Adaptation of translational machinery in malaria parasites to accommodate translation of poly-adenosine stretches throughout its life cycle

Malaria is caused by unicellular apicomplexan parasites of the genu

Translation efficiency is a determinant of the magnitude of miRNA-mediated repression

Abstract MicroRNAs are well known regulators of mRNA stability and translation. However, the magnitude of both translational repression and mRNA decay induced by miRNA binding varies greatly between miRNA targets. This can be the result of cis and trans factors that affect miRNA binding or action. We set out to address this issue by studying how various mRNA characteristics affect miRNA-mediated repression. Using a dual luciferase reporter system, we systematically analyzed the ability of selected mRNA elements to modulate miRNA-mediated repression. We found that changing the 3′UTR of a miRNA-targeted reporter modulates translational repression by affecting the translation efficiency. This 3′UTR dependent modulation can be further altered by changing the codon-optimality or 5′UTR of the luciferase reporter. We observed maximal repression with intermediate codon optimality and weak repression with very high or low codon optimality. Analysis of ribosome profiling and RNA-seq data for endogenous miRNA targets revealed translation efficiency as a key determinant of the magnitude of miRNA-mediated translational repression. Messages with high translation efficiency were more robustly repressed. Together our results reveal modulation of miRNA-mediated repression by characteristics and features of the 5′UTR, CDS and 3′UTR

PTRE-seq reveals mechanism and interactions of RNA binding proteins and miRNAs

A large number of RNA binding proteins (RBPs) and miRNAs bind to the 3′ untranslated regions of mRNA, but methods to dissect their function and interactions are lacking. Here the authors introduce post-transcriptional regulatory element sequencing (PTRE-seq) to dissect sequence preferences, interactions and consequences of RBP and miRNA binding

Association of the receptor for activated C-kinase 1 with ribosomes in Plasmodium falciparum

The receptor for activated C-kinase 1 (RACK1), a highly conserved eukaryotic protein, is known to have many varying biological roles and functions. Previous work has established RACK1 as a ribosomal protein, with defined regions important for ribosome binding in eukaryotic cells. In Plasmodium falciparum, RACK1 has been shown to be required for parasite growth, however, conflicting evidence has been presented about RACK1 ribosome binding and its role in mRNA translation. Given the importance of RACK1 as a regulatory component of mRNA translation and ribosome quality control, the case could be made in parasites that RACK1 either binds or does not bind the ribosome. Here, we used bioinformatics and transcription analyses to further characterize the P. falciparum RACK1 protein. Based on homology modeling and structural analyses, we generated a model of P. falciparum RACK1. We then explored mutant and chimeric human and P. falciparum RACK1 protein binding properties to the human and P. falciparum ribosome. We found that WT, chimeric, and mutant RACK1 exhibit distinct ribosome interactions suggesting different binding characteristics for P. falciparum and human RACK1 proteins. The ribosomal binding of RACK1 variants in human and parasite cells shown here demonstrates that although RACK1 proteins have highly conserved sequences and structures across species, ribosomal binding is affected by species-specific alterations to this protein. In conclusion, we show that in the case of P. falciparum, contrary to the structural data, RACK1 is found to bind ribosomes and actively translating polysomes in parasite cells

Urb-RIP - An adaptable and efficient approach for immunoprecipitation of RNAs and associated RNAs/proteins

Post-transcriptional regulation of gene expression is an important process that is mediated by interactions between mRNAs and RNA binding proteins (RBP), non-coding RNAs (ncRNA) or ribonucleoproteins (RNP). Key to the study of post-transcriptional regulation of mRNAs and the function of ncRNAs such as long non-coding RNAs (lncRNAs) is an understanding of what factors are interacting with these transcripts. While several techniques exist for the enrichment of a transcript whether it is an mRNA or an ncRNA, many of these techniques are cumbersome or limited in their application. Here we present a novel method for the immunoprecipitation of mRNAs and ncRNAs, Urb-RNA immunoprecipitation (Urb-RIP). This method employs the RRM1 domain of the "resurrected" snRNA-binding protein Urb to enrich messages containing a stem-loop tag. Unlike techniques which employ the MS2 protein, which require large repeats of the MS2 binding element, Urb-RIP requires only one stem-loop. This method routinely provides over ~100-fold enrichment of tagged messages. Using this technique we have shown enrichment of tagged mRNAs and lncRNAs as well as miRNAs and RNA-binding proteins bound to those messages. We have confirmed, using Urb-RIP, interaction between RNA PolIII transcribed lncRNA BC200 and polyA binding protein

