Discovery of two distinct aminoacyl-tRNA synthetase complexes anchored to the Plasmodium surface tRNA import protein.

Aminoacyl-tRNA synthetases (aaRSs) attach amino acids to their cognate transfer RNAs. In eukaryotes, a subset of cytosolic aaRSs is organized into a multisynthetase complex (MSC), along with specialized scaffolding proteins referred to as aaRS-interacting multifunctional proteins (AIMPs). In Plasmodium, the causative agent of malaria, the tRNA import protein (tRip), is a membrane protein that participates in tRNA trafficking; we show that tRip also functions as an AIMP. We identified three aaRSs, the glutamyl-tRNA synthetase (ERS), glutaminyl-tRNA synthetase (QRS), and methionyl-tRNA synthetase (MRS), which were specifically coimmunoprecipitated with tRip in Plasmodium berghei blood stage parasites. All four proteins contain an N-terminal glutathione-S-transferase (GST)-like domain that was demonstrated to be involved in MSC assembly. In contrast to previous studies, further dissection of GST-like interactions identified two exclusive heterotrimeric complexes: the Q-complex (tRip-ERS-QRS) and the M-complex (tRip-ERS-MRS). Gel filtration and light scattering suggest a 2:2:2 stoichiometry for both complexes but with distinct biophysical properties and mutational analysis further revealed that the GST-like domains of QRS and MRS use different strategies to bind ERS. Taken together, our results demonstrate that neither the singular homodimerization of tRip nor its localization in the parasite plasma membrane prevents the formation of MSCs in Plasmodium. Besides, the extracellular localization of the tRNA-binding module of tRip is compensated by the presence of additional tRNA-binding modules fused to MRS and QRS, providing each MSC with two spatially distinct functions: aminoacylation of intraparasitic tRNAs and binding of extracellular tRNAs. This unique host-pathogen interaction is discussed.

Elaborate uORF/IRES features control expression and localization of human glycyl-tRNA synthetase.

The canonical activity of glycyl-tRNA synthetase (GARS) is to charge glycine onto its cognate tRNAs. However, outside translation, GARS also participates in many other functions. A single gene encodes both the cytosolic and mitochondrial forms of GARS but 2 mRNA isoforms were identified. Using immunolocalization assays, in vitro translation assays and bicistronic constructs we provide experimental evidence that one of these mRNAs tightly controls expression and localization of human GARS. An intricate regulatory domain was found in its 5'-UTR which displays a functional Internal Ribosome Entry Site and an upstream Open Reading Frame. Together, these elements hinder the synthesis of the mitochondrial GARS and target the translation of the cytosolic enzyme to ER-bound ribosomes. This finding reveals a complex picture of GARS translation and localization in mammals. In this context, we discuss how human GARS expression could influence its moonlighting activities and its involvement in diseases.

Misfolded human tRNA isodecoder binds and neutralizes a 3' UTR-embedded Alu element.

Several classes of small noncoding RNAs are key players in cellular metabolism including mRNA decoding, RNA processing, and mRNA stability. Here we show that a tRNA(Asp) isodecoder, corresponding to a human tRNA-derived sequence, binds to an embedded Alu RNA element contained in the 3' UTR of the human aspartyl-tRNA synthetase mRNA. This interaction between two well-known classes of RNA molecules, tRNA and Alu RNA, is driven by an unexpected structural motif and induces a global rearrangement of the 3' UTR. Besides, this 3' UTR contains two functional polyadenylation signals. We propose a model where the tRNA/Alu interaction would modulate the accessibility of the two alternative polyadenylation sites and regulate the stability of the mRNA. This unique regulation mechanism would link gene expression to RNA polymerase III transcription and may have implications in a primate-specific signal pathway.

RNA aptamers developed against tRip: A preliminary approach targeting tRNA entry in Plasmodium.

