High-throughput Assessment of MicroRNA Activity and Function Using MicroRNA Sensor and Decoy Libraries

Overview

Journal Nat Methods

Specialties Biomedical Engineering
Pathology

Date 2012 Jul 4

PMID 22751203

Citations 256

Authors

Gavriel Mullokandov

Alessia Baccarini

Albert Ruzo

Anitha D Jayaprakash

Navpreet Tung

Benjamin Israelow

Matthew J Evans

Ravi Sachidanandam

Brian D Brown

Affiliations

Soon will be listed here.

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.

Citing Articles

Prenatal SARS-CoV-2 Infection Alters Human Milk-Derived Extracellular Vesicles.

Chutipongtanate S, Kongsomros S, Cetinkaya H, Zhang X, Kuhnell D, Benefield D Cells. 2025; 14(4).

PMID: 39996756 PMC: 11853888. DOI: 10.3390/cells14040284.

A comparative analysis of Marburg virus-infected bat and human models from public high-throughput sequencing data.

Xuan D, Yeh I, Liu H, Su C, Ko C, Ta H Int J Med Sci. 2025; 22(1):1-16.

PMID: 39744175 PMC: 11659840. DOI: 10.7150/ijms.100696.

Beyond the Bubble: A Debate on microRNA Sorting Into Extracellular Vesicles.

Liu X, Halushka M Lab Invest. 2024; 105(2):102206.

PMID: 39647608 PMC: 11842217. DOI: 10.1016/j.labinv.2024.102206.

Quantitative design of cell type-specific mRNA stability from microRNA expression data.

Oesinghaus L, Castillo-Hair S, Ludwig N, Keller A, Seelig G bioRxiv. 2024; .

PMID: 39554011 PMC: 11565874. DOI: 10.1101/2024.10.28.620728.

Ovarian cancer-derived IL-4 promotes immunotherapy resistance.

Mollaoglu G, Tepper A, Falcomata C, Potak H, Pia L, Amabile A Cell. 2024; 187(26):7492-7510.e22.

PMID: 39481380 PMC: 11682930. DOI: 10.1016/j.cell.2024.10.006.

References

Cleary M, Kilian K, Wang Y, Bradshaw J, Cavet G, Ge W . Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis. Nat Methods. 2005; 1(3):241-8. DOI: 10.1038/nmeth724. View

Grimson A, Farh K, Johnston W, Garrett-Engele P, Lim L, Bartel D . MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell. 2007; 27(1):91-105. PMC: 3800283. DOI: 10.1016/j.molcel.2007.06.017. View

Narbus C, Israelow B, Sourisseau M, Michta M, Hopcraft S, Zeiner G . HepG2 cells expressing microRNA miR-122 support the entire hepatitis C virus life cycle. J Virol. 2011; 85(22):12087-92. PMC: 3209320. DOI: 10.1128/JVI.05843-11. View

Brown B, Venneri M, Zingale A, Sergi Sergi L, Naldini L . Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nat Med. 2006; 12(5):585-91. DOI: 10.1038/nm1398. View

Mortazavi A, Williams B, McCue K, Schaeffer L, Wold B . Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008; 5(7):621-8. DOI: 10.1038/nmeth.1226. View

Kelly E, Hadac E, Greiner S, Russell S . Engineering microRNA responsiveness to decrease virus pathogenicity. Nat Med. 2008; 14(11):1278-83. DOI: 10.1038/nm.1776. View

Ebert M, Neilson J, Sharp P . MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods. 2007; 4(9):721-6. PMC: 3857099. DOI: 10.1038/nmeth1079. View

Hutvagner G, Zamore P . A microRNA in a multiple-turnover RNAi enzyme complex. Science. 2002; 297(5589):2056-60. DOI: 10.1126/science.1073827. View

Jayaprakash A, Jabado O, Brown B, Sachidanandam R . Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res. 2011; 39(21):e141. PMC: 3241666. DOI: 10.1093/nar/gkr693. View

10.

Pasquinelli A . MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship. Nat Rev Genet. 2012; 13(4):271-82. DOI: 10.1038/nrg3162. View

11.

Haley B, Zamore P . Kinetic analysis of the RNAi enzyme complex. Nat Struct Mol Biol. 2004; 11(7):599-606. DOI: 10.1038/nsmb780. View

12.

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. 2008; 4(3):239-48. PMC: 2605097. DOI: 10.1016/j.chom.2008.08.003. View

13.

Cesana M, Cacchiarelli D, Legnini I, Santini T, Sthandier O, Chinappi M . A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA. Cell. 2011; 147(2):358-69. PMC: 3234495. DOI: 10.1016/j.cell.2011.09.028. View

14.

Amendola M, Venneri M, Biffi A, Vigna E, Naldini L . Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters. Nat Biotechnol. 2004; 23(1):108-16. DOI: 10.1038/nbt1049. View

15.

Jopling C, Yi M, Lancaster A, Lemon S, Sarnow P . Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science. 2005; 309(5740):1577-81. DOI: 10.1126/science.1113329. View

16.

Hwang H, Wentzel E, Mendell J . A hexanucleotide element directs microRNA nuclear import. Science. 2007; 315(5808):97-100. DOI: 10.1126/science.1136235. View

17.

Brown B, Naldini L . Exploiting and antagonizing microRNA regulation for therapeutic and experimental applications. Nat Rev Genet. 2009; 10(8):578-85. DOI: 10.1038/nrg2628. View

18.

Xie J, Ameres S, Friedline R, Hung J, Zhang Y, Xie Q . Long-term, efficient inhibition of microRNA function in mice using rAAV vectors. Nat Methods. 2012; 9(4):403-9. PMC: 3420816. DOI: 10.1038/nmeth.1903. View

19.

Gottwein E, Cai X, Cullen B . A novel assay for viral microRNA function identifies a single nucleotide polymorphism that affects Drosha processing. J Virol. 2006; 80(11):5321-6. PMC: 1472151. DOI: 10.1128/JVI.02734-05. View

20.

Bassik M, Lebbink R, Churchman L, Ingolia N, Patena W, LeProust E . Rapid creation and quantitative monitoring of high coverage shRNA libraries. Nat Methods. 2009; 6(6):443-5. PMC: 2783737. DOI: 10.1038/nmeth.1330. View