13: miRNA targets and HITS-CLIP

microRNA Biogenesis

• The biogenesis process of microRNA is depicted

microRNA Targets: Identification Methods

• Bioinformatics helps in predicting mRNA targets for microRNAs
• Target predictions from bioinformatics can be verified through PCR or Western blotting
• Identifying all targets systematically needs experimental methods
• Transcriptomics can be used to identify microRNA targets

Measuring mRNA Changes with Transcriptomics

• Changes can be gauged in mRNA levels via transcriptomics, utilizing methods like mRNA microarrays (e.g., Affymetrix)
• Next Generation Sequencing (NGS) can also be used for transcriptomics
• Measuring the differences in mRNA levels is possible when a microRNA is overexpressed
• Measuring the differences in mRNA levels is possible during microRNA inhibition
• mRNAs that are differentially regulated are either direct targets or regulated by targets of a microRNA

CLIP as an Alternative Method

• Crosslinking and immunoprecipitation (CLIP) serves as an alternative for identifying microRNA targets
• CLIP is a go-to for pinpointing target sites for individual RNA-binding proteins
• High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation(HITS-CLIP)
• Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP)
• Individual nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) also exist

CLIP and RISC

• CLIP was initially for identifying direct RNA-binding protein (RBP)-RNA interactions
• CLIP is applicable in complex scenarios, like identifying mRNA targets of microRNAs (miRNAs)
• Target sites are parts of ternary complexes(RISC) made up of mRNA, Argonaute (Ago) protein and a guiding miRNA

HITS-CLIP

• High-Throughput Sequencing of RNAs Isolated by Crosslinking Immunoprecipitation is depicted in illustration

PAR-CLIP

• Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation is depicted in illustration

4SU in PAR-CLIP

• Thiouridine (4SU) replaces uridine (U) in RNA
• 4SU serves as a photo-crosslinker

CLIP Procedures and Considerations

• PAR-CLIP involves incubation with 4SU-like photoactivatable nucleoside analogs, succeeded by crosslinking cells with 365 nm UV light
• Regular CLIP omits modified nucleoside incubation; crosslinking happens at 254 nm
• Optimize ribonuclease treatment to prevent extreme RNA digestion which depletes binding sites
• Use nuclease post-immunoprecipitation to clear away non-ternary complex RNA from mRNA target
• Radiolabel resulting RNA with radioactive phosphorous

Final Steps in CLIP

• RNA-protein complexes that are radiolabeled are resolved via SDS-PAGE and blotted onto nitrocellulose membrane to reduce background from non-crosslinked RNA fragments
• Next, adaptor ligation and reverse transcription occur
• Conduct a pilot PCR to assess amplification cycles needed in the preparative-scale PCR
• Select the cycle count to allow for library amplification with PCR amplification bias
• Deep sequencing is performed to identify interactions

Ago-CLIP Specifics

• Uses an antibody against the protein Argonaught (Ago) to identify microRNA targets
• Binding sites present varied affinities to Ago
• High affinity binding sites, in the area of Ago, have the highest propensity to crosslink with Ago during the CLIP experiment resulting in a large number of CLIP reads
• Ranking binding sites by the number of reads allows discovery of the highest affinity and most relevant binding sites

Regulatory Networks also Explored

• Basis of the correlation between sites and Ago HITS-CLIP, authors had a look at regulatory networks

Specific Binding in Regulatory Networks

• Ago links to target transcripts at sites, 2.6 Ago-mRNA clusters for Ago regulated transcript

Further Research on Ago HITS-CLIP Maps

• To examine the function of Ago HITS-CLIP maps to define microRNA regulated transcripts, they looked at the functions encoded by 20

Importance of Ago HITS-CLIP

• Permits a different method to interpret target RNA expression
• Analysis of a single miRNA demonstrated that its expression led Ago sites
• Competition between a limited capacity for miRNA binding on a given UTR
• Ago occlusion includes experimental and clinical considerations manipulate microRNA levels

HITS-CLIP in Action

• Utilized to covalently crosslink native argonaute(Ago, also called Eif2c) protein-RNA complexes in brains
• This causes information which helps identify sites

Benefits of Genome-Wide Interaction Maps

• Interaction maps created through this method
• Provides a general platform for identifying sequences