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.

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.

The genome of Tetranychus urticae reveals herbivorous pest adaptations.

The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant-herbivore interactions, and provides unique opportunities for developing novel plant protection strategies.

Structure and activity of the N-terminal substrate recognition domains in proteasomal ATPases.

The proteasome forms the core of the protein quality control system in archaea and eukaryotes and also occurs in one bacterial lineage, the Actinobacteria. Access to its proteolytic compartment is controlled by AAA ATPases, whose N-terminal domains (N domains) are thought to mediate substrate recognition. The N domains of an archaeal proteasomal ATPase, Archaeoglobus fulgidus PAN, and of its actinobacterial homolog, Rhodococcus erythropolis ARC, form hexameric rings, whose subunits consist of an N-terminal coiled coil and a C-terminal OB domain. In ARC-N, the OB domains are duplicated and form separate rings. PAN-N and ARC-N can act as chaperones, preventing the aggregation of heterologous proteins in vitro, and this activity is preserved in various chimeras, even when these include coiled coils and OB domains from unrelated proteins. The structures suggest a molecular mechanism for substrate processing based on concerted radial motions of the coiled coils relative to the OB rings.

Regulation of Argonaute slicer activity by guide RNA 3' end interactions with the N-terminal lobe.

Structural studies indicate that binding of both the guide RNA (siRNA and miRNA) and the target mRNA trigger substantial conformational changes in the Argonaute proteins. Here we explore the role of the N-terminal lobe (and its PAZ domain) in these conformational changes using biochemical and cell culture-based approaches. In vitro, whereas deletion (or mutation) of the N-terminal lobe of DmAgo1 and DmAgo2 had no effect on binding affinity to guide RNAs, we observed a loss of protection of the 3' end of the guide RNA and decreased target RNA binding; consistent with this, in cells, loss of function DmAgo1 PAZ variant proteins (PAZ6 and ΔN-PAZ) still bind RNA, although the RNAs are shorter than normal. We also find that deletion of the N-terminal lobe results in constitutive activation of endogenous PIWI domain-based cleavage activity in vitro, providing insights into how cleavage activity may be regulated in vivo in response to different types of pairing interactions with the target mRNAs.

A Massively Parallel Screen of 5'UTR Mutations Identifies Variants Impacting Translation and Protein Production in Neurodevelopmental Disorder Genes.

De novo mutations cause a variety of neurodevelopmental disorders including autism. Recent whole genome sequencing from individuals with autism has shown that many de novo mutations also occur in untranslated regions (UTRs) of genes, but it is difficult to predict from sequence alone which mutations are functional, let alone causal. Therefore, we developed a high throughput assay to screen the transcriptional and translational effects of 997 variants from 5'UTR patient mutations. This assay successfully enriched for elements that alter reporter translation, identifying over 100 potentially functional mutations from probands. Studies in patient-derived cell lines further confirmed that these mutations can alter protein production in individuals with autism, and some variants fall in genes known to cause syndromic forms of autism, suggesting a diagnosis for these individual patients. Since UTR function varies by cell type, we further optimized this high throughput assay to enable assessment of mutations in neurons in vivo. First, comparing in cellulo to in vivo results, we demonstrate neurons have different principles of regulation by 5'UTRs, consistent with a more robust mechanism for reducing the impact of RNA secondary structure. Finally, we discovered patient mutations specifically altering the translational activity of additional known syndromic genes LRRC4 and ZNF644 in neurons of the brain. Overall our results highlight a new approach for assessing the impact of 5'UTR mutations across cell types and suggest that some cases of neurodevelopmental disorder may be caused by such variants.

Poly-basic peptides and polymers as new drug candidate against Plasmodium falciparum.

Plasmodium falciparum, the malaria-causing parasite, is a leading cause of infection-induced deaths worldwide. The preferred treatment approach is artemisinin-combination therapy, which couples fast-acting artemisinin derivatives with longer-acting drugs like lumefantrine, mefloquine, and amodiaquine. However, the urgency for new treatments has risen due to the parasite's growing resistance to existing therapies. Our study shows that a common characteristic of the P. falciparum proteome - stretches of poly-lysine residues such as those found in proteins related to adhesion and pathogenicity - can serve as an effective peptide treatment for infected erythrocytes. A single dose of these poly-basic peptides can successfully diminish parasitemia in human erythrocytes in vitro with minimal toxicity. The effectiveness of the treatment correlates with the length of the poly-lysine peptide, with 30 lysine peptides supporting the eradication of erythrocytic parasites within 72 hours. PEG-ylation of the poly-lysine peptides or utilizing poly-lysine dendrimers and polymers further increases parasite clearance efficiency and bolsters the stability of these potential new therapeutics. Lastly, our affinity pull-downs and mass-spectrometry identify P. falciparum's outer membrane proteins as likely targets for polybasic peptide medications. Since poly-lysine dendrimers are already FDA-approved for drug delivery, their adaptation as antimalarial drugs presents a promising new therapeutic strategy.

