{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T16:11:53Z","timestamp":1753891913236,"version":"3.41.2"},"reference-count":48,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,1,11]],"date-time":"2024-01-11T00:00:00Z","timestamp":1704931200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100011142","name":"Graduate School, University of Minnesota","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100011142","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000024","name":"Canadian Institutes of Health Research","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Parasitol."],"abstract":"<jats:p>RNA-specific nucleotidyltransferases (rNTrs) add nontemplated nucleotides to the 3<jats:sup>\u2032<\/jats:sup> end of RNA. Two noncanonical rNTRs that are thought to be poly(A) polymerases (PAPs) have been identified in the mitochondria of trypanosomes \u2013 KPAP1 and KPAP2. KPAP1 is the primary polymerase that adds adenines (As) to trypanosome mitochondrial mRNA 3<jats:sup>\u2032<\/jats:sup> tails, while KPAP2 is a non-essential putative polymerase whose role in the mitochondria is ambiguous. Here, we elucidate the effects of manipulations of KPAP1 and KPAP2 on the 5<jats:sup>\u2032<\/jats:sup> and 3<jats:sup>\u2032<\/jats:sup> termini of transcripts and their 3<jats:sup>\u2032<\/jats:sup> tails. Using glycerol gradients followed by immunoblotting, we present evidence that KPAP2 is found in protein complexes of up to about 1600 kDa. High-throughput sequencing of mRNA termini showed that KPAP2 overexpression subtly changes an edited transcript\u2019s 3<jats:sup>\u2032<\/jats:sup> tails, though not in a way consistent with general PAP activity. Next, to identify possible roles of posttranslational modifications on KPAP1 regulation, we mutated two KPAP1 arginine methylation sites to either mimic methylation or hypomethylation. We assessed their effect on 3<jats:sup>\u2032<\/jats:sup> mRNA tail characteristics and found that the two mutants generally had opposing effects, though some of these were transcript-specific. We present results suggesting that while methylation increases KPAP1 substrate binding and\/or initial nucleotide additions, unmethylated KPAP1is more processive. We also present a comprehensive review of UTR termini, and evidence that tail addition activity may change as mRNA editing is initiated. Together, this work furthers our understanding of the role of KPAP1 and KPAP2 on trypanosome mitochondrial mRNA 3<jats:sup>\u2032<\/jats:sup> tail addition, as well as provides more information on mRNA termini processing in general.<\/jats:p>","DOI":"10.3389\/fpara.2023.1298561","type":"journal-article","created":{"date-parts":[[2024,1,11]],"date-time":"2024-01-11T05:28:40Z","timestamp":1704950920000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Manipulation of mitochondrial poly(A) polymerase family proteins in Trypanosoma brucei impacts mRNA termini processing"],"prefix":"10.3389","volume":"2","author":[{"given":"Clara M.","family":"Smoniewski","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Poorya","family":"Mirzavand Borujeni","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marshall","family":"Hampton","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Austin","family":"Petersen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sean P.","family":"Faacks","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Reza","family":"Salavati","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sara L.","family":"Zimmer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2024,1,11]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"10617","DOI":"10.1073\/pnas.1833120100","article-title":"A tale of two TUTases","volume":"100","author":"Aphasizhev","year":"2003","journal-title":"Proc. Natl. Acad. Sci."},{"key":"B2","doi-asserted-by":"publisher","first-page":"637","DOI":"10.1016\/S0092-8674(02)00647-5","article-title":"Trypanosome mitochondrial 3\u2019 terminal uridylyl transferase (TUTase): The key enzyme in u-insertion\/deletion RNA editing","volume":"108","author":"Aphasizhev","year":"2002","journal-title":"Cell"},{"key":"B3","doi-asserted-by":"publisher","first-page":"337","DOI":"10.1016\/j.pt.2020.01.006","article-title":"Lexis and grammar of mitochondrial RNA processing in trypanosomes","volume":"36","author":"Aphasizheva","year":"2020","journal-title":"Trends Parasitol."},{"key":"B4","doi-asserted-by":"publisher","first-page":"1555","DOI":"10.1128\/MCB.01281-09","article-title":"RET1-catalyzed uridylylation shapes the mitochondrial transcriptome in trypanosoma brucei","volume":"30","author":"Aphasizheva","year":"2010","journal-title":"Mol. Cell. Biol."