RNA helicases involved in U-insertion/deletion-type RNA editing.

Mitochondrial pre-messenger RNAs in kinetoplastid protozoa such as the disease-causing African trypanosomes are substrates of a unique RNA editing reaction. The process is characterized by the site-specific insertion and deletion of exclusively U nucleotides and converts nonfunctional pre-mRNAs into translatable transcripts. Similar to other RNA-based metabolic pathways, RNA editing is catalyzed by a macromolecular protein complex, the editosome. Editosomes provide a reactive surface for the individual steps of the catalytic cycle and involve as key players a specific class of small, non-coding RNAs termed guide (g)RNAs. gRNAs basepair proximal to an editing site and act as quasi templates in the U-insertion/deletion reaction. Next to the editosome several accessory proteins and complexes have been identified, which contribute to different steps of the reaction. This includes matchmaking-type RNA/RNA annealing factors as well as RNA helicases of the archetypical DEAD- and DExH/D-box families. Here we summarize the current structural, genetic and biochemical knowledge of the two characterized "editing RNA helicases" and provide an outlook onto dynamic processes within the editing reaction cycle. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for Life

Multiple G-quartet structures in pre-edited mRNAs suggest evolutionary driving force for RNA editing in trypanosomes.

Mitochondrial transcript maturation in African trypanosomes requires a U-nucleotide specific RNA editing reaction. In its most extreme form hundreds of U's are inserted into and deleted from primary transcripts to generate functional mRNAs. Unfortunately, both origin and biological role of the process have remained enigmatic. Here we report a so far unrecognized structural feature of pre-edited mRNAs. We demonstrate that the cryptic pre-mRNAs contain numerous clustered G-nt, which fold into G-quadruplex (GQ) structures. We identified 27 GQ's in the different pre-mRNAs and demonstrate a positive correlation between the steady state abundance of guide (g)RNAs and the sequence position of GQ-elements. We postulate that the driving force for selecting G-rich sequences lies in the formation of DNA/RNA hybrid G-quadruplex (HQ) structures between the pre-edited transcripts and the non-template strands of mitochondrial DNA. HQ's are transcription termination/replication initiation sites and thus guarantee an unperturbed replication of the mt-genome. This is of special importance in the insect-stage of the parasite. In the transcription-on state, the identified GQ's require editing as a GQ-resolving activity indicating a link between replication, transcription and RNA editing. We propose that the different processes have coevolved and suggest the parasite life-cycle and the single mitochondrion as evolutionary driving forces

The RNA chaperone activity of the Trypanosoma brucei editosome raises the dynamic of bound pre-mRNAs.

Mitochondrial transcript maturation in African trypanosomes requires an RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. The reaction is catalyzed by editosomes and requires guide (g)RNAs as templates. Recent data demonstrate that the binding of pre-edited mRNAs to editosomes is followed by a chaperone-type RNA remodeling reaction. Here we map the changes in RNA folding using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). We demonstrate that pre-mRNAs in their free state adopt intricately folded, highly stable 2D-structures. Editosome binding renders the pre-mRNAs to adopt 2D-conformations of reduced stabilities. On average about 30% of the nucleotides in every pre-mRNA are affected with a prevalence for U-nucleotides. The data demonstrate that the chaperone activity acts by increasing the flexibility of U-residues to lower their base-pairing probability. This results in a simplified RNA folding landscape with a reduced energy barrier to facilitate the binding of gRNAs. The data provide a first rational for the enigmatic U-specificity of the editing reaction

Bioinspired Design of Lysolytic Triterpenoid-Peptide Conjugates that Kill African Trypanosomes.

Humans have evolved a natural immunity against Trypanosoma brucei infections, which is executed by two serum (lipo)protein complexes known as trypanolytic factors (TLF). Active TLF-ingredient is the primate-specific apolipoprotein L1 (ApoL1). The protein has a pore-forming activity that kills parasites by lysosomal and mitochondrial membrane fenestration. Of the many trypanosome subspecies only two are able to counteract the activity of ApoL1, which illustrates its evolutionary optimized design and trypanocidal potency. Here we ask the question whether a synthetic (syn)TLF can be synthesized using the design principles of the natural TLF-complexes but relying on different chemical building blocks. We demonstrate the stepwise development of triterpenoid-peptide conjugates, in which the triterpenoids act as a cell binding, uptake and lysosomal transport-moduls and the synthetic peptide GALA as a pH-sensitive, pore-forming lysolytic toxin. As designed, the conjugate kills infective-stage African trypanosomes through lysosomal lysis demonstrating proof-of-principle for the bioinspired, forward-design of a synTLF