Excretory/Secretory Proteome of Females and Males of the Hookworm Ancylostoma ceylanicum.

The dynamic host-parasite mechanisms underlying hookworm infection establishment and maintenance in mammalian hosts remain poorly understood but are primarily mediated by hookworm's excretory/secretory products (ESPs), which have a wide spectrum of biological functions. We used ultra-high performance mass spectrometry to comprehensively profile and compare female and male ESPs from the zoonotic human hookworm Ancylostoma ceylanicum, which is a natural parasite of dogs, cats, and humans. We improved the genome annotation, decreasing the number of protein-coding genes by 49% while improving completeness from 92 to 96%. Compared to the previous genome annotation, we detected 11% and 10% more spectra in female and male ESPs, respectively, using this improved version, identifying a total of 795 ESPs (70% in both sexes, with the remaining sex-specific). Using functional databases (KEGG, GO and Interpro), common and sex-specific enriched functions were identified. Comparisons with the exclusively human-infective hookworm Necator americanus identified species-specific and conserved ESPs. This is the first study identifying ESPs from female and male A. ceylanicum. The findings provide a deeper understanding of hookworm protein functions that assure long-term host survival and facilitate future engineering of transgenic hookworms and analysis of regulatory elements mediating the high-level expression of ESPs. Furthermore, the findings expand the list of potential vaccine and diagnostic targets and identify biologics that can be explored for anti-inflammatory potential.

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 various trans-acting factors that bind to specific sequences in the messenger RNA (mRNA). The binding of different trans factors influences various aspects of the mRNA such as degradation rate, translation efficiency, splicing, localization, etc. MicroRNAs (miRNAs) are short endogenous ncRNAs that combine with the Argonaute to form the microRNA-induced silencing complex (miRISC), which uses base-pair complementation to silence the target transcript. RNA-binding proteins (RBPs) contribute to post-transcriptional control by influencing the mRNA stability and translation upon binding to cis-elements within the mRNA transcript. RBPs have been shown to impact gene expression through influencing the miRISC biogenesis, composition, or miRISC-mRNA target interaction. While there is clear evidence that those interactions between RBPs, miRNAs, miRISC and target mRNAs influence the efficiency of miRISC-mediated gene silencing, the exact mechanism for most of them remains unclear. This review summarizes our current knowledge on gene expression regulation through interactions of miRNAs and RBPs.

N1-methylpseudouridine found within COVID-19 mRNA vaccines produces faithful protein products.

Synthetic mRNA technology is a promising avenue for treating and preventing disease. Key to the technology is the incorporation of modified nucleotides such as N1-methylpseudouridine (m1Ψ) to decrease immunogenicity of the RNA. However, relatively few studies have addressed the effects of modified nucleotides on the decoding process. Here, we investigate the effect of m1Ψ and the related modification pseudouridine (Ψ) on translation. In a reconstituted system, we find that m1Ψ does not significantly alter decoding accuracy. More importantly, we do not detect an increase in miscoded peptides when mRNA containing m1Ψ is translated in cell culture, compared with unmodified mRNA. We also find that m1Ψ does not stabilize mismatched RNA-duplex formation and only marginally promotes errors during reverse transcription. Overall, our results suggest that m1Ψ does not significantly impact translational fidelity, a welcome sign for future RNA therapeutics.

Gene dosage effects of poly(A) track-engineered hypomorphs.

