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WIREs Dev Biol
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Tiny giants of gene regulation: experimental strategies for microRNA functional studies

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The discovery over two decades ago of short regulatory microRNAs (miRNAs) has led to the inception of a vast biomedical research field dedicated to understanding these powerful orchestrators of gene expression. Here we aim to provide a comprehensive overview of the methods and techniques underpinning the experimental pipeline employed for exploratory miRNA studies in animals. Some of the greatest challenges in this field have been uncovering the identity of miRNA–target interactions and deciphering their significance with regard to particular physiological or pathological processes. These endeavors relied almost exclusively on the development of powerful research tools encompassing novel bioinformatics pipelines, high‐throughput target identification platforms, and functional target validation methodologies. Thus, in an unparalleled manner, the biomedical technology revolution unceasingly enhanced and refined our ability to dissect miRNA regulatory networks and understand their roles in vivo in the context of cells and organisms. Recurring motifs of target recognition have led to the creation of a large number of multifactorial bioinformatics analysis platforms, which have proved instrumental in guiding experimental miRNA studies. Subsequently, the need for discovery of miRNA–target binding events in vivo drove the emergence of a slew of high‐throughput multiplex strategies, which now provide a viable prospect for elucidating genome‐wide miRNA–target binding maps in a variety of cell types and tissues. Finally, deciphering the functional relevance of miRNA post‐transcriptional gene silencing under physiological conditions, prompted the evolution of a host of technologies enabling systemic manipulation of miRNA homeostasis as well as high‐precision interference with their direct, endogenous targets. WIREs Dev Biol 2016, 5:311–362. doi: 10.1002/wdev.223 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Gene Expression and Transcriptional Hierarchies > Regulatory RNA Technologies > Perturbing Genes and Generating Modified Animals
Strategies for discovery and validation of functional miRNA–target interactions. (a) miRNA‐activity‐sensors consist of a reporter construct (GFP) appended with a perfect target site for a miRNA of interest. In the presence of an active cognate miRNA, RNAi mediated slicing of the MRE results in loss of reporter expression. In the absence of the miRNA or in the presence of a control sensor with mutant MRE, expression of the reporter gene is detected. GFP‐based sensors allow spatial‐temporal detection of miRNA activity in vivo. (b) Target‐MRE‐sensors function on a similar principle as miRNA‐activity‐sensors, except that in this case an endogenous 3′UTR (or another part of the gene) containing a putative MRE of interest is fused to a reporter gene, and can be used to infer endogenous regulation of a candidate MRE in an intact organism or in cells in culture. (c) Luciferase‐based sensors provide a more quantitative readout of miRNA activity by measuring the relative expression of two distinct reporters. The 3′UTR of a gene of interest is cloned downstream of Renilla luciferase and codelivered to cells together with a control nontargeted Firefly luciferase construct. The ratio between the two reporters is used to quantitatively assess miRNA‐mediated repression of the MRE‐bearing sensor. (d) Target protectors (TPs) provide a direct in vivo approach for interfering with specific miRNA–target interactions. TPs are complementary with the seed region of a candidate MRE and the sequence immediately adjacent, which increases their specificity and decreases off‐target events. MRE, miRNA response element; GOI, gene of interest; R‐Luc, Renilla luciferase; FF, Firefly luciferase; AAA, poly‐A tail; Mo‐TP, morpholino target protector.
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miRNA loss‐of‐function approaches. (a) Under normal homeostasis, endogenous miRNA‐activity results in target repression. (b) Ablation of the genomic miRNA locus enables generation of a null miRNA mutant allele resulting in a global de‐repression of all its targets. (c) miRNA sponge (miR‐SP) competitive inhibitors are genetically encoded into the 3′UTR of a reporter genes and expressed from Pol II promoters. When deployed to in vivo they sequester a cognate miRNAs resulting in de‐repression of its endogenous targets. (d) Tough decoys (TuD), are also genetically encoded miRNA competitive inhibitors with enhanced RNA secondary structure for improved targeting and stability. (e) Modified synthetic antimiR oligonucleotides (such as antagomiRs) can be transfected into cells to sequester miRNAs away from their targets. (f) AntimiRs frequently contain chemical modifications which enhance their cellular stability and increase their miRNA binding affinity. Common ribose modifications include 2′‐O‐methyl nucleotides or locked nucleic acid (LNA) incorporations for enhanced affinity. The phosphate backbone can also carry a phosphorothioate modification for increased resistance to RNase degradation. Antagomirs are coupled to cholesterol to aid cellular uptake. LNA nucleotides have been incorporated in the center of long ASOs, spaced throughout the entire length of the ASO. In contrast, tiny LNAs are entirely LNA‐based short 8 bp ASOs complementary to the miRNA seed sequence. MRE, miRNA response element; Pol‐II/III, RNA Polymerase II/III promoters; t1,2,3, miRNA targets; AAA, poly‐A tail; miR‐SP, miRNA sponge; TuD, Tough decoy; ASO, antisense oligonucleotides; yellow box in MRE, seed match region.
