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      RNA Editing TUTase 1: structural foundation of substrate recognition, complex interactions and drug targeting

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          Abstract

          Terminal uridyltransferases (TUTases) execute 3′ RNA uridylation across protists, fungi, metazoan and plant species. Uridylation plays a particularly prominent role in RNA processing pathways of kinetoplastid protists typified by the causative agent of African sleeping sickness, Trypanosoma brucei. In mitochondria of this pathogen, most mRNAs are internally modified by U-insertion/deletion editing while guide RNAs and rRNAs are U-tailed. The founding member of TUTase family, RNA editing TUTase 1 (RET1), functions as a subunit of the 3′ processome in uridylation of gRNA precursors and mature guide RNAs. Along with KPAP1 poly(A) polymerase, RET1 also participates in mRNA translational activation. RET1 is divergent from human TUTases and is essential for parasite viability in the mammalian host and the insect vector. Given its robust in vitro activity, RET1 represents an attractive target for trypanocide development. Here, we report high-resolution crystal structures of the RET1 catalytic core alone and in complex with UTP analogs. These structures reveal a tight docking of the conserved nucleotidyl transferase bi-domain module with a RET1-specific C2H2 zinc finger and RNA recognition (RRM) domains. Furthermore, we define RET1 region required for incorporation into the 3′ processome, determinants for RNA binding, subunit oligomerization and processive UTP incorporation, and predict druggable pockets.

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          Most cited references55

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          A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei.

          First-generation inducible expression vectors for Trypanosoma brucei utilized a single tetracycline-responsive promoter to drive expression of an experimental gene, in tandem with a drug-resistance marker gene to select for integration (Wirtz E, Clayton CE. Science 1995; 268:1179-1183). Because drug resistance and experimental gene expression both depended upon the activity of the regulated promoter, this approach could not be used for inducible expression of toxic products. We have now developed a dual-promoter approach, for expressing highly toxic products and generating conditional gene knock-outs, using back-to-back constitutive T7 and tetracycline-responsive PARP promoters to drive expression of the selectable marker and test gene, respectively. Transformants are readily obtained with these vectors in the absence of tetracycline, in bloodstream or procyclic T. brucei cell lines co-expressing T7 RNA polymerase and Tet repressor, and consistently show tetracycline-responsive expression through a 10(3)-10(4)-fold range. Uninduced background expression of a luciferase reporter averages no more than one molecule per cell, enabling dominant-negative approaches relying upon inducible expression of toxic products. This tight regulation also permits the production of functional gene knock-outs through regulated expression of an experimental gene in a null-mutant background.
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            The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression.

            The RNA recognition motif (RRM), also known as RNA-binding domain (RBD) or ribonucleoprotein domain (RNP) is one of the most abundant protein domains in eukaryotes. Based on the comparison of more than 40 structures including 15 complexes (RRM-RNA or RRM-protein), we reviewed the structure-function relationships of this domain. We identified and classified the different structural elements of the RRM that are important for binding a multitude of RNA sequences and proteins. Common structural aspects were extracted that allowed us to define a structural leitmotif of the RRM-nucleic acid interface with its variations. Outside of the two conserved RNP motifs that lie in the center of the RRM beta-sheet, the two external beta-strands, the loops, the C- and N-termini, or even a second RRM domain allow high RNA-binding affinity and specific recognition. Protein-RRM interactions that have been found in several structures reinforce the notion of an extreme structural versatility of this domain supporting the numerous biological functions of the RRM-containing proteins.
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              Lin28 mediates the terminal uridylation of let-7 precursor MicroRNA.

              The precise control of microRNA (miRNA) biogenesis is critical for embryonic development and normal cellular functions, and its dysregulation is often associated with human diseases. Though the birth and maturation pathway of miRNA has been established, the regulation and death pathway remains largely unknown. Here, we report the RNA-binding proteins, Lin28a and Lin28b, as posttranscriptional repressors of let-7 miRNA biogenesis. We observe that the Lin28 proteins act mainly in the cytoplasm by inducing uridylation of precursor let-7 (pre-let-7) at its 3' end. The uridylated pre-let-7 (up-let-7) fails Dicer processing and undergoes degradation. We provide a mechanism for the posttranscriptional regulation of miRNA biogenesis by Lin28 which is highly expressed in undifferentiated cells and certain cancer cells. The Lin28-mediated downregulation of let-7 may play a key role in development, stem cell programming, and tumorigenesis.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                15 December 2016
                15 October 2016
                15 October 2016
                : 44
                : 22
                : 10862-10878
                Affiliations
                [1 ]Department of Molecular Biology, University of Geneva, 1211 Geneva, Switzerland
                [2 ]Department of Molecular and Cell Biology, Boston University School of Dental Medicine, Boston, MA 02118, USA
                [3 ]Department of Chemistry & Biochemistry and the National Biomedical Computation Resource, University of California, San Diego, La Jolla, CA 92093, USA
                [4 ]Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA
                [5 ]Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
                [6 ]INSERM, U1212, ARNA Laboratory, Bordeaux 33000, France
                [7 ]CNRS UMR5320, ARNA Laboratory, Bordeaux 33000, France
                [8 ]University of Bordeaux, ARNA Laboratory, Bordeaux 33000, France
                Author notes
                [* ]To whom correspondence should be addressed. Tel: +33 5 57 57 15 11; Email: stephane.thore@ 123456inserm.fr
                Correspondence may also be addressed to Ruslan Aphasizhev. Tel: +1 617 414 1055; Email: ruslana@ 123456bu.edu
                []These authors contributed equally to this work as the first authors.
                Article
                10.1093/nar/gkw917
                5159558
                27744351
                027e1818-5305-41f9-b0ef-225dd3b37ff0
                © 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 journals.permissions@ 123456oup.com

                History
                : 04 October 2016
                : 27 September 2016
                : 10 May 2016
                Page count
                Pages: 17
                Categories
                Nucleic Acid Enzymes
                Custom metadata
                15 December 2016

                Genetics
                Genetics

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