},{"key":"B5","doi-asserted-by":"publisher","first-page":"106","DOI":"10.1016\/j.molcel.2011.02.021","article-title":"Pentatricopeptide repeat proteins stimulate mRNA adenylation\/uridylation to activate mitochondrial translation in trypanosomes","volume":"42","author":"Aphasizheva","year":"2011","journal-title":"Mol. Cell"},{"key":"B6","doi-asserted-by":"publisher","first-page":"4329","DOI":"10.1128\/MCB.01075-14","article-title":"RNA binding and core complexes constitute the u-insertion\/deletion editosome","volume":"34","author":"Aphasizheva","year":"2014","journal-title":"Mol. Cell. Biol."},{"key":"B7","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.molcel.2008.12.013","article-title":"Protein arginine methylation in mammals: Who, what, and why","volume":"33","author":"Bedford","year":"2009","journal-title":"Mol. Cell"},{"key":"B8","doi-asserted-by":"publisher","first-page":"4667","DOI":"10.1093\/nar\/gkx116","article-title":"In vivo cleavage specificity of trypanosoma brucei editosome endonucleases","volume":"45","author":"Carnes","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"B9","doi-asserted-by":"publisher","first-page":"1632","DOI":"10.1101\/gad.7.8.1632","article-title":"A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation","volume":"7","author":"Decker","year":"1993","journal-title":"Genes Dev."},{"key":"B10","doi-asserted-by":"publisher","first-page":"27872","DOI":"10.1074\/jbc.M113.491092","article-title":"Automethylation of protein arginine methyltransferase 8 (PRMT8) regulates activity by impeding s-adenosylmethionine sensitivity","volume":"288","author":"Dillon","year":"2013","journal-title":"J. Biol. Chem."},{"key":"B11","doi-asserted-by":"publisher","first-page":"1596","DOI":"10.1038\/emboj.2008.87","article-title":"3\u2019 adenylation determines mRNA abundance and monitors completion of RNA editing in t. brucei mitochondria","volume":"27","author":"Etheridge","year":"2008","journal-title":"EMBO J."},{"key":"B12","doi-asserted-by":"publisher","first-page":"23016","DOI":"10.1074\/jbc.M801021200","article-title":"TbRGG2, an essential RNA editing accessory factor in two trypanosoma brucei life cycle stages","volume":"283","author":"Fisk","year":"2008","journal-title":"J. Biol. Chem."},{"key":"B13","doi-asserted-by":"publisher","first-page":"866","DOI":"10.1128\/EC.00018-10","article-title":"TbPRMT6 is a type i protein arginine methyltransferase that contributes to cytokinesis in trypanosoma brucei","volume":"9","author":"Fisk","year":"2010","journal-title":"Eukaryotic Cell"},{"key":"B14","doi-asserted-by":"publisher","first-page":"477","DOI":"10.1261\/rna.054494.115","article-title":"circTAIL-seq, a targeted method for deep analysis of RNA 3\u2019 tails, reveals transcript-specific differences by multiple metrics","volume":"22","author":"Gazestani","year":"2016","journal-title":"RNA"},{"key":"B15","doi-asserted-by":"publisher","first-page":"179","DOI":"10.1016\/j.ijpara.2017.08.012","article-title":"Tail characteristics of trypanosoma brucei mitochondrial transcripts are developmentally altered in a transcript-specific manner","volume":"48","author":"Gazestani","year":"2018","journal-title":"Int. J. Parasitol."},{"key":"B16","doi-asserted-by":"publisher","first-page":"1545","DOI":"10.1261\/rna.90106","article-title":"Arginine methylation regulates mitochondrial gene expression in trypanosoma brucei through multiple effector proteins","volume":"12","author":"Goulah","year":"2006","journal-title":"RNA"},{"key":"B17","doi-asserted-by":"publisher","first-page":"7181","DOI":"10.1074\/jbc.M609485200","article-title":"Differential effects of arginine methylation on RBP16 mRNA binding, guide RNA (gRNA) binding, and gRNA-containing ribonucleoprotein complex (gRNP) formation","volume":"282","author":"Goulah","year":"2007","journal-title":"J. Biol. Chem."},{"key":"B18","doi-asserted-by":"publisher","first-page":"6577","DOI":"10.1093\/nar\/gkt369","article-title":"The hnRNP f\/h homologue of trypanosoma brucei is differentially expressed in the two life cycle stages of the parasite and regulates splicing and mRNA stability","volume":"41","author":"Gupta","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0244858","article-title":"Probabilistic models of biological enzymatic polymerization","volume":"16","author":"Hampton","year":"2021","journal-title":"PloS One"},{"key":"B20","doi-asserted-by":"publisher","first-page":"11368","DOI":"10.1016\/S0021-9258(19)38602-8","article-title":"Addition of uridines to edited RNAs in trypanosome mitochondria occurs independently of transcription","volume":"265","author":"Harris","year":"1990","journal-title":"J. Biol. Chem."