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Abstract

RNA-binding proteins (RBPs) are central players in cell biology and respond to a multitude of cellular cues and environmental stimuli. Identification of RBPs associated with specific transcripts in a cell is a challenging task; and the available strategies to purify specific transcripts and their bound proteome face numerous limitations. Thus, methods to determine the composition of proteins on a given RNA are required to further understand the regulation and biological function of any given RNA. Therefore, the focus of my PhD project was to develop a highly specific and selective method, “specific Ribonucleoprotein (RNP) capture”, to isolate a specific RNA species together with its bound proteome. Following irradiation with ultraviolet (UV) light that creates a covalent bond between RNA and protein, RNAs are captured using short LNA (locked nucleic acid)/DNA mixmer antisense probes coupled to a solid support. The proteins covalently linked to the isolated RNA are then identified by quantitative mass spectrometry. First, I successfully established the method for this application in vitro. Mass spectrometry data revealed that the protein Sister of Sex lethal (Ssx) has similar binding preferences to a mRNA derived from male-specific lethal (msl)2 mRNA as its paralog Sxl in Drosophila melanogaster embryo extracts. This demonstrated the specificity and selectivity of the method and provided direct experimental evidence for Ssx-RNA binding. Following these experiments, I extended the protocol to in-cell applications, focusing on the 18S and 28S ribosomal RNA (rRNA) of HeLa cells. Compared to bacteria, eukaryotic rRNAs possess “expansion segments” with much to be learnt about their bound RBPs and function. The high specificity of the method allowed me to generate distinct proteomic datasets for these two rRNAs. The method’s excellent biochemical performance is reflected by the overlap of these datasets with previous literature information on the cytoplasmic ribosome and system-wide screens of ribosomal biogenesis. Notably, my data revealed a strong connection between heterogeneous ribonucleoproteins (HNRNPs) and ribosomal RNA biogenesis, which is an unexplored area of research. In summary, “specific RNP capture” allows identification of a given RNAs proteome and can be applied to both in vitro and cultured cells systems.