Manipulation of gene activity through creation of hypomorphic mutants has been a long-standing tool in examining gene function. Our previous studies have indicated that hypomorphic mutants could be created by inserting cis-regulatory sequences composed of consecutive adenosine nucleotides called poly(A) tracks. Here we use poly(A) tracks to create hypomorphic mutants and functional characterization of membrane, secretory, and endogenous proteins. Insertion of poly(A) tracks into the sequences of interleukin-2 and membrane protein CD20 results in a programmable reduction of mRNA stability and attenuation of protein expression regardless of the presence of a signaling sequence. Likewise, CRISPR-Cas9 targeted insertion of poly(A) tracks into the coding sequence of the endogenous human genes AUF1 and TP53 results in a programmable reduction of targeted protein and mRNA levels. Functional analyses of AUF1-engineered hypomorphs indicate a direct correlation between AUF1 gene levels and the stability of AUF1-regulated mRNAs. Hypomorphs of TP53 affect expression of the target genes differentially depending on the severity of the hypomorphic mutation. Finally, decreases in TP53 protein affect the same cellular pathways in poly(A) track-engineered cells as in cancer cells, indicating these variants' biological relevance. These results highlight this technology's power to create predictable, stable hypomorphs in recombinant or endogenous genes in combination with CRISPR-Cas9 engineering tools.

Optimized measurement temperature gives access to the solution structure of a 49 kDa homohexameric ß-propeller.

Ph1500 is a homohexameric, two-domain protein of unknown function from the hyperthermophilic archaeon Pyrococcus horikoshii. The C-terminal hexamerization domain (Ph1500C) is of particular interest, as it lacks sequence homology to proteins of known structure. However, it resisted crystallization for X-ray analysis, and proteins of this size (49 kDa) present a considerable challenge to NMR structure determination in solution. We solved the high-resolution structure of Ph1500C, exploiting the hyperthermophilic nature of the protein to minimize unfavorable relaxation properties by high-temperature measurement. Thus, the side chain assignment (97%) and structure determination became possible at full proton density. To our knowledge, Ph1500C is the largest protein for which this has been achieved. To minimize detrimental fast water exchange of amide protons at increased temperature, we employed a strategy where the temperature was optimized separately for backbone and side chain experiments.

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.

A CTP-dependent archaeal riboflavin kinase forms a bridge in the evolution of cradle-loop barrels.

Proteins of the cradle-loop barrel metafold are formed by duplication of a conserved betaalphabeta-element, suggesting a common evolutionary origin from an ancestral group of nucleic acid-binding proteins. The basal fold within this metafold, the RIFT barrel, is also found in a wide range of enzymes, whose homologous relationship with the nucleic acid-binding group is unclear. We have characterized a protein family that is intermediate in sequence and structure between the basal group of cradle-loop barrels and one family of RIFT-barrel enzymes, the riboflavin kinases. We report the structure, substrate-binding mode, and catalytic activity for one of these proteins, Methanocaldococcus jannaschii Mj0056, which is an archaeal riboflavin kinase. Mj0056 is unusual in utilizing CTP rather than ATP as the donor nucleotide, and sequence conservation in the relevant residues suggests that this is a general feature of archaeal riboflavin kinases.

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 genus Plasmodium, which includes the major human parasite Plasmodium falciparum. The complex cycle of the malaria parasite in both mosquito and human hosts has been studied extensively. There is tight control of gene expression in each developmental stage, and at every level of gene synthesis: from RNA transcription, to its subsequent translation, and finally post-translational modifications of the resulting protein. Whole-genome sequencing of P. falciparum has laid the foundation for significant biological advances by revealing surprising genomic information. The P. falciparum genome is extremely AT-rich (∼80%), with a substantial portion of genes encoding intragenic polyadenosine (polyA) tracks being expressed throughout the entire parasite life cycle. In most eukaryotes, intragenic polyA runs act as negative regulators of gene expression. Recent studies have shown that translation of mRNAs containing 12 or more consecutive adenosines results in ribosomal stalling and frameshifting; activating mRNA surveillance mechanisms. In contrast, P. falciparum translational machinery can efficiently and accurately translate polyA tracks without activating mRNA surveillance pathways. This unique feature of P. falciparum raises interesting questions: (1) How is P. falciparum able to efficiently and correctly translate polyA track transcripts, and (2) What are the specifics of the translational machinery and mRNA surveillance mechanisms that separate P. falciparum from other organisms? In this review, we analyze possible evolutionary shifts in P. falciparum protein synthesis machinery that allow efficient translation of an AU rich-transcriptome. We focus on physiological and structural differences of P. falciparum stage specific ribosomes, ribosome-associated proteins, and changes in mRNA surveillance mechanisms throughout the complete parasite life cycle, with an emphasis on the mosquito and liver stages.

A short translational ramp determines the efficiency of protein synthesis.