},{"key":"B21","doi-asserted-by":"publisher","first-page":"2089","DOI":"10.1074\/jbc.M116.757112","article-title":"The major protein arginine methyltransferase in trypanosoma brucei functions as an enzyme-prozyme complex","volume":"292","author":"Kafkov\u00e1","year":"2017","journal-title":"J. Biol. Chem."},{"key":"B22","doi-asserted-by":"publisher","first-page":"65","DOI":"10.1038\/cdd.2017.186","article-title":"BCL-2 family proteins: changing partners in the dance towards death","volume":"25","author":"Kale","year":"2018","journal-title":"Cell Death Differentiation"},{"key":"B23","doi-asserted-by":"publisher","first-page":"158","DOI":"10.1016\/j.molbiopara.2007.04.014","article-title":"Targeted depletion of a mitochondrial nucleotidyltransferase suggests the presence of multiple enzymes that polymerize mRNA 3\u2019 tails in trypanosoma brucei mitochondria","volume":"154","author":"Kao","year":"2007","journal-title":"Mol. Biochem. Parasitol."},{"key":"B24","doi-asserted-by":"publisher","first-page":"238","DOI":"10.1128\/MCB.00945-14","article-title":"PRMT4-mediated arginine methylation negatively regulates retinoblastoma tumor suppressor protein and promotes e2f-1 dissociation","volume":"35","author":"Kim","year":"2015","journal-title":"Mol. Cell. Biol."},{"key":"B25","doi-asserted-by":"crossref","first-page":"879","DOI":"10.1038\/s41592-019-0503-y","article-title":"FLAM-seq: full-length mRNA sequencing reveals principles of poly(a) tail length control","volume":"16","author":"Legnini","year":"2019","journal-title":"Nat Methods"},{"key":"B26","doi-asserted-by":"publisher","first-page":"210","DOI":"10.1016\/j.jprot.2013.07.010","article-title":"Global proteomic analysis in trypanosomes reveals unique proteins and conserved cellular processes impacted by arginine methylation","volume":"91","author":"Lott","year":"2013","journal-title":"J. Proteomics"},{"key":"B27","doi-asserted-by":"publisher","first-page":"5501","DOI":"10.1093\/nar\/gkv428","article-title":"Arginine methylation of DRBD18 differentially impacts its opposing effects on the trypanosome transcriptome","volume":"43","author":"Lott","year":"2015","journal-title":"Nucleic Acids Res."},{"key":"B28","doi-asserted-by":"publisher","first-page":"595","DOI":"10.1002\/mbo3.191","article-title":"Functional interplay between protein arginine methyltransferases in trypanosoma brucei","volume":"3","author":"Lott","year":"2014","journal-title":"MicrobiologyOpen"},{"key":"B29","doi-asserted-by":"publisher","first-page":"1834","DOI":"10.1261\/rna.652807","article-title":"RNA-specific ribonucleotidyl transferases","volume":"13","author":"Martin","year":"2007","journal-title":"RNA"},{"key":"B30","doi-asserted-by":"publisher","first-page":"149","DOI":"10.1128\/EC.00232-14","article-title":"An arginineglycine-rich RNA binding protein impacts the abundance of specific mRNAs in the mitochondria of trypanosoma brucei","volume":"14","author":"McAdams","year":"2015","journal-title":"Eukaryotic Cell"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-08137-2","article-title":"Pentatricopeptide repeat poly(a) binding protein KPAF4 stabilizes mitochondrial mRNAs in trypanosoma brucei","volume":"10","author":"Mesitov","year":"2019","journal-title":"Nat. Commun."},{"key":"B32","doi-asserted-by":"publisher","first-page":"1807","DOI":"10.1073\/pnas.92.6.1807","article-title":"Polyadenylylation helps regulate mRNA decay in escherichia coli","volume":"92","author":"O\u2019Hara","year":"1995","journal-title":"Proc. Natl. Acad. Sci. United States America"},{"key":"B33","doi-asserted-by":"publisher","first-page":"728","DOI":"10.1016\/S1044-0305(03)00126-0","article-title":"Mass spectrometric analysis of the editosome and other multiprotein complexes in trypanosoma brucei","volume":"14","author":"Panigrahi","year":"2003","journal-title":"J. Am. Soc. Mass Spectrometry"},{"key":"B34","doi-asserted-by":"publisher","first-page":"6833","DOI":"10.1128\/MCB.21.20.6833-6840.2001","article-title":"Four related proteins of the trypanosoma brucei RNA editing complex","volume":"21","author":"Panigrahi","year":"2001","journal-title":"Mol. Cell. Biol."