Tailoring CLIP-Based Methods for Exploring the miRNA Targetome

Abstract

MicroRNAs (miRNAs) are post-transcriptional regulators of protein-coding and other RNAs. Their main (canonical) function is the suppression of target RNAs which are recognised as such through its binding to an often conserved and partially complementary sequence. The miRNA is responsible for target recognition and guides the miRNA induced silencing complex (RISC) machinery to the targets, where posttranscriptional regulations are effected. Since targeting only requires partial complementarity in the interaction site, a single miRNA is able to regulate many transcripts. Given that miRNAs are involved in many important physiological- and disease-development processes, the RNA field has invested significantly in methods to increase the understanding of these small RNAs. Some targeting rules have been described and established. Most of the canonical targeting motives have been confirmed by many different groups, explaining a major part of miRNA functions and targeting rules. Nevertheless, more recent findings of less biased target identification methods have confirmed the importance of non-canonical targeting. In this thesis, we confirmed the applicability of a previously developed technique - miRNA crosslinking and immunoprecipitation (miR-CLIP). The protocol uses psoralen and biotin bis-modified pre-miRNAs and was shown to be useful for identification of canonical and non-canonical targets. In one of the projects, we used miR-CLIP to capture the targetome of miR-124 and miR-132, two brain-enriched 3p miRNAs. In course of these studies, we optimized the protocol and validated many of the captured miR-124 and miR-132 specific targets. In addition, we confirmed the suitability of the protocol for capturing targetomes of 3p miRNAs. We also compared the targetomes from two differently modified probes. Finally, our data revealed a biased processing of miR-124 in non-neuronal cells. Recent developments of chimera-generating methods have uncovered new insights into miRNA-target recognition. By physically ligating the miRNA to its target sites, non-canonical interaction-sites are captured as covalently attached sequences to the miRNA. We attempted to develop a new chimera-generating method - cyCLIP - based on a ligase, which joins 2'3-cyclic phosphate to the 5'-OH of a target RNA. We established the ligation of chemically-synthesized 2'3'-cyclic phosphate miRNAs and optimized several steps of the protocol. However, first attempts to sequence a cyCLIP library revealed very few chimeras bearing the full length miRNAs. This new method will be further investigated and pursued.