Ghareeb, Ali Essam; (2025) Endogenous RNA editing records the transcriptional history of human cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Obtaining continuous measurements of transcriptional activity across the transcriptome would unveil a granularity of information about cell regulation not currently possible. RNA sequencing, however, takes a snapshot of the transcriptome at an instant and is destructive, preventing further sampling. From the point of transcription until degradation, mRNA gradually accumulates adenosine-to-inosine edits due to the activity of ADAR enzymes, potentially providing an endogenous record of mRNA age. Determining the age distribution of transcripts present within a cell would allow one to infer the cell’s recent transcriptional record. In this work, I find that endogenous A-to-I editing of RNA transcripts in unmodified human cells serves as a molecular recorder, allowing us to infer the ages of endogenous RNA transcripts at the single molecule level with precision on the order of hours. Using a controlled gene expression system, I show that the distribution of ages of a transcript encode past transcriptional history, information which is not found in static RNA sequencing. I show that Timestamps can recover the transcriptional dynamics of hundreds of genes in diRerentiating primary human monocytes, even identifying new putative regulators of human monocyte diRerentiation. In dividing single cells, I show that the periodic transcription of S- and G2/M-phase associated genes, as recorded by Timestamps, tracks the period of the cell cycle. By sorting the single neurons, Timestamps could enable RNA sequencing to identify which neurones respond to diverse but temporally separated stimuli, thus enabling highthroughput functional brain mapping. I begin developing an in vivo experiment to show that Timestamps can trace the production of immediate early genes and thereby infer the past activity of neurons. Finally, to improve Timestamps and extend its functionality to non-primate model organisms, I invent a new class of promiscuous, hyperactive RNA editors. Timestamps is an endogenous molecular recorder of transcriptional history.

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