Smoniewski, Clara2024-01-052024-01-052023-08https://hdl.handle.net/11299/259657University of Minnesota Ph.D. dissertation. August 2023. Major: Integrated Biosciences. Advisor: Sara Zimmer. 1 computer file (PDF); x, 130 pages.A large and ever-expanding field, RNA biology is the study of the RNA lifecycle, how RNAs function, and their diverse roles in cellular processes. The most well-known RNA type are the protein-coding mRNAs transcribed from genes encoded in DNA. mRNAs progress through multiple steps during their lifecycle, one of which is maturation in which a 3' nonencoded nucleotide tail is added. These tails vary widely across organisms and organelles, both in composition and function. This dissertation examines the 3' tail addition and termini processing of mRNA transcripts in the mitochondrion of a parasitic protozoan <i>Trypanosoma brucei</i>. Trypanosomes have a single mitochondrion in which RNA regulation is crucial for gene expression changes necessary for host-switching. Although a working model for mitochondrial mRNA processing in <i>T. brucei</i> has been developed, transcript-specific gene regulation remains undefined. Differences in mRNA editing, stability, and translation remain unexplained, as do differences in 3' mRNA tail characteristics between transcripts and life stages. Here, I present several studies of the RNA lifecycle and mRNA termini processing in the mitochondrion of <i>T. brucei</i>. Chapter 1 contains an introduction to RNA, 3' mRNA tails, and trypanosomes. In Chapter 2, I explain our refinement of circTAIL-seq, a tool used to high-throughput sequence 3' and 5' termini. I define the circTAIL-seq error rate and describe a newly developed alignment protocol that allows for greater nuance in aligning reads. The discovery and characterization of circular mitochondrial mRNAs (circRNAs) is presented in Chapter 3. We show that circRNAs are present in both <i>T. brucei</i> life stages and are a subpopulation of total mRNAs with distinct characteristics. Chapter 4 contains a description of the effects of manipulating two poly(A) polymerase (PAP)-family proteins found in <i>T. brucei</i>’s mitochondria &mdash; KPAP1 and KPAP2. KPAP2 is found in unknown complexes of various sizes, and mutations of KPAP1 arginine methylation sites are shown to have transcript-specific effects on 3' tail characteristics. I then summarize my major conclusions and present a discussion of limitations and future directions of this work. Overall, this dissertation expands our understanding of the RNA lifecycle by further elucidating mitochondrial mRNA termini processing in <i>T. brucei</i>.en3' mRNA tailsbioinformaticscircular RNAnoncoding RNAparasitologypoly(A) polymerasesThesis or Dissertation