Translation initiation is a major rate-limiting step for protein synthesis. However, recent studies strongly suggest that the efficiency of protein synthesis is additionally regulated by multiple factors that impact the elongation phase. To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays. Here we report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally, introduction of preferred sequence motifs only at specific codon positions improves protein synthesis efficiency for recombinant proteins. Collectively, our data underscore the critical role of early elongation events in translational control of gene expression.

prlF and yhaV encode a new toxin-antitoxin system in Escherichia coli.

Toxin-antitoxin systems consist of a stable toxin, frequently with endonuclease activity, and a small, labile antitoxin, which sequesters the toxin into an inactive complex. Under unfavorable conditions, the antitoxin is degraded, leading to activation of the toxin and resulting in growth arrest, possibly also in bacterial programmed cell death. Correspondingly, these systems are generally viewed as agents of the stress response in prokaryotes. Here we show that prlF and yhaV encode a novel toxin-antitoxin system in Escherichia coli. YhaV, a ribonuclease of the RelE superfamily, causes reversible bacteriostasis that is counteracted by PrlF, a swapped-hairpin transcription factor homologous to MazE. The two proteins form a tight, hexameric complex, which binds with high specificity to a conserved sequence in the promoter region of the prlF-yhaV operon. As homologs of MazE and RelE, respectively, PrlF and YhaV provide an evolutionary connection between the two best-characterized toxin-antitoxin systems in E. coli, mazEF and relEB.

Common evolutionary origin of swapped-hairpin and double-psi beta barrels.

The core of swapped-hairpin and double-psi beta barrels is formed by duplication of a conserved betaalphabeta element, suggesting a common evolutionary origin. The path connecting the two folds is unclear as the two barrels are not interconvertible by a simple topological modification, such as circular permutation. We have identified a protein family whose sequence properties are intermediate to the two folds. The structure of one of these proteins, Pyrococcus horikoshii PhS018, is also built by duplication of the conserved betaalphabeta element but shows yet a third topology, which we name the RIFT barrel. This topology is widespread in the structure database and spans three folds of the SCOP classification, including the middle domain of EF-Tu and the N domain of F1-ATPase. We propose that swapped-hairpin beta barrels arose from an ancestral RIFT barrel by strand invasion and double-psi beta barrels by a strand swap. We group the three barrel types into a metafold, the cradle-loop barrels.

PolyA tracks, polybasic peptides, poly-translational hurdles.

The abundance of messenger RNA (mRNA) is one of the major determinants of protein synthesis. As such, factors that influence mRNA stability often contribute to gene regulation. Polyadenylation of the 3' end of mRNA transcripts, the poly(A) tail, has long been recognized as one of these regulatory elements given its influence on translation efficiency and mRNA stability. Unwanted translation of the poly(A) tail signals to the cell an aberrant polyadenylation event or the lack of stop codons, which makes this sequence an important element in translation fidelity and mRNA surveillance response. Consequently, investigations into the effects of the poly(A) tail lead to the discoveries that poly-lysine as well as other polybasic peptide sequences and, to a much greater extent, polyA mRNA sequences within the open reading frame influence mRNA stability and translational efficiency. Conservation and evolutionary selection of codon usage in polyA track sequences across multiple organisms suggests a biological significance for coding polyA tracks in the regulation of gene expression. Here, we discuss the cellular responses and consequences of coding polyA track translation and synthesis of polybasic peptides. This article is categorized under: Translation > Translation Mechanisms Translation > Translation Regulation RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms.

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

RNA binding proteins (RBP) and microRNAs (miRNAs) often bind sequences in 3' untranslated regions (UTRs) of mRNAs, and regulate stability and translation efficiency. With the identification of numerous RBPs and miRNAs, there is an urgent need for new technologies to dissect the function of the cis-acting elements of RBPs and miRNAs. We describe post-transcriptional regulatory element sequencing (PTRE-seq), a massively parallel method for assaying the target sequences of miRNAs and RBPs. We use PTRE-seq to dissect sequence preferences and interactions between miRNAs and RBPs. The binding sites for these effector molecules influenced different aspects of the RNA lifecycle: RNA stability, translation efficiency, and translation initiation. In some cases, post-transcriptional control is modular, with different factors acting independently of each other, while in other cases factors show specific epistatic interactions. The throughput, flexibility, and reproducibility of PTRE-seq make it a valuable tool to study post-transcriptional regulation by 3'UTR elements.