Computational Pipelines for Single Molecule Mapping of Post-Transcriptional Modifications

Abstract

Post-transcriptional regulation shapes gene expression by influencing RNA stability, translation, and decay. Two central mechanisms, RNA modifications, like N6-methyladenosine (m6A), and poly(A) tail length dynamics, serve as key determinants of RNA fate. Yet, conventional short-read sequencing lacks the resolution to fully capture these features at isoform or single-nucleotide precision. This thesis combines high-resolution and nanopore-based approaches to map and characterize m6A modifications and poly(A) tail lengths across diverse systems. In the first aim, nucleotide-resolution m6A maps were generated in the malaria-causing parasite Plasmodium falciparum using DART-seq, establishing one of the most detailed modification landscapes reported for this pathogen. To examine how heat shock affects the P. falciparum post-transcriptional landscape and to elucidate adaptive strategies employed by the parasite, the second aim leveraged Oxford Nanopore Technologies (ONT) direct RNA sequencing (DRS) to independently profile m6A modifications and poly(A) tail lengths at single-molecule resolution, providing insights into their context-specific regulatory roles. Finally, the third aim utilized polyA+mod, a modular Nextflow pipeline developed to simultaneously predict m6A modifications and poly(A) tail lengths from ONT DRS data, enabling investigation of the crosstalk between m6A and polyadenylation in human cell lines. This work advances understanding of RNA regulation across species and introduces innovative methods for RNA research. It lays the foundation for future technologies capable of capturing multiple RNA modifications, poly(A) tails, and other RNA features in a single experiment, reducing time and cost. Such multi-dimensional views will enable new RNA-centered diagnostics and therapeutics, providing strategies to study and treat disease.