African trypanosomes are the causative agent of sleeping sickness. The therapeutics used to control and treat the disease are very ineffective and thus, the development of improved drugs is urgently ...needed. Recently, new strategies for the design of novel trypanocidals have been put forward. Among them are techniques that rely on parasite-specific RNA aptamers. One approach involves the aptamer-directed transport of lytic compounds to the lysosome of the parasite. The aptamer has been termed 2-16 RNA and here we report the optimization of the RNA for its applications in vivo. To convert aptamer 2-16 into a serum-stable reagent 2'-deoxy-2'-F- and/or 2'-deoxy-2'-NH(2)-uridine- and cytidine-substituted RNAs were generated. While 2'-NH(2)-dC/dU-modified RNAs were RNase-resistant, they were functionally inactive. By contrast, 2'-F-dC/dU-substituted 2-16 RNA retained its ability to bind to live trypanosomes (K(d)=45 nM) and was routed to the lysosome identically to unmodified RNA. 2'-F-dC/dU-substituted 2-16 RNA is thermostable (T(m)=75 degrees C) and has a serum half-life of 3.4 days. Furthermore, aptamer 2-16 was site-specifically PEGylated to increase its serum retention time. Conjugation with PEG polymers < or = 10 kDa only marginally impacted the binding characteristics of the RNA, while the addition of higher molecular mass PEG molecules resulted in non-functional aptamers. Together, the data provide optimized conjugation chemistries for the large-scale production of substituted aptamer 2-16 preparations with improved in vivo functionality.
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.
•We discuss dynamic aspects of the RNA editing reaction that involve RNA helicases.•RNA editing helicases interact transiently with the editosome.•mHel61p catalyzes the unwinding of gRNA/pre-mRNA hybrid RNAs.
Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene expression post-transcriptionally and not at the level of transcription initiation. This involves all ...known cellular RNA circuits, from mRNA processing to mRNA decay, to translation, in addition to a large panel of RNA-interacting proteins that modulate mRNA abundance. However, other forms of gene regulation, for example by lncRNAs, cannot be excluded. LncRNAs are poorly studied in trypanosomatids, with only a single lncRNA characterized to date. Furthermore, it is not clear whether the complete inventory of trypanosomatid lncRNAs is known, because of the inherent cDNA-recoding and DNA-amplification limitations of short-read RNA sequencing. Here, we overcome these limitations by using long-read direct RNA sequencing (DRS) on nanopore arrays. We analyze the native RNA pool of the two main lifecycle stages of the African trypanosome
with a special emphasis on the inventory of lncRNAs. We identify 207 previously unknown lncRNAs, 32 of which are stage-specifically expressed. We also present insights into the complexity of the
transcriptome, including alternative transcriptional start and stop sites and potential transcript isoforms, to provide a bias-free understanding of the intricate RNA landscape in
.
RNA editing describes a chemically diverse set of biomolecular reactions in which the nucleotide sequence of RNA molecules is altered. Editing reactions have been identified in many organisms and ...frequently contribute to the maturation of organellar transcripts. A special editing reaction has evolved within the mitochondria of the kinetoplastid protozoa. The process is characterized by the insertion and deletion of uridine nucleotides into otherwise nontranslatable messenger RNAs. Kinetoplastid RNA editing involves an exclusive class of small, noncoding RNAs known as guide RNAs. Furthermore, a unique molecular machinery, the editosome, catalyzes the process. Editosomes are megadalton multienzyme assemblies that provide a catalytic surface for the individual steps of the reaction cycle. Here I review the current mechanistic understanding and molecular inventory of kinetoplastid RNA editing and the editosome machinery. Special emphasis is placed on the molecular morphology of the editing complex in order to correlate structural features with functional characteristics.
African trypanosomiasis is a parasitic disease caused by a specific class of protozoan organisms. The best-studied representative of that group is Trypanosoma brucei which is transmitted by tsetse ...flies and multiplies in the blood of many mammals. Trypanosomes evade the immune system by altering their surface structure which is dominated by a layer of a variant surface glycoprotein (VSG). Although invariant surface proteins exist, they are inaccessible to the humoral immune response. Using a combinatorial selection method in conjunction with live trypanosomes as the binding target, we show that short RNA ligands (aptamers) for constant surface components can be isolated. We describe the selection of three classes of RNA aptamers that crosslink to a single 42 kDa protein located within the flagellar pocket of the parasite. The RNAs associate rapidly and with high affinity. They do not discriminate between two different trypanosome VSG variant strains and, furthermore, are able to bind to other trypanosome strains not used in the selection protocol. Thus, the aptamers have the potential to function as markers on the surface of the extracellular parasite and as such they might be modified to function as novel drugs against African trypanosomiasis.