Modulation of miRISC-mediated gene silencing in eukaryotes

Gene expression is regulated at multiple levels in eukaryotic cells. Regulation at the post-transcriptional level is modulated by variou

Evolution of substrate recognition domains of the AAA proteins

AAA Proteine gehören zur großen Überfamilie der AAA+ Proteine. Diese sind ringförmige P-loop NTPasen, deren gemeinsame Funktion das engerieabhängige Entfalten von Makromolekülen ist. AAA Proteine bestehen im Allgemeinen aus einer N-terminalen Domäne und einer oder zwei ATPase Domänen, die mit D1 und D2 bezeichnet werden. Die ATPase Domänen innerhalb der Familie der AAA Proteine sind verhältnismäßig konserviert. Sie bewirken wahrscheinlich eine Hexamerisierung. Die N-terminalen Domänen sind wichtig für die Erkennung und Bindung des Substrats und unterscheiden sich im Gegensatz zu den ATPase Domänen strukturell. Auf der Grundlage veröffentlichter Daten und zusätzlicher bioinformatischer Analyse der AAA Proteine wurden mehrere verschiedene N-terminale Domänen von AAA Proteinen aus Archaebakterien für eine funktionelle und strukturelle Charakterisierung ausgewählt. Es wurden weiterhin Proteine charakterisiert, die ähnliche oder verwandte Domänen wie die AAA Proteine haben. Dabei wurde darauf Wert gelegt, Zusammenhänge und Gemeinsamkeiten herauszustellen. Es wurden Hitze- und chemische Aggregations Tests mit verschiedenen Substratproteinen durchgeführt, um die N-terminalen Domänen oder die gesamten AAA Proteine auf intrinsische Chaperon-Aktivität zu untersuchen. Die Proteinstrukturen wurden mit Röntgenstrukturanalyse oder mit NMR Spektroskopie bestimmt. Die Ergebnisse dieser Arbeit zeigen, dass die barrel-fömigen N-terminalen Domänen der AAA Proteine aus bauart-ähnlichen, Nukleinsäure bindenden Domänen hervorgingen. Die Substrataffinität hat sich wahrscheinlich durch verschiedene evolutionäre Mechanismen von Nukleinsäuren zu Proteinen gewandelt. Im Fall der doppelten Psi-Barrels ist dies vermutlich durch die Evolution eines einfachen beta-alpha-beta-Motifs geschehen, das man auch in den RIFT und in den swapped hairpin-Barrels findet. Letztere sind Transkriptionsfaktoren, d.h. sie binden DNA. Die Struktur von Mj0056 und SpoVT legt nahe, dass RIFT und swapped hairpin-Barrels entweder durch eine Insertion verschiedener Strukturelemente (Mj0056) oder durch die Rekrutierung einer Domäne (SpoVT) weiter evolviert sind. Ähnlichkeiten zwischen den N-terminalen Domänen von PAN und ARC wurden sowohl in Struktur und Funktion gefunden. Beide Domänen beinhalten eine coiled-coil Faltung, auf die eine oder zwei OB Faltungen folgen. OB steht für Oligosaccharid-Bindung und weist darauf hin, dass diese Domäne ebenfalls aus einer DNA-bindenden Struktur entstand. Die Struktur der ARC-Nc Subdomäne und eine umfassende Analyse von Chimären aus coiled-coil und OB Faltungen zeigen auf, dass sich die N-terminalen Domänen von ARC und PAN evolutionär durch die Rekrutierung von Domänen entwickelt haben. Die strikte strukturelle Zusammensetzung der Subdomänen ist wichtig für die Chaperon-Funktion und wird durch die konservierten PP-Linker, die die beiden Subdomänen verbinden, erhalten. Die strukturelle und funktionale Charakterisierung von AfAMA, das zu der neuen Gruppe der AMA AAA Proteine gehört, zeigte, dass die Substratbindefunktion und die Chaperon-Aktivität dieser Proteine in ihrer beta-clam ähnlichen N-terminalen Domäne liegen. Diese Domäne kann die genannten Funktionen unabhängig voneinander erfüllen, was sie von anderen homologen Domänen unterscheidet. Es war uns möglich, die Bedeutung der Oligomerisierung für die Aktivität dieser Domänen zu zeigen. Des Weiteren haben wir gezeigt, dass die Oligomerisierung durch ein kleines GYPL-Motif vermittelt wird, das auf einem Loop liegt, der vermutlich in das Zentrum