Charge Reduction and Thermodynamic Stabilization of Substrate RNAs Inhibit RNA Editing (original) (raw)
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Targeting RNA Structure to Inhibit Editing in Trypanosomes
International Journal of Molecular Sciences
Mitochondrial RNA editing in trypanosomes represents an attractive target for developing safer and more efficient drugs for treating infections with trypanosomes because this RNA editing pathway is not found in humans. Other workers have targeted several enzymes in this editing system, but not the RNA. Here, we target a universal domain of the RNA editing substrate, which is the U-helix formed between the oligo-U tail of the guide RNA and the target mRNA. We selected a part of the U-helix that is rich in G-U wobble base pairs as the target site for the virtual screening of 262,000 compounds. After chemoinformatic filtering of the top 5000 leads, we subjected 50 representative complexes to 50 nanoseconds of molecular dynamics simulations. We identified 15 compounds that retained stable interactions in the deep groove of the U-helix. The microscale thermophoresis binding experiments on these five compounds show low-micromolar to nanomolar binding affinities. The UV melting studies sho...
Elements of Nucleotide Specificity in the Trypanosoma brucei Mitochondrial RNA Editing Enzyme RET2
Journal of Chemical Information and Modeling, 2012
The causative agent of African sleeping sickness, Trypanosoma brucei, undergoes an unusual mitochondrial RNA editing process that is essential for its survival. RNA editing terminal uridylyl transferase 2 of T. brucei (TbRET2) is an indispensable component of the editosome machinery that performs this editing. TbRET2 is required to maintain the vitality of both the insect and bloodstream forms of the parasite, and, with its high-resolution crystal structure, it poses as a promising pharmaceutical target. Neither the exclusive requirement of UTP for catalysis, nor the RNA primer preference of TbRET2 is well understood. Using all-atom explicitly solvated molecular dynamics (MD) simulations, we investigated the effect of UTP binding on TbRET2 structure and dynamics, as well as the determinants governing TbRET2's exclusive UTP preference. Through our investigations of various nucleoside triphosphate substrates (NTPs), we show that UTP pre-organizes the binding site through an extensive water-mediated H-bonding network, bringing Glu424 and Arg144 sidechains to an optimum position for RNA primer binding. In contrast, CTP and ATP cannot achieve this pre-organization and thus preclude productive RNA primer binding. Additionally, we have located ligand-binding "hot spots" of TbRET2 based on the MD conformational ensembles and computational fragment mapping. TbRET2 reveals different binding pockets in the apo and UTP-bound MD simulations, which could be targeted for inhibitor design.
Journal of Molecular Biology, 2008
U-insertion/deletion RNA editing in the single mitochondrion of ancient kinetoplastids is a unique mRNA maturation process needed for translation. Multi-subunit editing complexes recognize many pre-mRNA sites and modify them via cycles of three catalytic steps: guide-RNA (gRNA) mediated cleavage, insertion or deletion of uridylates at the 3' terminus of the upstream cleaved piece, and ligation of the two mRNA pieces. While catalytic and many structural protein subunits of these complexes have been identified, the mechanisms and basic determinants of substrate recognition are still poorly understood. The current study defined relatively simple single-and double-stranded determinants for association and gRNA-directed cleavage. To this end, we used an electrophoretic mobility shift assay to directly score the association of purified editing complexes with RNA ligands, in parallel with U.V. photo-crosslinking and functional studies. The cleaved strand required a minimal 5' overhang of 12-nt and a ~15-bp duplex with gRNA to direct the cleavage site. A second protruding element in either the cleaved or the guide strand was required unless longer duplexes were used. Importantly, the single-stranded RNA requirement for association can be upstream or downstream of the duplex, and the binding and cleavage activities of purified editing complexes could be uncoupled. The current observations together with our previous reports (Cifuentes-Rojas et al., 2005 and 2006) show that association, cleavage and full-round editing by purified editing complexes have distinct determinants that increase in complexity as these editing stages progress. Finally, we found that the endonuclease KREN1 in purified complexes photo-crosslinks with a targeted editing site. A model is proposed whereby one or more RNase III-type endonucleases in editing complexes mediate the initial binding and scrutiny of potential ligands, and subsequent catalytic selectivity triggers either insertion or deletion editing enzymes.