Trypanosoma brucei is the causal infectious agent of African trypanosomiasis in humans and Nagana in livestock. Both diseases are currently treated with a small number of chemotherapeutics, which are ...hampered by a variety of limitations reaching from efficacy and toxicity complications to drug‐resistance problems. Here, we explore the forward design of a new class of synthetic trypanocides based on nanostructured, core‐shell DNA‐lipid particles. In aqueous solution, the particles self‐assemble into micelle‐type structures consisting of a solvent‐exposed, hydrophilic DNA shell and a hydrophobic lipid core. DNA‐lipid nanoparticles have membrane‐adhesive qualities and can permeabilize lipid membranes. We report the synthesis of DNA‐cholesterol nanoparticles, which specifically subvert the membrane integrity of the T. brucei lysosome, killing the parasite with nanomolar potencies. Furthermore, we provide an example of the programmability of the nanoparticles. By functionalizing the DNA shell with a spliced leader (SL)‐RNA‐specific DNAzyme, we target a second trypanosome‐specific pathway (dual‐target approach). The DNAzyme provides a backup to counteract the recovery of compromised parasites, which reduces the risk of developing drug resistance.
The bioengineering of synthetic nanoparticles with therapeutic potential for the treatment of African sleeping sickness is reported. The molecules are DNA‐cholesterol amphiphiles, which self‐assemble into micelle‐type nanoparticles with an exterior DNA shell and a lipid core. The particles are constructed to attack two parasite‐specific targets thereby reducing the risk of developing drug resistance.
The guide RNA-binding protein gBP21 has been characterized as a mitochondrial RNA/RNA annealing factor. The protein co-immunoprecipitates with RNA editing ribonucleoprotein complexes, which suggests ...that gBP21 contributes its annealing activity to the RNA editing machinery. In support of this view, gBP21 was found to accelerate the hybridization of cognate guide (g)RNA/pre-edited mRNA pairs. Here we analyze the mechanism of the gBP21-mediated RNA annealing reaction. Three possible modes of action are considered: chaperone function, matchmaker function and product stabilization. We conclude that gBP21 works as a matchmaker by binding to gRNAs as one of the two RNA annealing reactants. Three lines of evidence substantiate this model. First, gBP21 and gRNAs form a thermodynamically and kinetically stable complex in a 1 + 1 stoichiometry. Secondly, gRNA-bound gBP21 stabilizes single-stranded RNA, which can be considered the transition state in the annealing reaction. Thirdly, gBP21 has a low affinity for double-stranded RNAs, suggesting the release of the annealed reaction product after the hybridization step. In the process, up to six ionic bonds are formed between gBP21 and a gRNA, which decreases the net negative charge of the RNA. As a consequence, the electrostatic repulsion between the two annealing reactants is reduced favoring the hybridization reaction.
Abstract
Sequence-deficient mitochondrial pre-mRNAs in African trypanosomes are substrates of a U-nucleotide-specific RNA editing reaction to generate translation-competent mRNAs. The reaction is ...catalyzed by a macromolecular protein complex termed the editosome. Editosomes execute RNA-chaperone activity to overcome the highly folded nature of pre-edited substrate mRNAs. The molecular basis for this activity is unknown. Here we test five of the OB-fold proteins of the Trypanosoma brucei editosome as candidates. We demonstrate that all proteins execute RNA-chaperone activity albeit to different degrees. We further show that the activities correlate to the surface areas of the proteins and we map the protein-induced RNA-structure changes using SHAPE-chemical probing. To provide a structural context for our findings we calculate a coarse-grained model of the editosome. The model has a shell-like structure: Structurally well-defined protein domains are separated from an outer shell of intrinsically disordered protein domains, which suggests a surface-driven mechanism for the chaperone activity.
Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing ...mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3′ extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3′ adenylation, uridine insertion/deletion editing, and 3′ A/U-tailing. The rRNAs and gRNAs are 3′ uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.
Mitochondrial RNA processing events in kinetoplastid protists include 5′ modification, 3′–5′ degradation, internal sequence changes by U-insertion/deletion mRNA editing, and nontemplated 3′ extensions.The specificity of mRNA editing is dictated by gRNAs while 5′ modifications and 3′ extensions are controlled by diverse pentatricopeptide repeat (PPR) RNA-binding factors.Antisense transcription plays a central role in delimiting 3′ termini of mature RNAs.Macromolecular protein and ribonucleoprotein (RNP) complexes and auxiliary factors involved in these processes have been identified and characterized to varying degrees. This review discusses recent developments and introduces a consensus nomenclature for mitochondrial RNA-processing complexes and factors in Trypanosoma brucei.
PDF