des Hexamers zeigt. Die N-terminale Domäne von AMA bewirkt die Hexamerisierung des ganzen Proteins unabhängig von dem AAA Teil des Proteins und unabhängig von ATP Verbrauch. Darin weicht sie stark von anderen Familien der AAA Proteinen ab. Funktionelle Daten von anderen beta-clam Domänen (z.B. VAT-Nc, Ph1500N und Hm-clam) würden darauf hinweisen, dass diese Domänen universelle Protein-Protein Wechselwirkungsmodule darstellen.AAA proteins are part of the large superfamily of AAA+ proteins, which are ringshaped P loop NTPases, whose common function is unfolding macromolecules in an energy-dependant manner. AAA proteins usually consist of an N-terminal domain, and one or two ATPase domains named D1 and D2. ATPase domains are relatively conserved within the family of AAA proteins and they are also thought to mediate hexamerization. N-terminal domains are important for substrate recognition and binding and, in contrast to the ATPase domains, they vary in their folds. Based on published data and additional bioinformatic analysis of AAA proteins, we selected several different N-terminal domains from archaeal AAA proteins for functional and structural characterization. We also characterized proteins which share similar or related domains to the ones found in AAA proteins, making an important link between them. Heat and chemical aggregation assays of different substrate proteins were used to assay N-terminal domains, or full AAA proteins, for intrinsic chaperone activity. Protein structures were determined by crystallography or NMR spectroscopy. Results of this study indicate that the barrel-like N-terminal domains of AAA proteins originated from the similar nucleic acid binding domains. A change in the affinity for substrate, from nucleic acid to protein, may have occurred through different mechanisms in the evolution. In the case of double-psi barrels this has probably happened through the evolution of a simple beta-alpha-beta-motif found also in the RIFT and swapped hairpin barrels which are transcription factors, i.e. DNA binders. Structures of Mj0056 and SpoVT indicate that RIFT and swapped hairpin barrels have evolved further either by insertion of different structure elements (Mj0056) or by domain recruitment (SpoVT). Similarity between the PAN and ARC N-domains was found to be both in structure and function. Both domains comprise a coiled-coil followed by one or two OB folds. OB stands for oligosaccharide binding and indicates that also this domain originated from a DNA-binding fold. Structure of the ARC-Nc subdomain and a comprehensive analysis of chimeric constructs of the coiled coils and OB folds indicate that ARC and PAN N-domains have arisen through evolution by domain recruitment. Strict structural composition of the subdomains important in the chaperone function is maintained through the conserved PP-linker connecting the two subdomains. Structural and functional characterization of the AfAMA, a member of the novel group of AMA AAA proteins, showed that substrate binding function and chaperone activity of these proteins resides in its beta-clam like N-terminal domain. This domain can fulfill these functions independently, in contrast to the other homologous domains. We were able to show the importance of oligomerization for activity of these domains and that oligomerization is mediated by a small GYPL-motif found in a loop that presumably projects to the center of the hexamer. The N-terminal domain of AMA mediates hexamerization of the full protein independently from the AAA part of the protein and ATP utilization, which differs largely from other families of AAA proteins. Functional data on other beta-clam domains: VAT-Nc, Ph1500N and Hm-clam would indicate that these domains represent universal protein-protein interaction modules