Molecular Cell, 2005
the formation of an antiparallel RNA/RNA duplex structure between the preedited mRNA and a cognate gRNA Ulrich F. Müller, 1,3 Ken Stuart, 2 molecule. It is assumed that the base pairing interacand H. Ulrich Göringer 1, * tion is catalyzed by the RNA annealing factors gBP21 1 Department of Microbiology and Genetics and gBP27, which have been identified in Trypanosoma Darmstadt University of Technology brucei, Leishmania tarentolae, and Crithidia fasciculata Schnittspahnstraße 10 (Müller et al., 2001; Blom et al., 2001; Aphasizhev et 64287 Darmstadt al., 2003b). The gRNA/pre-mRNA duplex positions an Germany editing site 5# of the helical element, thereby defining 2 Seattle Biomedical Research Institute the endoribonucleolytic cleavage site of the preedited 307 Westlake Avenue N, Suite 500 mRNA. An endoribonucleolytic enzyme activity has Seattle, Washington 98109 been identified in editing-active mitochondrial fractions (Adler and Hajduk, 1997; Piller et al., 1997; Salavati et al., 2002); however, no candidate protein has yet been Summary characterized. During deletion-type RNA editing, uridylate residues are exonucleolytically removed from the RNA editing in trypanosomatids is catalyzed by a 3# end of the 5# mRNA cleavage product and released high molecular mass RNP complex, which is only paras UMP. This requires a U-specific 3# to 5# exoribotially characterized. TbMP42 is a 42 kDa protein of unnuclease (exoUase). As for the endoribonuclease, mitoknown function that copurifies with the editing comchondrial extracts contain exoUase activity (Aphaplex. The polypeptide is characterized by two Zn sizhev and Simpson, 2001, Igo et al., 2002), but no fingers and a potential barrel structure/OB-fold at its candidate protein has been identified to date. Insertion-C terminus. Using recombinant TbMP42, we show type editing requires the addition of U nucleotides to that the protein can bind to dsRNA and dsDNA but the 3# end of the 5# mRNA cleavage product. This reacfails to recognize DNA/RNA hybrids. rTbMP42 detion step is catalyzed by a 3# terminal uridylyl transfergrades ssRNA by a 3 to 5 exoribonuclease activity. ase (TUTase). The enzyme has recently been cloned In addition, rTbMP42 has endoribonuclease activfrom both Leishmania and trypanosomes and was ity, which preferentially hydrolyzes non-base-paired characterized as a member of the DNA polymerase β uridylate-containing sequences. Gene silencing of superfamily of nucleotidyltransferases (Aphasizhev et TbMP42 inhibits cell growth and is ultimately lethal al., 2003c; Ernst et al., 2003). to the parasite. Mitochondrial extracts from TbMP42-An editing reaction cycle is completed by the ligation minus trypanosomes have only residual RNA editing of the processed 5# fragment to the 3# fragment of the activity and strongly reduced endo-exoribonuclease pre-mRNA. Two editing-specific RNA ligases (REL1, activity. However, all three activities can be restored REL2) have been identified and were biochemically by the addition of rTbMP42. Together, the data sugand genetically characterized (McManus et al., 2001; gest that TbMP42 contributes both endo-and exoribo-Schnaufer et al., 2001; Huang et al., 2001). nuclease activity to the editing reaction cycle. Aside from these core activities, evidence exists that several auxiliary factors add to the reaction cycle. Introduction Among these factors are proteins which interact and stabilize preedited mRNA, such as REAP1 (Madison-The RNA editing reaction of mitochondrial mRNAs in Antenucci and Hajduk, 2001), polypeptides which can kinetoplastid protozoa is characterized by an enzybind to the 3# oligo(U) extensions of gRNAs (TbRGG1) matic reaction cycle that inserts and deletes uridylate (Vanhamme et al., 1998), or proteins such as mHel61p, nucleotides into otherwise incomplete primary trana complex-associated putative RNA helicase (Missel et scripts. The process is catalyzed by a high molecular al., 1997; Stuart et al., 2002), which may catalyze the mass ribonucleoprotein complex, which is composed unwinding of fully base-paired gRNAs from edited of preedited mRNAs, guide (g) RNAs, and an uncertain mRNAs. number of proteins (Madison-Antenucci et al., 2002; Potential candidates for the yet unidentified catalytic Worthey et al., 2003; Simpson et al., 2004). Depending components of the editing machinery are proteins that on the enrichment protocol, active RNA editing comcopurify with the complex (Stuart et al., 2002; Worthey plexes contain as little as 7 (Rusché et al., 1997), 13 et al., 2003; Simpson et al., 2004). They include poly-(Aphasizhev et al., 2003a), or up to 20 polypeptides peptides that have been shown to contain Zn finger (Panigrahi et al., 2001). motifs, suggesting direct contact points to nucleic acid Although not all contributing enzyme activities of a ligand molecules (Panigrahi et al., 2001, Huang et al., full reaction cycle are currently known, it is generally 2002; Lu et al., 2003). One such protein is TbMP42. The accepted that the initiation step of the process involves mitochondrial polypeptide has a molecular mass of 42 kDa and was first identified in African trypanosomes (Panigrahi et al., 2001). It shares sequence homology
RNA editing complex interactions with a site for full-round U deletion in Trypanosoma brucei
RNA, 2006
Trypanosome U insertion and U deletion RNA editing of mitochondrial pre-mRNAs is catalyzed by multisubunit editing complexes as directed by partially complementary guide RNAs. The basic enzymatic activities and protein composition of these high-molecular mass complexes have been under intense study, but their specific protein interactions with functional pre-mRNA/gRNA substrates remains unknown. We show that editing complexes purified through extensive ion-exchange chromatography and immunoprecipitation make specific cross-linking interactions with A6 pre-mRNA containing a single 32 P and photoreactive 4-thioU at the scissile bond of a functional site for full-round U deletion. At least four direct protein-RNA contacts are detected at this site by cross-linking. All four interactions are stimulated by unpaired residues just 59 of the pre-mRNA/gRNA anchor duplex, but strongly inhibited by pairing of the editing site region. Furthermore, competition analysis with homologous and heterologous transcripts suggests preferential contacts of the editing complex with the mRNA/gRNA duplex substrate. This apparent structural selectivity suggests that the RNA-protein interactions we observe may be involved in recognition of editing sites and/or catalysis in assembled complexes.
