1. Methods for identifying microRNA binding motifs
Wenfa Ng
Yong Loo Lin School of Medicine
National University of Singapore

2. RNA interference (RNAi)
Highly conserved pathway across species and domains
Mammalian (RNAi); Bacteria (CRISPR)
Post-transcriptional modulation of gene expression
Mediated by formation of RNA-induced silencing complex (RISC) with small non-coding (ncRNA) as guide RNA
Binding of microRNA to mRNA either results in mRNA cleavage or reduce mRNA stability through deadenylation and exonuclease degradation
NCBI

3. Types of miRNA-mRNA interactions
Current Genomics, Vol. 14, pp

4. Search for canonical base-pairing rules in RNAi
Significant effort devoted to searching conserved binding motifs between miRNAs and mRNA, which may inform miRNA biological functions and regulatory mechanisms
Ideal case of exact template-dependent base-pairing between non-coding RNA and mRNA
But, structural effects such as stem-loop and hairpin structures in mRNA prevent exact base-pairing
Thus, partial sequence complementarity is the norm rather than the exception
Manifest as graduation in inhibition, cleavage and translation block

5. But non-canonical interactions abound
Bulges and G:U wobbles
Non seed pairing
Seedless interactions
Non-canonical interactions could occur in 3’ UTR, 5’ UTR, promoter and coding DNA sequence (CDS)
Generally, non-canonical interactions are “typical,” while only a quarter of miRNA-mRNA interactions follow canonical rules

6. Additional caveats
Uninterrupted, bulge-free base-pairing to the 5’ end of miRNA may not lead to substantial repression
Coincidence of multiple G:U pairs with ≥1 single nucleotide bulges in the seed region could result in significant repression
Asymmetric, single nucleotide seed-region bulges on miRNA side does not necessarily abolish repression

7. Site accessibility versus sequence complementarily
Intra- and intermolecular base pairing accounts for large fraction of miRNA-mRNA interactions
Tight secondary structures found to hinder access of RNA-induced silencing complex (RISC) to binding site
Efficient interactions between miRNA and target require unpairing of short sequence flanking binding motif
Equal proportion of identified seed regions in random regions and genomic areas with open conformation
Nature Genetics, Vol. 39, No. 10, pp

8. Genome-wide isolation of miRNA targets and determination of binding motifs
Ago pull-down, crosslinking and RNase digestion (HTIS-CLIP)
Crosslinking and immunoprecipitation followed by high throughput sequencing (CLIP-Seq)
Photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP)
Crosslinking, ligation and sequencing of hybrids (CLASH)
Computational sequence and energetics based miRNA target prediction for deciphering immunoprecipitation cum sequencing data

9. Computational miRNA target prediction
Sequence complementarily between 5’ seed of miRNA and 3’ UTR of mRNA
Conservation of the miRNA binding site
Topology of base-pairing at 5’ seed region of miRNA
Structural accessibility of target sequences and the flanking regions
Favourable minimum free energy of miRNA-target hybridisation (-15 to -10 kcal/mol, -25 kcal/mol stringent cut-off)

10. Overview of crosslinking-precipitation approaches
Commonalities
Formation of miRISC, crosslinking Ago protein (nestled with miRNA) to mRNA, immunoprecipitation of complex, release of bound RNA for identification of recognition sequence, readout by short-read sequencing
Amenable to conduct large-scale analysis but unable to differentiate between direct and indirect miRNA-target interactions
Nature Reviews Genetics, Vol. 15, No. 9, pp

11. HITS-CLIP (High throughput sequencing of crosslinking immunoprecipitation)
Formation of Ago-miRNA-mRNA complex
UV crosslinking and ligation
Isolation of binding motif via enzymatic digestion
Dissociation of RNA from protein complex
Reverse transcription, PCR amplification, deep sequencing
Unable to identify specific target site (resolution of ~100 nt)