},{"key":"B35","first-page":"75","article-title":"Chapter 4 - isolation of RNA binding proteins involved in insertion\/deletion editing","volume-title":"Methods in Enzymology","author":"Pelletier","year":"2007"},{"key":"B36","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1007\/978-1-61779-018-8_2","article-title":"iCODA: RNAi-based inducible knock-in system in trypanosoma brucei","volume-title":"RNA and DNA Editing","author":"Ringpis","year":"2011"},{"key":"B37","doi-asserted-by":"publisher","first-page":"E10323","DOI":"10.1073\/pnas.1808981115","article-title":"Transcription initiation defines kinetoplast RNA boundaries","volume":"115","author":"Sement","year":"2018","journal-title":"Proc. Natl. Acad. Sci."},{"key":"B38","doi-asserted-by":"publisher","first-page":"e00051","DOI":"10.1128\/mSphere.00051-16","article-title":"Mitochondrial gene expression is responsive to starvation stress and developmental transition in trypanosoma cruzi","volume":"1","author":"Shaw","year":"2016","journal-title":"mSphere"},{"key":"B39","doi-asserted-by":"publisher","first-page":"677","DOI":"10.1261\/rna.055160.115","article-title":"High-throughput sequencing of partially edited trypanosome mRNAs reveals barriers to editing progression and evidence for alternative editing","volume":"22","author":"Simpson","year":"2016","journal-title":"RNA"},{"key":"B40","doi-asserted-by":"publisher","first-page":"2071","DOI":"10.1093\/oxfordjournals.molbev.a004032","article-title":"The evolutionary history of kinetoplastids and their kinetoplasts","volume":"19","author":"Simpson","year":"2002","journal-title":"Mol. Biol. Evol."},{"key":"B41","doi-asserted-by":"publisher","first-page":"7825","DOI":"10.1038\/s41598-023-34255-z","article-title":"Circular mitochondrial-encoded mRNAs are a distinct subpopulation of mitochondrial mRNA in trypanosoma brucei","volume":"13","author":"Smoniewski","year":"2023","journal-title":"Sci. Rep."},{"key":"B42","doi-asserted-by":"publisher","first-page":"3640","DOI":"10.1093\/nar\/gkz012","article-title":"Intrinsic and regulated properties of minimally edited trypanosome mRNAs","volume":"47","author":"Tylec","year":"2019","journal-title":"Nucleic Acids Res"},{"key":"B43","doi-asserted-by":"publisher","first-page":"2429","DOI":"10.1074\/jbc.M405933200","article-title":"RNA interference analyses suggest a transcript-specific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in trypanosoma brucei","volume":"280","author":"Vondru\u0161kov\u00e1","year":"2005","journal-title":"J. Biol. Chem."},{"key":"B44","doi-asserted-by":"publisher","first-page":"211","DOI":"10.1016\/S0166-6851(02)00238-4","article-title":"Targeting of a tetracycline-inducible expression system to the transcriptionally silent minichromosomes of trypanosoma brucei","volume":"125","author":"Wickstead","year":"2002","journal-title":"Mol. Biochem. Parasitol."},{"key":"B45","doi-asserted-by":"publisher","first-page":"2433","DOI":"10.1093\/nar\/9.11.2433","article-title":"Terminal uridylyl transferase of vigna unguiculata: purification and characterization of an enzyme catalyzing the addition of a single UMP residue to the 3\u2019-end of an RNA primer","volume":"9","author":"Zabel","year":"1981","journal-title":"Nucleic Acids Res."},{"key":"B46","doi-asserted-by":"publisher","first-page":"2435","DOI":"10.15252\/embj.201796808","article-title":"PPR polyadenylation factor defines mitochondrial mRNA identity and stability in trypanosomes","volume":"36","author":"Zhang","year":"2017","journal-title":"EMBO J."},{"key":"B47","doi-asserted-by":"publisher","first-page":"10329","DOI":"10.1074\/jbc.M110.152439","article-title":"A novel member of the RNase d exoribonuclease family functions in mitochondrial guide RNA metabolism in trypanosoma brucei","volume":"286","author":"Zimmer","year":"2011","journal-title":"J. Biol. Chem."},{"key":"B48","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0037639","article-title":"Additive and transcript-specific effects of KPAP1 and TbRND activities on 3\u2019 non-encoded tail characteristics and mRNA stability in trypanosoma brucei","volume":"7","author":"Zimmer","year":"2012","journal-title":"PloS One"}],"container-title":["Frontiers in Parasitology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fpara.2023.1298561\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,11]],"date-time":"2024-01-11T05:28:46Z","timestamp":1704950926000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fpara.2023.1298561\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,1,11]]},"references-count":48,"alternative-id":["10.3389\/fpara.2023.1298561"],"URL":"https:\/\/doi.org\/10.3389\/fpara.2023.1298561","relation":{},"ISSN":["2813-2424"],"issn-type":[{"type":"electronic","value":"2813-2424"}],"subject":[],"published":{"date-parts":[[2024,1,11]]},"article-number":"1298561"}}