Funktionelle Charakterisierung von Aquaporinen aus Plasmodium falciparum, Toxoplasma gondii und Trypanosoma brucei

P. falciparum, T. gondii and T. brucei parasites cause infectious diseases that affect more than 300-400 million people world-wide and, thus, represent a major health problem. The rapid spreading of drug resistant parasite strains initiated the search for new drug targets. Aquaporins at the parasite host interface have the potential to be used as a novel targets. I characterised biochemically a single T. gondii aquaporin (TgAQP) which has only 28% sequence similarity to the P. falciparum aquaporin (PfAQP) and 47% sequence similarity to water specific plant aquaporins (TIP 1;1) and three T. brucei aquaporins (TbAQP1-3) which show 40-45% similarity to mammalian AQP3. The pore constriction region of TgAQP has a valine instead of a highly conserved arginine. This exchange is rather typical for above-mentioned plant aquaporins. Thus, it supports the hypothesis that apicomplexans have plant ancestors. TbAQP1 and 3 have a similar amino acid composition in the pore constriction as AQP3. TbAQP2, however, has a leucine instead of the pore arginine, and NSA and NPS motifs instead of the canonical NPA motifs. I established that TgAQP and the TbAQPs are bifunctional pores with high glycerol and intermediate water permeability whereas in PfAQP both, water and glycerol permeability are high. Further testing of TgAQP permeability showed that besides urea, which passes the pore equally well as glycerol, only erythritol and D-arabitol reasonably permeate TgAQP. Other larger and/or charged compounds do not pass. Strikingly, hydroxyurea, an anti-neoplastic agent with an inhibitory effect on parasite proliferation, could be identified as a permeant of TgAQP. Solute permeability TbAQPs is more restricted. Only TbAQP3 passed erythrytol and ribitol well. TbAQP2 showed permeability for pyruvate. All TbAQPs passed urea. The TbAQPs and PfAQP were further highly permeable for dihydroxyacetone (DHA). I could show that DHA is toxic for plasmodia parasites (IC50 2.5 mM). PfAQP was also tested for methylglyoxal (MG) permeability. MG is a side product of glycolysis and has a similar structure as DHA. MG was conducted by PfAQP and showed a toxic effect on malaria parasites (IC50 200 µM). P. falciparum glyceraldehydes phosphate dehydrogenase (PfGAPDH) was fully inhibited by 2.5 mM DHA after 6 h whereas MG did not have an inhibitory effect. Rabbit GAPDH as a control was fully inhibited by DHA after 3 h and by MG after 20h. In addition, PfAQP is permeable for ammonia. I showed that one liter of packed plasmodia red cells produces 8.8 mmol of ammonia of ammonia per hour. PfAQP may thus be vital for malaria parasites as a pathway for ammonia release.P. falciparum, T. gondii und T brucei Parasiten verursachen Infektionskrankheiten, die mehr als 300-400 Millionen Menschen auf der Welt betreffen und stellen ein großes Gesundheitsproblem dar. Die schnelle Ausbreitung von resistenten Stämmen löste die Suche nach neuen Angriffspunkten für Medikamente aus. Aquaporine im Membransystem zwischen Parasit und Wirt könnten als neue Angriffspunkte für Medikamente genutzt werden. In der vorliegenden Arbeit wurden ein Aquaporin aus T. gondii (TgAQP) sowie drei Aquaporine aus T. brucei (TbAQP1-3) biochemisch charakterisiert. TgAQP zeigt 28% Sequenzähnhichkeit zu dem Auqaglyceroporin aus P. falciparum (PfAQP) und 47% Sequenzähnhichkeit zu wasserspezifischen Pflanzenaquaporinen (TIP 1;1). TbAQP1-3 weisen 40-45% Ähnhichkeit zu AQP3 aus Säugern auf. In der Porenregion von TgAQP, befindet sich ein Valin anstelle eines hochkonservierten Arginins. Dieser Aminosäurenaustausch ist typisch für die TIPs der Pflanzen und stützt die Hypothese, dass Apicomplexa pflanzliche Vorfahren haben. TbAQP1 und 3 haben eine ähnliche Aminosäurenzusammensetzung der Porenregion wie AQP3 und NPA Motive in der Region, die den Porenengpass bildet. TbAQP2 bildet eine Ausnahme, da es ein Leucin anstelle des Arginins hat und NSA und NPS Motive aufweist. Es wurde gezeigt, dass TgAQP und TbAQPs bifunktionale Poren sind, die durch eine hohe Permeabilität für Glycerin und eine mittlere Wasserpermeabilität gekennzeichnet sind. PfAQP hingegen hat eine hohe Permeabilität für sowohl Glycerin als auch Wasser. Die weitere Untersuchung der Permeabilität von TgAQP ergab, dass neben Harnstoff, der die Pore ebenso gut wie Wasser passiert, auch Erythritol und D-Arabitol eine gewisse Durchgangigkeit besitzen. Fur andere Verbindungen, die groser und/oder geladen sind, ist TgAQP nicht durchlässig. Interessanterweise kann Hydroxyharnstoff, ein anti-neoplastischer Wirkstoff, der die Parasitenproliferation inhibiert, durch die Pore dringen. Die Solutleitfähigkeit der TbAQPe ist eingeschränkt. Nur TbAQP3 lässt Erythritol und Ribitol pasieren. TbAQP2 hat eine geringe Permeabilität fur Pyruvat. Alle TbAQPe sind permeabel fur Harnstoff. Sowohl die TbAQPs als auch PfAQP haben eine hohe Durchlässigkeit fur Dihydroxyaceton (DHA). Es wurde nachgewiesen, dass DHA fur die Plasmodienparasiten toxisch ist (IC50 2,5 mM). PfAQP wurde positiv auf Permeabilität von Methylglyoxal (MG) getestet. MG ist ein Nebenprodukt der Glykolyse und hat eine ähnliche Struktur wie DHA. Das von PfAQP eingeschleuste DHA wirkte toxisch auf die Malariaparasiten (IC50 200 µM). Des weiterem wurde die Auswirkung von 2,5 mM DHA bzw. MG auf P. falciparum Glycerolaldehyd-Dehydrogenase (PfGAPDH) untersucht. PfGAPDH wird nach 6 h vollständig von DHA inhibiert, wohingegen MG keinen inhibitorischen Effekt zeigt. Als Kontrolle wurde GAPDH aus dem Kaninchen verwendet, das von DHA nach 3 h und von MG nach 20 h vollständig inhibiert wird. PfAQP ist auch durchlässig für Ammoniak. Es wurde gezeigt, dass ein Liter gepackter malariainfizierter Erythrozyten 8,8 mmol Ammoniak pro Stunde produzieren. PfAQP konnte demnach überlebenswichtig fur Plasmodien sein und als Anlass für Ammoniak fungieren