RNA editing: complexity and complications
2002
led most scientists to consider it a bizarre, isolated process. However, recent findings showing that much of editing entails conventional enzymes and processes suggest that a reconsideration of that view may be useful. It is uncertain how and when editing arose and survived evolutionary selection, but it does appear that it functions in the regulation of energy-generating processes during the life cycle of trypanosomatids.
Wiley interdisciplinary reviews. RNA, 2018
RNA editing causes massive remodeling of the mitochondrial mRNA transcriptome in trypanosomes and related kinetoplastid protozoa. This type of editing involves the specific insertion or deletion of uridylates (U) directed by small noncoding guide RNAs (gRNAs). Because U-insertion exceeds U-deletion by a factor of 10, editing increases the nascent mRNA size by up to 55%. In Trypanosoma brucei, the editing apparatus uses ~40 proteins and >1,200 gRNAs to create the functional open reading frame in 12 mRNAs. Thousands of sites are specifically recognized in the pre-edited mRNAs and a myriad of partially edited transcript intermediates accumulates in mitochondria. The control of editing is poorly understood, but past work suggests that it occurs during substrate recognition, the initiation and progression of editing, and during the life-cycle in different hosts. The growing understanding of the editing proteins offers clues about editing control. Most editing proteins reside in the &q...
Journal of Biological Chemistry, 2012
Background: Mitochondrial transcripts in African trypanosomes undergo U insertion/deletion-type RNA editing that is catalyzed by a protein complex known as the editosome. Results: Editosomes have a single RNA substrate-binding site and catalyze RNA unwinding. Conclusion: Both U insertion and U deletion are executed within a single, multifunctional reaction center. Significance: RNA binding is followed by RNA unwinding, which represents a novel activity of the editing machinery. Editing of mitochondrial pre-mRNAs in African trypanosomes generates full-length transcripts by the site-specific insertion and deletion of uridylate nucleotides. The reaction is catalyzed by a 0.8 MDa multienzyme complex, the editosome. Although the binding of substrate pre-edited mRNAs and cognate guide RNAs (gRNAs) represents the first step in the reaction cycle, the biochemical and biophysical details of the editosome/RNA interaction are not understood. Here we show that editosomes bind full-length substrate mRNAs with nanomolar affinity in a nonselective fashion. The complexes do not discriminate-neither kinetically nor thermodynamically-between different mitochondrial pre-mRNAs or between edited and unedited versions of the same transcript. They also bind gRNAs and gRNA/pre-mRNA hybrid RNAs with similar affinities and association rate constants. Gold labeling of editosome-bound RNA in combination with transmission electron microscopy identified a single RNA-binding site per editosome. However, atomic force microscopy of individual pre-mRNA-editosome complexes revealed that multiple editosomes can interact with one pre-mRNA. Lastly, we demonstrate a so far unknown activity of the editing machinery: editosome-bound RNA becomes unfolded by a chaperone-type RNA unwinding activity.
Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo
The EMBO Journal, 2009
Mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of uridylate-specific RNA editing. RNA editing converts non-functional pre-mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of B20S and B35-40S and present the three-dimensional structures of both complexes by electron microscopy. The B35-40S complexes consist of a platform density packed against a semispherical element. The B20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The B20S editosomes contain an RNA-binding site, which binds gRNA, pre-mRNA and gRNA/pre-mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.