host Microbe 4 239 248 2008 18779050 PMC2605097
  9. [10]Gentner B Identification of hematopoietic stem cell-specific miRNAs enables gene therapy of globoid cell leukodystrophy Sci Transl Med 2 58ra84 2010 21084719
  10. [11]Care A MicroRNA-133 controls cardiac hypertrophy Nat Med 13 613 618 2007 17468766
  11. [12]Ebert MS Neilson JR Sharp PA MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells Nat Methods 4 721 726 2007 17694064 PMC3857099
  12. [13]Gentner B Stable knockdown of microRNA in vivo by lentiviral vectors Nat Methods 6 63 66 2009 19043411
  13. [15]Brown BD Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state Nat Biotechnol 25 1457 1467 2007 18026085
  14. [17]Baccarini A Kinetic analysis reveals the fate of a microRNA following target regulation in mammalian cells Curr Biol 21 369 376 2011 21353554 PMC3088433
  15. [18]Hafner M Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP Cell 141 129 141 2010 20371350 PMC2861495
  16. [19]Jayaprakash AD Jabado O Brown BD Sachidanandam R Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing Nucleic Acids Res 39 e141 2011 21890899 PMC3241666
  17. [20]Franco-Zorrilla JM Target mimicry provides a new mechanism for regulation of microRNA activity Nat Genet 39 1033 1037 2007 17643101
  18. [21]Jones MR Zcchc11-dependent uridylation of microRNA directs cytokine expression Nat Cell Biol 11 1157 1163 2009 19701194 PMC2759306
  19. [22]Cesana M A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA Cell 147 358 369 2011 22000014 PMC3234495
  20. [23]Arvey A Larsson E Sander C Leslie CS Marks DS Target mRNA abundance dilutes microRNA and siRNA activity Mol Syst Biol 6 363 2010 20404830 PMC2872614
  21. [24]Garcia DM Weak seed-pairing stability and high target-site abundance decrease the proficiency of lsy-6 and other microRNAs Nat Struct Mol Biol 18 1139 1146 2011 21909094 PMC3190056
  22. [26]Loya CM Lu CS Van Vactor D Fulga TA Transgenic microRNA inhibition with spatiotemporal specificity in intact organisms Nat Methods 6 897 903 2009 19915559 PMC3183579
  23. [27]Xie J Long-term, efficient inhibition of microRNA function in mice using rAAV vectors Nat Methods 9 403 409 2012 22388288 PMC3420816
  24. [28]Jopling CL Yi M Lancaster AM Lemon SM Sarnow P Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA Science 309 1577 1581 2005 16141076
  25. [29]Pasquinelli AE MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship Nat Rev Genet 13 271 282 2012 22411466
  26. [30]Seitz H Redefining microRNA targets Curr Biol 19 870 873 2009 19375315
  27. [31]Hutvagner G Zamore PD A microRNA in a multiple-turnover RNAi enzyme complex Science 297 2056 2060 2002 12154197
  28. [32]Haley B Zamore PD Kinetic analysis of the RNAi enzyme complex Nat Struct Mol Biol 11 599 606 2004 15170178
  29. [33]Hwang HW Wentzel EA Mendell JT A hexanucleotide element directs microRNA nuclear import Science 315 97 100 2007 17204650
  30. [36]Ni JQ A genome-scale shRNA resource for transgenic RNAi in Drosophila Nat Methods 8 405 407 2011 21460824 PMC3489273
  31. [37]Cleary MA Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis Nat Methods 1 241 248 2004 15782200
  32. [38]Bassik MC Rapid creation and quantitative monitoring of high coverage shRNA libraries Nat Methods 6 443 445 2009 19448642 PMC2783737
  33. [39]Amendola M Venneri MA Biffi A Vigna E Naldini L Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters Nat Biotechnol 23 108 116 2005 15619618
  34. [40]Baccarini A Brown BD Monitoring microRNA activity and validating microRNA targets by reporter-based approaches Methods Mol Biol 667 215 233 2010 20827537
  35. [41]Malone CD Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary Cell 137 522 535 2009 19395010 PMC2882632
  36. [42]Jayaprakash AD Jabado O Brown BD Sachidanandam R Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing Nucleic Acids Res 2011 PMC3241666 21890899
  37. [43]Brennecke J Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila Cell 128 1089 1103 2007 17346786
  38. [44]Girard A Sachidanandam R Hannon GJ Carmell MA A germline-specific class of small RNAs binds mammalian Piwi proteins Nature 442 199 202 2006 16751776
  39. [45]Grimson A MicroRNA targeting specificity in mammals: determinants beyond seed pairing Mol Cell 27 91 105 2007 17612493 PMC3800283
  40. [47]Lindenbach BD Complete replication of hepatitis C virus in cell culture Science 309 623 626 2005 15947137
  41. [48]Narbus CM HepG2 cells expressing microRNA miR-122 support the entire hepatitis C virus life cycle J Virol 85 12087 12092 2011 21917968 PMC3209320

Authors
Mullokandov, G., Baccarini, A., Ruzo, A., Jayaprakash, A. D., Tung, N., Israelow, B., Evans, M. J., Sachidanandam, R., & Brown, B. D.
Affiliations
Icahn School of Medicine at Mount Sinai (US)
Corresponding author
Brown, Brian D
ORCID
Venue
Nature Methods
Publisher
Springer Science and Business Media LLC
Published
2012-07-01
Volume
9
Issue
8
Pages
840-846
ISSN
  • 1548-7091 (ISSN-L)
  • 1548-7105
DOI
10.1038/nmeth.2078
PMC
PMC3518396
OpenAlex
W1973401140
Cited by
394
References
47
Citations by year
2026: 16 · 2025: 24 · 2024: 13 · 2023: 20 · 2022: 16 · 2021: 25 · 2020: 22 · 2019: 19 · 2018: 31 · 2017: 49 · 2016: 44 · 2015: 48 · 2014: 35 · 2013: 28 · 2012: 4
FWCI
12.21
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature Methods (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institutes of Health (1dp2dk083052-01)
  • National Institute of Allergy and Infectious Diseases (R56AI091792)
  • National Institute of Diabetes and Digestive and Kidney Diseases (DP2DK083052‑01, R01DK095125, DP2DK083052)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
Keywords
microRNA function, loss-of-function screen, miRNome, miRNA abundance, miRNA-based therapy
MeSH terms
Animals, Genetic Vectors, Humans, Biosensing Techniques, Gene Library, MicroRNAs, High-Throughput Screening Assays

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