Download Methods for identifying microRNA binding motifs

Methods for identifying microRNA
binding motifs
Wenfa Ng
Email: [email protected]
[email protected]
RNA interference (RNAi)
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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
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Types of miRNA-mRNA interactions
Current Genomics, Vol. 14, pp. 127136
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Search for canonical base-pairing
rules in RNAi
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Significant effort devoted to searching conserved binding motifs
between miRNAs and mRNA, which may inform miRNA biological
functions and regulatory mechanisms
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Ideal case of exact template dependent base pairing between noncoding RNA and mRNA
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But, structural effects such as stem-loop and hairpin structures in
mRNA prevent exact base-pairing
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Thus, partial sequence complementarity is the norm rather than the
exception
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Manifest as graduation in inhibition, cleavage and translation block
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But non-canonical interactions abound
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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
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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
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Site accessibility versus sequence
complementarily
• Intra- and intermolecular base pairing accounts for a 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
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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
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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)
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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 large-scale analysis but unable to differentiate between
direct and indirect miRNA-target interactions
Nature Reviews Genetics,
Vol. 15, No. 9, pp.. 599 - 612
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HITS-CLIP (High throughput sequencing
of crosslinking immunoprecipitation)
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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)
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PAR-CLIP (Photo-activated ribonucleoside
enhanced crosslinking and immunoprecipitation)
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Improved CLIP for identifying RNAbinding 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 (4thiouridine) 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. 129141
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CLASH (Crosslinking, Ligation and
Sequencing of Hybrids)
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Genome-wide unbiased assay for
miRNA-mRNA interactions
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UV-crosslinking of miRNA-mRNA duplex
in complex with Ago1 followed by
sequencing
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Most miRNA binding includes the 5’
seed, but sequence analysis reveals
that 60% of binding sites are noncanonical
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18% of miRNA-mRNA involves 3’ end of
miRNA with little contribution from the 5’
“seed”
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miRNA-species specific effect in
miRNA-mRNA interactions observed in
dataset (>18K), postulated to affect
RISC complex formation
Cell, Vol. 153, pp. 654665
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Detailed workflow of CLASH
Cell, Vol. 153, pp. 654665
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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
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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
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Improved protocol for systems level
identification of MREs
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Replaced microarray with
deep sequencing (Pulldownseq)
Addition of RNase step for
identifying MREs (IMPACTSeq: Identification of MREs by
pull-down and alignment of
captive transcriptssequencing)
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. 17
1225-1239
miRanda
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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- 18
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TargetScan
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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. 787798
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Summary
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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