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RNA Editing Tutase 1 10862–10878 Nucleic Acids Research, 2016, Vol. 44, No. 22 Published online 15 October 2016 doi: 10.1093/nar/gkw917 RNA Editing TUTase 1: structural foundation of substrate recognition, complex interactions and drug targeting Lional Rajappa-Titu1,†, Takuma Suematsu2,†, Paola Munoz-Tello1, Marius Long1, Ozlem¨ Demir3, Kevin J. Cheng3, Jason R. Stagno4, Hartmut Luecke4, Rommie E. Amaro3, Inna Aphasizheva2, Ruslan Aphasizhev2,5,* and Stephane´ Thore1,6,7,8,* 1Department of Molecular Biology, University of Geneva, 1211 Geneva, Switzerland, 2Department of Molecular and Cell Biology, Boston University School of Dental Medicine, Boston, MA 02118, USA, 3Department of Chemistry & Biochemistry and the National Biomedical Computation Resource, University of California, San Diego, La Jolla, CA 92093, USA, 4Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA, 5Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA, 6INSERM, U1212, ARNA Laboratory, Bordeaux 33000, France, 7CNRS UMR5320, ARNA Laboratory, Bordeaux 33000, France and 8University of Bordeaux, ARNA Laboratory, Bordeaux 33000, France Received May 10, 2016; Revised September 27, 2016; Accepted October 04, 2016 ABSTRACT thermore, we define RET1 region required for incor- poration into the 3 processome, determinants for Terminal uridyltransferases (TUTases) execute 3 RNA binding, subunit oligomerization and proces- RNA uridylation across protists, fungi, metazoan and sive UTP incorporation, and predict druggable pock- plant species. Uridylation plays a particularly promi- ets. nent role in RNA processing pathways of kineto- plastid protists typified by the causative agent of African sleeping sickness, Trypanosoma brucei.In INTRODUCTION mitochondria of this pathogen, most mRNAs are Parasitic protists from the order of Kinetoplastidae cause internally modified by U-insertion/deletion editing devastating human and animal infections such as African while guide RNAs and rRNAs are U-tailed. The found- sleeping sickness, Nagana, Chagas disease, leishmaniasis ing member of TUTase family, RNA editing TUTase and others (1). These early branching eukaryotes share 1 (RET1), functions as a subunit of the 3 proces- many unique mechanisms of gene expression including mul- some in uridylation of gRNA precursors and mature ticistronic transcription of nuclear protein-encoding genes guide RNAs. Along with KPAP1 poly(A) polymerase, by RNA polymerase II and intricate mitochondrial RNA RET1 also participates in mRNA translational activa- processing pathways (2,3). To enable synthesis of 18 pre- tion. RET1 is divergent from human TUTases and is dicted mitochondrial proteins, messenger RNA precursors essential for parasite viability in the mammalian host must be transcribed from maxicircle DNA, 3 adenylated, / and the insect vector. Given its robust in vitro activity, often subjected to extensive U-insertion deletion editing, / RET1 represents an attractive target for trypanocide and further modified by 3 adenylation uridylation prior to translation (4–6). The principal role of uridylation in development. Here, we report high-resolution crys- trypanosomal mitochondrial RNA metabolism motivated tal structures of the RET1 catalytic core alone and in identification of the first terminal uridyltransferase (TU- complex with UTP analogs. These structures reveal Tase) RET1 (7). In the following decade, the perception of a tight docking of the conserved nucleotidyl trans- RNA uridylation has evolved from an unusual reaction in ferase bi-domain module with a RET1-specific C2H2 an enigmatic organelle into a recognition of U-tailing as the zinc finger and RNA recognition (RRM) domains. Fur- major transcriptome-shaping force in eukaryotes (8). *To whom correspondence should be addressed. Tel: +33 5 57 57 15 11; Email: [email protected] Correspondence may also be addressed to Ruslan Aphasizhev. Tel: +1 617 414 1055; Email: [email protected] †These authors contributed equally to this work as the first authors. Present addresses: Paola Munoz-Tello, Department of Molecular Therapeutics, The Scripps Research Institute-Scripps Florida, FL 33458, USA. Jason R. Stagno, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA. C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2016, Vol. 44, No. 22 10863 Earlier studies implicated RET1 TUTase in adding 10– unclear (33). The critical and well-understood roles in at 15 nt-long U-tails to guide RNAs (gRNAs) and ribo- least two essential RNA processing pathways, and robust somal RNAs (9,10). The 50–60 nt-long gRNAs bind to in vitro UTP polymerization activity render RET1 an at- and direct the editing of 12 among 18 mitochondrial pre- tractive target for trypanocide development. To that end, mRNAs (11). In addition, observed UTP-stimulated in or- screening of ∼3000-compound library led to identification ganello mRNA degradation hinted at possible involvement of competitive inhibitors with Ki constants in a low micro- of RET1-catalyzed uridylation in mRNA decay (12,13). Fi- molar range (34). nally, sequencing of mRNA 3 termini revealed that most Here, we report the high-resolution atomic structure of mRNAs in the actively respiring insect (procyclic) devel- the RET1 core region obtained by deleting N- and C- opmental stage of the parasite are modified by addition of terminal extensions that show no similarity to proteins 200–300 nt-long A/U-heteropolymers (14), which indicated outside Kinetoplastidae. The acquired structures of binary RET1’s involvement in mRNA 3 processing and transla- complexes with UTP and UTP analogs encompass the en- tional activation (15). Subsequent work demonstrated that zymatic module plus the zinc finger domain and the puta- RET1 is not only required for 3 uridylation on mature tive RRM fold inserted within the catalytic domain. Struc- gRNAs, ribosomal RNAs and some mRNAs, but also tural comparison of the zinc finger domain localization and participates in nucleolytic processing of precursors tran- orientation suggests its capacity to bind double-stranded scribed from maxicircle and minicircle genomes (16,17). RNA. We also solved the structure of a minimal TUT4 In-depth investigation of mitochondrial high molecular TUTase (24,25) in ternary complex with UTP and RNA- mass TUTase-containing complexes revealed that RET1 is imitating UpU dinucleotide. The TUT4-UTP-UpU struc- mostly sequestered into a stable particle along with DSS1 ture was used in molecular modeling experiments to reveal 3’-5’ exonuclease and three proteins lacking annotated mo- the RET1’s RNA binding determinants in the vicinity of tifs (16). This ∼900 kDa complex, termed the mitochon- the active site. In addition, we applied molecular dynamics drial 3’ processome (MPsome), is responsible for recog- simulations to determine potentially druggable cavities in nition, uridylation and exonucleolytic processing of 800– UTP-bound RET1. The mutational and biochemical anal- 1200 nt-long gRNA precursors, and U-tailing of mature yses demonstrate that deleting the N-terminal extension gRNAs. Thus, the enzymes with seemingly opposing enzy- does not affect RET1 activity in vitro, but hinders protein matic activities, 3’ addition and 3’-5’ degradation, function oligomerization and in vivo function. We demonstrate that as subunits of a single protein complex. This is a profound the C-terminal region and the RRM domains contribute to example of coupling between TUTase and RNase II-like RNA binding and, therefore, processivity of UTP polymer- exonuclease, an apparently evolutionarily conserved RNA ization reaction in vitro. We further show that the N-, C- and decay pathway (18–20). Apart from binding to the MP- N-C terminally truncated RET1 variants successfully in- some, RET1 transiently interacts with a complex of kine- corporate into the mitochondrial 3’ processome, but cause toplast polyadenylation factors 1 and 2 (KPAF1/2) and mi- a dominant negative phenotype. In summary, our findings tochondrial poly(A) polymerase KPAP1. Pentatricopeptide reveal the high-resolution structure of the key mitochon- repeat (PPR)-containing RNA binding factors KPAF1/2 drial RNA processing enzyme in trypanosomes and deepen coordinate TUTase and poly(A) polymerase activities in the structure-function understanding of the TUTase fam- adding A/U-heteropolymers to the mature mRNA 3’ ter- ily.This work also provides a foundation for structure-based mini, which is required for translational activation (15,21). design of TUTase inhibitors with potential trypanocidal ac- High resolution atomic structures of apo forms and bi- tivities. nary complexes with substrate or non-substrate nucleotides have been determined for three TUTases from T. bru- MATERIALS AND METHODS cei. These include mitochondrial RET2 (22) and MEAT1 Protein expression and purification (23), and cytosolic TUT4 (24,25) proteins. In addition, the atomic mechanism of UTP recognition by Cid1 TU- All reagents and materials were purchased from Thermo Tase from Schizosaccharomyces pombe has been reported Fisher Scientific
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