Simon Horskjær Rasmussen:
|
MicroRNAs (miRNAs) are small non-coding RNAs that mediate degradation and translational repression of target mRNAs. miRNAs rely on RNA-binding proteins (RBPs) in every step of their biogenesis, localization, targeting and execution of the regulatory effect. With the recent introduction and diversification of genomewide Next Generation Sequencing (NGS) technologies, it has become possible to study interaction of miRNAs and RBPs in greater detail. miRNA-binding to an mRNA can have different outcomes, which depends on the RBPs they interact with. In two papers in this thesis, the link between RBPs and miRNA regulation was studied by cross-linking immunoprecipitation (CLIP) and RBP double knockdown experiments. A comprehensive analysis of 107 CLIP datasets of 49 RBPs demonstrated that RBPs modulate miRNA regulation. Results suggest it is mediated by RBP-binding hotspots that likely are the “switches” of combinatorial regulation. RBP hotspots are highly accessible AU-rich regions that are more frequently bound by RBPs and they are frequently in the vicinity of miRNA target sites. To further investigate this, an experimental design and analysis method, to further unravel combinatorial regulation of pairs of factors, were developed. From analyses of HuR and AGO2 knockdown experiments, several putative targets of cooperative destabilization, stabilization and HuR antagonization of AGO2, were identified. In another paper the functions of the RBP IMP in Drosophila Melanogaster, were investigated using high-throughput data. Analysis of IMP RIP-seq, iCLIP and RNA-seq datasets identified transcripts associated with cytoplasmic IMP ribonucleoproteins. Many of these transcripts were functionally involved in actin cytoskeletal remodeling. Further analyses of this data permitted estimation of a bipartite motif, composed of an AU-rich and a CA-rich domain. In addition, a regulatory motif discovery method was developed and applied to identify motifs using differential expression data and CLIP-data in the above investigations. This thesis increased the understanding of the role of RBPs in miRNA regulation, further uncovered the binding motif and functions of Drosophila Melanogaster IMP, introduced the biological concept of RBP hotspots and contributed with new methodologies to the Post-transcriptional Genomics field of research.