Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Pyle, Anna

Abstract

The relationship between RNA structure and function has challenged generations of scientists trying to unravel the ways that RNA functions in health and disease. RNA is known to adopt elaborate 2-D and 3-D folds that extend their regulatory possibilities way beyond those enabled by their primary sequence. The discovery, more than a decade ago, that most of our genome is pervasively transcribed has largely expanded the repertoire of known structured RNAs and their modes of action. Adding to the complexity of this picture, posttranscriptional RNA modifications chemically change the building blocks of RNA polymers and can also affect their structural dynamics and functional interactions. Here I develop new tools and methodologies to address some of the pressing challenges in the structural and functional analysis of long RNAs, a class of molecules spanning from long noncoding RNAs, or lncRNAs, to viral RNA genomes. I first develop a genetic strategy to investigate the functionality of human lincRNA-p21 in the mammalian DNA damage response, and show that the transcript expressed from this locus is important for proper levels of stress-induced apoptosis. On the structural side, I show that this molecule has an elaborate secondary structure containing well-defined motifs of potential functional relevance. Next, I delve into the problem of assessing structural conservation in lncRNAs. After identifying several features inherent to this class of transcripts that challenge previous conclusions about their conservation, I adapt existing methods to enable detection of conserved base pairs within their complex structures. I then focus my attention to the large RNA genome of the SARS-CoV-2 virus, a long RNA with unparalleled structural stability. I develop a pipeline to quantify and visualize the secondary structure content across such a large sequence and show that sequence context differences between the viral genome and sub-genomes might produce different sub-structures within the same coding sequence, which has important implications to our understanding of the molecular interactions within the life cycle of this and other RNA viruses. Lastly, I devote a special effort to the analysis of posttranscriptional RNA modifications, a more recently explored fine layer of structural and functional regulation. I use the highly processive Marathon Reverse Transcriptase to establish a new method to detect RNA modifications in cellular RNAs. A dual divalent cofactor strategy significantly expands the obtained cDNA mutational signatures to enable detection of several types of modified nucleotides and opens new possibilities for fast discovery of chemical RNA modifications in both population-level and single-molecule sequencing studies.

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