The lifecycle of messenger RNAs is regulated by multiple layers beyond their primary sequence. In addition to carrying the information for protein synthesis, mRNAs are decorated with RNA binding proteins, marked with covalent chemical modifications, and fold into intricate secondary structures. However, the full set of information encoded by these “epitranscriptomic” layers is only partially understood, and is often only characterized for select transcripts. Thus, it is crucial to develop and apply transcriptome-wide analytical tools to probe the location and functional relevance of epitranscriptome features. In this dissertation, I focus on applying such methods toward better understanding determinants of mRNA stability, through using 1) High Throughput Annotation of Modified Nucleotides, 2) nuclease-mediated probing of RNA secondary structure, and 3) detection of partial mRNA degradation from RNA sequencing. I observe that chemical modifications tend to mark uncapped and small RNA fragments derived from mRNAs in plants and humans, suggesting a link between modifications and mRNA stability. I then show this link is direct through showing differential stability at Arabidopsis transcripts that change modification status during long-term salt stress. By probing secondary structure, I show a link between structure, smRNA production, and co-translational RNA decay. Finally, I develop a new in silico method to detect partial RNA degradation in mouse oocytes, and identify sequence elements that appear to block complete exonucleolytic transcript cleavage during meiosis. I then identify putative RNA binding proteins that might mediate this partial decay. In summary, I apply transcriptome-wide sequencing-based methods to survey the effects of covalent modifications, secondary structure, and RNA binding proteins on mRNA stability.