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CLIP‐based high‐throughput miRNA target identification strategies. Specificity is achieved in all CLIP technologies by UV crosslinking (red X denote crosslinking sites) and size selection of Ago‐mRNA‐miRNA ternary complexes (SDS‐PAGE). All approaches use high‐throughput sequencing to quantify the purified RNA. (a) HITS‐CLIP yields two separate sets of data, one for mRNAs and one for miRNAs. (b) In PAR‐CLIP 4SU or 6SG is incorporated into RNA to enhance crosslinking and pull‐down efficiency. Only the coprecipitated mRNA is used to map the Ago‐binding sites with high resolution. (c) In iCLIP a special barcoded primer allows for the recovery of RT fragments that have been terminated at crosslinking sites due to protein remnants (diamond close to red X) resulting from incomplete crosslink reversal. The primer allows for circularization of fragments and subsequent linearization, via an internal restriction site, generating adapters on both ends of the fragments. These fragments are used to map Ago‐binding sites with high resolution. (d) The CLASH protocol introduces an additional Ago‐mRNA‐miRNA purification step on Ni‐NTA beads and ligates the 3′ end of the miRNA to the 5′ end of target mRNA to obtain miRNA–target chimeras. (e) In iPAR‐CLIP 4SU is used to increase crosslinking efficiency and an Ago‐GFP fusion protein for the IP. This method also includes a ligation step to link the 3′ end of the miRNA to the 5′ end of the target mRNA generating miRNA–target chimeras. UV, ultraviolet light; 4SU, 4‐thiouridine; 6SG, 6‐thioguanosine; NGS, next‐generation sequencing; RT, reverse transcription; CIP, calf intestinal alkaline phosphatase; IP, immunoprecipitation; Ni‐NTA, nickel‐charged affinity resin (nitrilotriacetic acid); TCA, trichloroacetic acid; PTH, protein A + TEV protease cleavage site + 6xHis tag; P, phosphate; OH, hydroxyl; PNK, polynucleotide kinase; T4 PNK (P‐ase ‐), T4 polynucleotide kinase (3′ phosphatase minus); prot. K, proteinase K; TSAP, thermosensitive alkaline phosphatase; GFP, green fluorescence protein; SDS‐PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Pmn, puromycin.
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Profiling and pull‐down‐based miRNA target identification techniques. (a) RNAseq yields short sequencing reads from all transcribed genes including 5′UTRs, exons, introns, and 3′UTRs. Intron‐exon split analysis (EISA) has the potential to distinguish between primary and secondary miRNA targets based on intron read counts differences. (b) In SILAC all proteins of the experimental condition are labeled with a heavy (H) isotope version while all proteins of control cells contain a normal (N) isotope version. The ratio between isotope versions indicates differential expression of proteins. In pSILAC a medium (M) and a heavy (H) isotope version are added to the control and experimental condition (=pulse) and differences in newly synthesized proteins are quantified. (c) Ribosome profiling yields all transcripts that are bound by ribosomes and the position of each ribosome with nucleotide resolution. (d) mRNA baits consist of a 3′UTR from the gene of interest (GOI) and a tag (M2‐loops or biotin) that allows for pull‐down via bead coupled protein moieties (MCP or streptavidin). Copurified miRNAs are analyzed by targeted qRT‐PCR or RNAseq. Transduction of the mRNA bait enables association of bait and miRNAs prior to cell lysis in vivo, while in vitro transcribed baits rely on proper target recognition ex vivo after cell lysis. (e) In mir‐CATCH miRNA–target complexes are crosslinked in vivo and the mRNA of interest is affinity purified via antisense capture oligonucleotides (oligo). After crosslinking reversal, copurified miRNAs are analyzed by targeted qRT‐PCR or nanostring. (f) In miR‐CLIP UV crosslinking is enhanced via a psoralen group. To reduce background a two‐step purification protocol is performed prior to quantification of copurified RNAs by RNAseq. MCP, MS2 coat protein; MBP, maltose binding protein; RT, reverse transcription; prot. K, proteinase K; SDS‐PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.
