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Investigation Of In Vivo RNA-Protein Interactions Using Individual Nucleotide Resolution Cross-Linking Immunoprecipitation (ICLIP)


Eukaryotic gene expression involves a complex system of checkpoints that regulate RNA biogenesis, maturation, and localization. Along the way, these RNA will encounter a host of trans-acting factors, collectively known as RNA binding proteins (RBPs), which will govern the fate of RNA at each stage of gene expression. These RNA-protein interactions are the principal regulators of post-transcriptional control and are critical to the accurate expression of human genes. Misregulation of these interactions has been seen to be highly associated with several neurological disorders, cancer and inflammatory diseases. However, the regulatory mechanisms by which RBP function in the context of their RNA associations remains less understood. For my dissertation, I studied the RNA-protein interactions of a small set of proteins involved in the global regulation of transcription, processing and stability of the cellular transcriptome.

In order to interrogate the global RNA interactions of the proteins of interest, I performed a technique called individual nucleotide resolution crosslinking immunoprecipitation, or iCLIP. This technique takes advantage of photoreactive amino acids and nucleic acids in order to capture the in situ RNA-protein interactions of endogenous factors from both cell culture and tissue samples. Partial RNase digestion, stringent purification conditions, and advantageous use of reverse transcriptase difficulty reading through protein adducts allow for direct identification both the genomic origin of the CLIP RNA and the exact crosslinking site of the RNA-binding protein of interest at high specificity. These data form a functional RNA-protein interaction map to elucidate putative molecular roles for any RBP. I have used this technique on three separate projects in order to elucidate the mechanisms by which these RNA binding proteins regulate their targets and interact and modulate the binding of other RNA-binding proteins. First, I studied the effects of modulating the cellular levels of a splicing repressor, hnRNP A1, on the RNA recognition and binding of two splicing enhancer, U2AF65 and SRSF1, and the subsequent changes in pre-mRNA splicing. Secondly, in two concurrent collaborations, I elucidated the endogenous RNA binding targets of IGF2BP3, an oncofetal RBP whose up-regulation is associated with aggressive pancreatic cancer and B-cell leukemia. Finally, I elucidated the effects of human disease-associated genetic mutations on RNA-protein interactions in the context of pre-mRNA splicing. These studies are discussed herein.

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