A Comparative Approach to Studying RNA Biochemistry using TimeLapse Chemistry and Labeling in Cell Culture (TILAC)

Date of Award

Spring 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Simon, Matthew

Abstract

RNA sequencing is a sensitive, transcriptome-wide analysis of RNA levels, valuable for assessing changes in gene expression, or measuring RNA enrichment after immunoprecipitation or cellular fractionation. However, accurate quantification of reads in RNA sequencing datasets is challenging due to variance from variable handling during RNA purification, and biases introduced in downstream processing, like shearing and amplification. To address these challenges, I developed TimeLapse Chemistry Labeling in Cell Culture (TILAC), an internally controlled and normalized approach to compare RNA content between samples using RNA metabolic labeling that is analogous to the SILAC method from protein biochemistry. TILAC utilizes the metabolic labels 4-thiouridine (s4U) and 6-thioguanisine (s6G) to differentially label RNA populations, allowing the samples to be pooled at the beginning of the experiment, prior to any of the handling steps that can introduce noise. TimeLapse chemistry recodes s4U into a C analogue, and s6G into an A analogue, thereby inducing mutations in sequencing reads. The ratios of RNAs from the two samples can be determined using the mutational content of the sequencing library. I have used TILAC to study perturbations to both transcription and translation of RNA. First, I show that TILAC can capture the global downregulation that occurs when cells are treated with the RNA polymerase II inhibitor, flavopiridol. Then, in the context of the Drosophila heat shock system, I show that TILAC captures both the high upregulation of heat shock responsive genes, as well as the more subtle downregulation across much of the transcriptome. I then turn my attention to translation, which can be studied by isolating polysomes by velocity sedimentation over a sucrose gradient. This method fractionates total cell lysate, and so polysome fractions contain contamination from non-ribosomal RNPs with similar coefficients of sedimentation. I use TILAC to characterize the background in sucrose sedimentation under normal conditions, and conditions of sodium arsenite stress. Armed with this knowledge, I used TILAC to study changes in translation, as well as in the total RNA pool in response to stress. Surprisingly, I found that there were few changes in the total RNA pool, but there was increased translation of a set of RNA helicases that are also found in stress granules. This puts forth an exciting hypothesis that the cell may need to upregulate these proteins to mitigate the potential damage caused by aggregating RNA in the cytoplasm. Through this set of experiments, I explore the advantages of using TILAC in RNA sequencing experiments and demonstrate how it can be used to advance biochemical studies, particularly in the context of global changes in RNA levels and in fractionations.

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