Methods for identifying microRNA binding motifs Wenfa Ng Yong Loo Lin School of Medicine National University of Singapore Email: ngwenfa@alumni.nus.edu.sg a0015092@u.nus.edu

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

Types of miRNA-mRNA interactions Current Genomics, Vol. 14, pp. 127-136

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

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

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

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. 1278-1284

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

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)

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.. 599 - 612

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)

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 100-1000 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. 129-141

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. 654-665

Detailed workflow of CLASH Cell, Vol. 153, pp. 654-665

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

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

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. 1225-1239

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. 1203-1217

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. 787-798

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