Optimization of a translating ribosomal affinity purification method

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2021-2-10

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Gürcüoğlu, Irmak

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Novel technologies revealed an immense transcriptome complexity in normal and in cancer cells. One of the reasons for this complexity is isoform variability in cancer cells. Transcript isoforms can arise due to alternative processing of the pre-mRNAs through alternative transcription start sites, alternative splicing, and alternative polyadenylation. To begin understanding how these isoforms may functionally contribute to the cancer phenotype, translation efficiency and coding potential of isoforms need to be established. Therefore, here, we optimized an assay where we captured translated mRNAs by immunoprecipitating active ribosome complexes that stably express a GFP tagged Ribosomal Protein L10A (RPL10A). We used magnetic bead conjugated Llama Anti-GFP VHH Single Domain Monoclonal Antibody to minimize unspecific interactions. Following immunoprecipitation, we isolated RNAs, cleaned DNA contamination, synthesized cDNA, and performed RT-qPCR. The optimized protocol was tested with non-coding RNAs and coding mRNAs. Hence, the optimization of this protocol allows individual and/or high throughput analysis of ribosome-associated mRNAs.

Subject Keywords

RPL10A, TRAP, Ribosome, Polysome, Immunoprecipitation, eGFP

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https://hdl.handle.net/11511/89800

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Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats

I. Gürcüoğlu, “Optimization of a translating ribosomal affinity purification method,” M.S. - Master of Science, Middle East Technical University, 2021.