Parallelism and Epistasis in Skeletal Evolution Identified through Use of Phylogenomic Mapping Strategies

The identification of genetic mechanisms underlying evolutionary change is critical to our understanding of natural diversity, but is presently limited by the lack of genetic and genomic resources for most species. Here, we present a new comparative genomic approach that can be applied to a broad taxonomic sampling of nonmodel species to investigate the genetic basis of evolutionary change. Using our analysis pipeline, we show that duplication and divergence of fgfr1a is correlated with the reduction of scales within fishes of the genus Phoxinellus. As a parallel genetic mechanism is observed in scale-reduction within independent lineages of cypriniforms, our finding exposes significant developmental constraint guiding morphological evolution. In addition, we identified fixed variation in fgf20a within Phoxinellus and demonstrated that combinatorial loss-of-function of fgfr1a and fgf20a within zebrafish phenocopies the evolved scalation pattern. Together, these findings reveal epistatic interactions between fgfr1a and fgf20a as a developmental mechanism regulating skeletal variation among fishes

Dihydroxyacetone and methylglyoxal as permeants of the Plasmodium aquaglyceroporin inhibit parasite proliferation

AbstractThe aquaglyceroporin of Plasmodium falciparum (PfAQP) is a bi-functional channel with permeability for water and solutes. Its functions supposedly are in osmotic protection of parasites and in facilitation of glycerol permeation for glycerolipid biosynthesis. Here, we show PfAQP permeability for the glycolysis-related metabolites methylglyoxal, a cytotoxic byproduct, and dihydroxyacetone, a ketotriose. AQP3, the red cell aquaglyceroporin, also passed dihydroxacetone but excluded methylglyoxal. Proliferation of malaria parasites was inhibited by methylglyoxal with an IC50 around 200 μM. Surprisingly, also dihydroxyacetone, which is an energy source in human cells, was antiproliferative in chloroquine-sensitive and resistant strains with an IC50 around 3 mM. We expressed P. falciparum glyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) to examine whether it is inhibited by either carbonyl compound. Methylglyoxal did not affect PfGAPDH on incubation with 2.5 mM for 20 h. Treatment with 2.5 mM dihydroxyacetone, however, abolished PfGAPDH activity within 6 h. Aquaglyceroporin permeability for glycolytic metabolites may thus be of physiological significance