12. PAR-CLIP (Photo-activated ribonucleoside enhanced crosslinking and immunoprecipitation)
Improved CLIP for identifying RNA-binding protein and miRNA targets with more efficient UV crosslinking
Transcriptome-wide crosslinking method for RNA-binding proteins with fold improvement in RNA recovery relative to HITS-CLIP
Based on incorporation of photoactivable nucleoside (4-thiouridine) analogs into nascent RNA during co-incubation in cell culture
Site of crosslinking near centre of AGO-miRNA-mRNA complex; home in on specific target sequence
Characteristic sequence transition (thymidine to cytidine) in prepared cDNA helps reveal the precise binding site between Ago-miRNA complex and mRNA
Cell, Vol. 141, pp

13. CLASH (Crosslinking, Ligation and Sequencing of Hybrids)
Genome-wide unbiased assay for miRNA-mRNA interactions
UV-crosslinking of miRNA-mRNA duplex in complex with Ago1 followed by sequencing
Most miRNA binding includes the 5’ seed, but sequence analysis reveals that 60% of binding sites are non-canonical
18% of miRNA-mRNA involves 3’ end of miRNA with little contribution from the 5’ “seed”
miRNA-species specific effect in miRNA-mRNA interactions observed in dataset (>18K), postulated to affect RISC complex formation
Cell, Vol. 153, pp

14. Detailed workflow of CLASH
Cell, Vol. 153, pp

15. Biotinylated miRNA mimic
Biotinylation of the 3’ end of active strand
Able to form miRNA-induced silencing complex (miRISC) for downregulating mRNA expression
Does not require crosslinking and require 20 fold lower cell number relative to crosslinking methods
High specificity (~90%)
But dependent on mRNA microarray to identify enriched candidates and is not a direct binding assay for miRNA-response elements (MREs)
Reduces background as the strong streptavidin binding affords the use of harsher (but more effective) washing conditions

16. Deficiencies of current methods
Reliant on seed-based (canonical) analysis or predictive algorithms for assigning isolated sequence to miRNA
Require large numbers of cells (20 million cells)
More suitable for miRNA with high expression levels
UV crosslinking introduces background and is inefficient
Photoactivable nucleotides increase specificity but may introduce sequence bias

17. Improved protocol for systems-level identification of MREs
Replaced microarray with deep sequencing (Pulldown-seq)
Addition of RNase step for identifying MREs (IMPACT-Seq: Identification of MREs by pull-down and alignment of captive transcripts-sequencing)
Analysis of transcription factors that regulate target genes via gene ontology and interactome approach
Method enabled the rapid identification of target genes and biological functions of a specific miRNA at global level
Cell Reports, Vol. 8, pp

18. miRanda
Cross-species conservation filter free identification of miRNA binding sites
Identifies putative miRNA binding sites, and subsequently, deduces targeting miRNA
No prior knowledge of miRNA needed
Approach reveals that some miRNAs could have several thousand targets
Significantly larger number of miRNA precursors, miRNA binding sites and targets than hypothesized
Binding sites occur in 5’ UTR, CDS and 3’ UTR
Cell, Vol. 126, pp

19. TargetScan
Checks for perfect Watson-Crick base-pairing between the 5’ miRNA seed sequence (7 nt) and 3’ UTR sequence of mRNA
Algorithm also considers the free energy of miRNA-mRNA interactions for evaluating heteroduplex stability
Scan for homologous binding motif in UTRs of other species
A variation of TargetScan, TargetScan S, considers the flanking region of the seed region but does not take into account free energy of binding
Cell, Vol. 115, pp

20. Summary
Functional importance of miRNA binding in post-transcriptional gene regulation drives effort in identifying conserved binding motifs
But non-canonical motifs dominate over those based on seed sequence
Experimental approaches based on RISC complex formation and immunoprecipitation help isolate miRNA-mRNA-Ago complex for downstream readout via next-generation sequencing
Computational predictive tools (e.g., miRanda, TargetScan) help deduce binding motif from sequence reads
Multiple drawbacks such as inefficiencies of UV crosslinking, sequence bias and large cell mass requirement motivate the development of improved techniques
One example replaces microarray with deep sequencing, added an RNase step, and used biotinylated miRNA mimic (for abrogating use of crosslinking)
Identification of miRNA binding motif together with characterizing the nature of miRNA-mRNA interactions remain outstanding problems in the field