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

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

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

Ribosomes slide on lysine-encoding homopolymeric A stretches.

Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation. DOI: http://dx.doi.org/10.7554/eLife.05534.00

Plasmodium falciparum translational machinery condones polyadenosine repeats

Plasmodium falciparum is a causative agent of human malaria. Sixty percent of mRNAs from its extremely AT-rich (81%) genome harbor long polyadenosine (polyA) runs within their ORFs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate polyA runs cause ribosome stalling and frameshifting, triggering mRNA surveillance pathways and attenuating protein synthesis. Here, we show that P. falciparum is an exception to this rule. We demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately translated in P. falciparum cells. We show that polyA runs do not elicit any response from No Go Decay (NGD) or result in the production of frameshifted proteins. This is in stark contrast to what we observe in human cells or T. thermophila, an organism with similar AT-content. Finally, using stalling reporters we show that Plasmodium cells evolved not to have a fully functional NGD pathway