Talk abstracts
Talk on Saturday 10:00-10:15am submitted by Clara Smoniewski
The role of KPAP1 methylation in moderating mitochondrial mRNA 3’ tail addition in Trypanosoma brucei
Clara M. Smoniewski (Dept. of Biomedical Sciences, U of MN Medical School, Duluth campus), Poorya Mirzavand Borujeni (Institute of Parasitology,McGill University), Marshall Hampton (Department of Mathematics and Statistics, University of Minnesota Duluth), Reza Salavati (Institute of Parasitology,McGill University), Sara L. Zimmer (Dept. of Biomedical Sciences, U of MN Medical School, Duluth campus)
Abstract:
Trypanosoma brucei is a protozoan parasite with unique molecular mechanisms to control expression of its nuclear and mitochondrial (mt) genomes. Its mt mRNAs have regulatory non-templated heterogenous adenine/uridine tails added post-transcriptionally to their 3’ ends. We previously found population level differences in tail characteristics, such as length and composition, between transcripts. The mechanism for imparting these differences is unknown. Tail adenines are added by kinetoplast poly(A) polymerase I (KPAP1), possessing two previously identified arginine methylation sites. Methylation of protein arginine residues is a known mechanism for altering protein:protein and protein:nucleic acid associations. We have mutated KPAP1 at its arginine methylation sites to mimic constant methylation (methyl-mimic) or no methylation (methyl-mutant). One of these mutated versions of KPAP1 or a wildtype version were inducibly expressed in procyclic T. brucei along with simultaneous silencing of native KPAP1. The three cell lines were compared using circTAILseq, which combines enzymatic circularization of RNA, RT-PCR, and Illumina sequencing. We found that tail length and composition are impacted by mutation of KPAP1 arginine methylation sites. Addition of adenine to pre-edited mRNA tails seems more robust but less processive under activity of methyl-mimic KPAP1; the reverse can sometimes be observed for never-edited mRNA. This suggests a role for methylation in KPAP1 regulation. Surprisingly, some transcripts yielded circTAILseq libraries when the ligase responsible for RNA circularization was omitted, although the size of the library’s molecules was smaller. These libraries consist of cDNAs possessing many 3’ and 5’ UTRs ligated together, sometimes possessing a short tail between the two UTRs. Further experiments suggest that these products reflect a minor sub-population of mt mRNAs that are intracellularly circularized. To our knowledge, this is the first circRNA to be identified in a Kinetoplastid species.
References:
Aphasizheva, I. & Aphasizhev, R. (2010). Mol Cell Bio 30, 1555-1567.; Etheridge, R.D. et al. (2008). EMBO J 27, 1596–1608.; Fisk, J.C. et al. (2013). Mol Cell Proteomics 12, 302–311.; Gazestani, V.H. et al. (2016). RNA 22, 477-86.; Gazestani, V.H. et al. (2018). Int J Parasitol 48, 179-189.; Hampton, M. et al., (2021). PLOS ONE 16: e0244858.; Zhang, L. et al. (2017). EMBO J 36, 2435–2454.
Keywords: Trypanosomes, Mitochondria, mRNA 3 tails