Key Points
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Experimental approaches such as genetic screening, mRNA expression profiling, and Argonaute crosslinking and immunoprecipitation aim to identify microRNA (miRNA) targets and/or individual binding sites within them. Computational analyses of the resultant high-throughput data reveal the miRNA targets with high accuracy and resolution.
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Factors that define functional miRNA interaction sites include miRNA 'seed' complementarity, structural accessibility, and sequence and positional biases. These factors support modulatory interactions with RNA-binding proteins.
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miRNA seed families and clusters of co-expressed miRNAs are prevalent and may contribute to regulation of individual pathways across tissues and developmental stages.
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'Non-canonical' miRNA-binding sites seem to be prevalent, and their functionality should be further investigated.
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Outcomes of miRNA–target interactions include repression and increased precision of target gene expression, as well as induction of correlations in the expression levels of different targets.
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Computational modelling of miRNA–target interactions has provided insights into their consequences on target expression.
Abstract
Comparative genomics analyses and high-throughput experimental studies indicate that a microRNA (miRNA) binds to hundreds of sites across the transcriptome. Although the knockout of components of the miRNA biogenesis pathway has profound phenotypic consequences, most predicted miRNA targets undergo small changes at the mRNA and protein levels when the expression of the miRNA is perturbed. Alternatively, miRNAs can establish thresholds in and increase the coherence of the expression of their target genes, as well as reduce the cell-to-cell variability in target gene expression. Here, we review the recent progress in identifying miRNA targets and the emerging paradigms of how miRNAs shape the dynamics of target gene expression.
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Change history
16 September 2014
In Box 3 of this article, equation 3 was given incorrectly. The article has been corrected online. The authors apologize for this error.
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Acknowledgements
The authors thank P. Pemberton-Ross for help with the figure in Box 2 and members of M.Z.'s laboratory for comments on the manuscript.
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Glossary
- Argonaute
-
(AGO). A protein that, together with a microRNA (miRNA), forms a minimal miRNA-induced silencing complex (miRISC). Although the number of AGO proteins varies across species, four paralogues (which are thought to have overlapping activities) are known in humans and mice.
- miRNA-induced silencing complex
-
(miRISC). A ribonucleoprotein complex that includes Argonaute (AGO), a microRNA (miRNA) and additional proteins such as Dicer and trinucleotide repeat-containing gene 6A protein (TNRC6A). The miRNA in this complex guides the AGO protein to targets.
- CRISPR–Cas
-
(Clustered regularly interspaced short palindromic repeat–CRISPR-associated). A system that mediates the RNA-based immune defence of bacteria and archea to viruses and plasmids. It is composed of a genomic CRISPR array (which contains virus- or plasmid- complementary sequences that are interspersed with repeat elements) and a Cas protein (which carries the endonuclease activity).
- Stable isotope labelling by amino acids in cell culture
-
(SILAC). A mass spectrometry-based experimental technique that is used to compare protein abundance in different experimental conditions. Cells of one sample are grown in a medium containing amino acids labelled with light isotopes, and cells of another sample are grown in medium with heavy-isotope-labelled amino acids. The samples are then mixed, and changes in protein abundance are determined from the ratio between the signal from the light and heavy isotopes.
- Ribosome profiling
-
An experimental method to quantify the translation efficiency of individual mRNAs. Sucrose gradients are used to separate mRNAs that are actively translated and that are therefore associated with multiple ribosomes. Quantification can be done either at the level of the whole transcript (as in polysome profiling) or of the precise regions in the mRNA that are bound by ribosomes, which are protected from nuclease digestion (as in ribosome profiling or footprinting).
- Crosslinking and immunoprecipitation
-
(CLIP). An experimental method to map the binding sites of RNA-binding proteins across the transcriptome. Proteins are crosslinked to RNA using ultraviolet light, and an antibody is used to specifically isolate the RNA-binding protein of interest together with its RNA interaction partners, which are subjected to sequencing.
- Photoactivatable ribonucleoside-enhanced CLIP
-
(PAR-CLIP). A variant of CLIP in which photoreactive ribonucleoside analogues such as 4-thiouridine are incorporated into RNAs before crosslinking with ultraviolet A light (the wavelength of which is 365 nm).
- High-throughput sequencing of RNAs isolated by CLIP
-
(HITS-CLIP). A variant of CLIP in which the crosslinking is achieved using ultraviolet C light (the wavelength of which is 254 nm).
- Individual-nucleotide resolution CLIP
-
(iCLIP). A variant of CLIP that, in contrast to the other methods that rely on the reverse transcriptase to polymerize beyond the crosslinked nucleotides, aims to sequence the cDNAs at which the reverse transcriptase stopped at the crosslinked nucleotides, thereby achieving individual-nucleotide resolution in the mapping of sites of RNA-binding proteins.
- Crosslinking, immunoprecipitation and sequencing of hybrids
-
(CLASH). An experimental method to isolate RNAs that interact by hybridization in a ribonucleoprotein complex. The complex is immunoprecipitated with a specific antibody, the RNA is partially fragmented, interacting RNAs are ligated and the resulting chimeric products are sequenced.
- Long non-coding RNA
-
(lncRNA). An RNA molecule that is generally longer than structural RNAs (such as tRNAs, small nuclear RNAs and small nucleolar RNAs) and that does not encode proteins. Further subcategories are distinguished depending on the type of genomic regions from which they derive. One example is the long intergenic non-coding RNAs (lincRNAs) that are transcribed from genomic regions between protein-coding gene loci.
- Circular RNAs
-
(circRNAs). Very stable RNAs with circular structures that result from the ligation of the 3′ ends to the 5′ ends, for example, of exons. The circRNA CDR1 antisense RNA (CDR1as) has recently been found to function as a miRNA 'sponge'.
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Hausser, J., Zavolan, M. Identification and consequences of miRNA–target interactions — beyond repression of gene expression. Nat Rev Genet 15, 599–612 (2014). https://doi.org/10.1038/nrg3765
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DOI: https://doi.org/10.1038/nrg3765
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