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Discovery and characterization of functional MREs by genome engineering. (a) Injection of in vitro transcribed RNA encoding a TALEN pair targeting approximately 20 bp flanking a predicted MRE site into single cell zebrafish embryos can be used to generate NHEJ‐mediated precise deletions of the MRE seed sequence. High efficiency editing events enable direct functional analysis of miRNA–target axes in mosaic animals without the necessity of establishing clonal lines. (b) Taking advantage of the simplicity and superior versatility of CRISPR/Cas9 genome engineering strategies, germline‐transmissible precise MRE deletions can be rapidly generated by injection of the Cas9 nuclease and a synthetic guide RNA (sgRNA) targeting the MRE of interest in Drosophila syncytial blastoderm embryos. Homozygous MRE mutant lines can then be used to study the consequence of interfering with a specific miRNA–target axis during development and adult life of an organism. (c) CRISPR/Cas9 genome engineering can also be adapted to assess the endogenous activity of specific MREs in human cells without the necessity of establishing clonal lines. This system relies on altering an MRE of interest by CRISPR‐mediated homology‐directed repair (HDR) using two user‐defined ssDNA oligonucleotide repair templates. The first template deletes the MRE and replaces it with a T7 ‘barcode’, while the second maintains the MRE and adds a T3 ‘barcode’. This allows the activity of the MRE to be analyzed in a heterogeneous mixture of cells by comparing the levels of mRNA containing the T7 ‘barcode’ where the MRE has been deleted, to the T3 ‘barcode’, where the MRE remains intact. This can be accurately quantified by measuring the mRNA levels (cDNA) normalized for HDR integration efficiency to genomic DNA (gDNA) from the same sample. TALEN, Transcription activator‐like effector nuclease; CRISPR/Cas9, clustered regularly interspaced short palindromic repeats (CRISPR)‐associated nuclease (Cas9); MRE, miRNA response element; NHEJ, nonhomologous end joining; HDR, homology‐directed repair; FokI, FokI nuclease; ssODN, single stranded DNA oligonucleotides; cDNA, complementary DNA; gDNA, genomic DNA.
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Schematic representation of different MRE types. (a) Canonical sites are defined by perfect complementarity with the miRNA seed sequence. 8mers are matched from position 1–8 and confer the strongest repression. 7mer‐m8 sites are matched at position 8 in addition to position 2–7. 7mer‐1A sites bear a 6mer seed as well as an A‐U pair at position 1. 3′ compensatory sites compensate a G:U wobble (or mismatch) within the seed by complementarity outside the seed. CLASH class II and III are both seed matched but display recurring complementarity at position 13–16 and 17–21, respectively. (b) Noncanonical sites are defined by mismatches within the seed region. Sites with single nt mismatches in the seed were often reported in multiple high‐throughput studies. A G bulged pivot nucleotide was frequently found between position 5 and 6 of the miRNA in Ago‐CLIP datasets. Centered sites display longer consecutive complementarity with only partial involvement of the seed. Cleavage sites possess extensive complementarity leading to slicing of the target. CLASH class IV sites have minimum 9 nt consecutive pairings outside the seed region. CLASH class V are orphan clusters without recurring motifs. Gray boxes = miRNA ‘seed’ region (nucleotides 2–7); green boxes denote characteristic motifs for each class; bold = base paired nucleotides; : = G:U wobble; red bar = complementarity at position 1 of the miRNA (unlikely to allow base pairing in vivo since this position is anchored inside Ago).
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Suggested experimental guideline for systematic interrogation of miRNA functions. A typical miRNA functional study entails a multipronged approach encompassing several layers of analysis. These include but are not limited to: in silico target prediction, unbiased in vivo interrogation of putative target networks, establishing phenotypic consequences of interfering with miRNA homeostasis (LOF), and discovery of physiologically relevant downstream targets and pathways. However, the order in which each of these phases is implemented can vary, and frequently some analyses can be carried out in parallel or are synergistically reinforcing each other. Similarly, the experimental strategies supporting such a pipeline are also subject to bias. The techniques outlined here reflect our preference regarding the current state‐of‐the‐art strategies underlying each of these steps.
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