<Review> Maurice Cornforth ; Science and Idealism

African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcript maturation in these organisms requires a RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. Editing represents an ideal target for a parasite-specific therapeutic intervention since the reaction cycle is absent in the infected host. In addition, editing relies on a macromolecular protein complex, the editosome, that only exists in the parasite. Therefore, all attempts to search for editing interfering compounds have been focused on molecules that bind to proteins of the editing machinery. However, in analogy to other RNA-driven biochemical pathways it should be possible to stall the reaction by targeting its substrate RNAs. Here we demonstrate inhibition of editing by specific aminoglycosides. The molecules bind into the major groove of the gRNA/pre-mRNA editing substrates thereby causing a stabilization of the RNA molecules through charge compensation and an increase in stacking. The data shed light on mechanistic details of the editing process and identify critical parameters for the development of new trypanocidal compounds

Salt dependence of UV-melting profiles.

<p>Sodium ion dependence (10–250mM) of melting temperatures of the U-insertion (A/B) and U-deletion (C/D) gRNA/pre-mRNA editing substrate RNAs (1μM) in the absence (open squares) and presence (filled squares) of neomycin B (13μM). Plots of the melting temperature (T_m) <i>versus</i> log of the Na⁺-ion concentration (log c_Na+) for the 4 individual helices of the two editing RNAs. Solid lines: linear regressions of the data points. (E) Summary of the derived data.</p

CD-spectra of RNA editing substrate RNAs at a concentration of 12μM.

<p>(A) U-insertion gRNA/pre-mRNA hybrid RNA (red trace). (B) U-deletion gRNA/pre-mRNA hybrid RNA (dark blue trace). Both spectra show typical A-form characteristics. Adding increasing concentrations of neomycin B (1.3μM-0.5mM) yields the spectra shown in orange to yellow (U-insertion) and light blue to cyan (U-deletion). (C/D) Plotting the spectral changes at 273nm as a function of the molar neomycinB/RNA ratio results in 3.1 (U-insertion) and 3.3 (U-deletion) neomycin binding sites per editing RNA.</p

Trypanosome-specific U-insertion and U-deletion RNA editing.

<p>Depicted are two gRNA/pre-mRNA pairs emphasizing the helical domains of the U-insertion RNA substrate in red and yellow (A) and the helices of the U-deletion RNA in dark and light blue (B). <i>In vitro</i> U-insertion monitors the insertion of 3 U’s; <i>in vitro</i> U-deletion the removal of 4 U nt. The reaction is catalyzed by the 20S editosome. Primary sequences of the individual RNAs are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118940#pone.0118940.s002" target="_blank">S2 Fig</a>. Inhibition of U-insertion (C) and U-deletion RNA editing (D) by neamin. Radioactively labelled (5’-³²P) gRNA/pre-mRNA substrate RNAs were incubated with 20S editosomes in the presence of increasing concentrations of neamin (1.6μM-1.7mM, left to right). RNA reactants, intermediates and edited products (annotated to the right of the two gels) were electrophoretically separated and densitometrically quantified to yield dose response curves (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118940#pone.0118940.s003" target="_blank">S3 Fig</a>) from which IC₅₀-values were derived (E). Errors are standard deviations (s.d.). M: mock treated sample. *: position of the radioactive label (³²P). Arrows indicate the position of the fully edited mRNA products.</p

ITC-titration profiles of U-insertion (A) and U-deletion (B) gRNA/pre-mRNA hybrid RNAs with neomycin B.

<p>(C) Summary of the derived thermodynamic characteristics of the binding reaction: equilibrium dissociation constant (K_d), number of binding sites (n), enthalpy (ΔH), entropy (ΔS) and Gibbs free energy (ΔG). Errors are standard deviations (s.d.).</p

UV-melting profiles of U-insertion (A) and U-deletion (B) gRNA/pre-mRNA substrate RNAs (1μM) in the absence (grey trace) and presence (black trace) of neomycin B.

<p>Both RNAs exhibit two distinct helix/coil transitions indicating the independent melting of the two helical domains in both RNAs. In the presence of neomycin B (13μM; 13-fold over K_d) both melting curves shift to higher temperatures indicating a stabilization of the editing RNAs. (C)/(D) Plotting the T_m-value changes of the individual helices as a function of the molar neomycin B/bp ratio results in 3.1 (U-insertion) and 3.3 (U-deletion) neomycin binding sites per editing RNA. (E) Summery of the measured/calculated T_m, ΔT_m and thermodynamic values for the individual helices in both gRNA/pre-mRNA substrate RNAs.</p