Malaria is caused by Plasmodium parasites that multiply inside host cells and can be lethal when P. falciparum is involved. We identified tRip as a membrane protein that facilitates the import of exogenous transfer RNA (tRNA) into the parasite. tRip encompasses a tRNA binding domain exposed on the parasite surface. We used the SELEX approach to isolate high-affinity and specific tRip-binding RNA motifs from a library of random 25 nucleotide-long sequences. In five rounds of combined negative and positive selections, an enriched pool of aptamers was obtained; sequencing revealed that they were all different in their primary sequence; only by comparing their structure predictions did most of the selected aptamers reveal a conserved 5-nucleotide motif sequence. We showed that the integral motif is essential for tRip-binding while the rest of the molecule can be significantly reduced or mutated as long as the motif is presented in a single-stranded region. Such RNA aptamers bind in place of the original tRNA substrate and act as an efficient competitor, suggesting that they can block tRip function and slow parasite development.

Solution X-ray scattering highlights discrepancies in Plasmodium multi-aminoacyl-tRNA synthetase complexes.

tRip is a tRNA import protein specific to Plasmodium, the causative agent of malaria. In addition to its membrane localization and tRNA trafficking properties, tRip has the capacity to associate with three aminoacyl-tRNA synthetases (aaRS), the glutamyl- (ERS), glutaminyl- (QRS), and methionyl- (MRS) tRNA synthetases. In eukaryotes, such multi-aaRSs complexes (MSC) regulate the moonlighting activities of aaRSs. In Plasmodium, tRip and the three aaRSs all contain an N-terminal GST-like domain involved in the assembly of two independent complexes: the Q-complex (tRip:ERS:QRS) and the M-complex (tRip:ERS:MRS) with a 2:2:2 stoichiometry and in which the association of the GST-like domains of tRip and ERS (tRip-N:ERS-N) is central. In this study, the crystal structure of the N-terminal GST-like domain of ERS was solved and made possible further investigation of the solution architecture of the Q- and M-complexes by small-angle x-ray scattering (SAXS). This strategy relied on the engineering of a tRip-N-ERS-N chimeric protein to study the structural scaffold of both Plasmodium MSCs and confirm the unique homodimerization pattern of tRip in solution. The biological impact of these structural arrangements is discussed.

Spatio-temporal based deep learning for rapid detection and identification of bacterial colonies through lens-free microscopy time-lapses.

Detection and identification of pathogenic bacteria isolated from biological samples (blood, urine, sputum, etc.) are crucial steps in accelerated clinical diagnosis. However, accurate and rapid identification remain difficult to achieve due to the challenge of having to analyse complex and large samples. Current solutions (mass spectrometry, automated biochemical testing, etc.) propose a trade-off between time and accuracy, achieving satisfactory results at the expense of time-consuming processes, which can also be intrusive, destructive and costly. Moreover, those techniques tend to require an overnight subculture on solid agar medium delaying bacteria identification by 12-48 hours, thus preventing rapid prescription of appropriate treatment as it hinders antibiotic susceptibility testing. In this study, lens-free imaging is presented as a possible solution to achieve a quick and accurate wide range, non-destructive, label-free pathogenic bacteria detection and identification in real-time using micro colonies (10-500 µm) kinetic growth pattern combined with a two-stage deep learning architecture. Bacterial colonies growth time-lapses were acquired thanks to a live-cell lens-free imaging system and a thin-layer agar media made of 20 µl BHI (Brain Heart Infusion) to train our deep learning networks. Our architecture proposal achieved interesting results on a dataset constituted of seven different pathogenic bacteria-Staphylococcus aureus (S. aureus), Enterococcus faecium (E. faecium), Enterococcus faecalis (E. faecalis), Staphylococcus epidermidis (S. epidermidis), Streptococcus pneumoniae R6 (S. pneumoniae), Streptococcus pyogenes (S. pyogenes), Lactococcus Lactis (L. Lactis). At T = 8h, our detection network reached an average 96.0% detection rate while our classification network precision and sensitivity averaged around 93.1% and 94.0% respectively, both were tested on 1908 colonies. Our classification network even obtained a perfect score for E. faecalis (60 colonies) and very high score for S. epidermidis at 99.7% (647 colonies). Our method achieved those results thanks to a novel technique coupling convolutional and recurrent neural networks together to extract spatio-temporal patterns from unreconstructed lens-free microscopy time-lapses.

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip.

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

Structural Analysis of RNA by Small-Angle X-ray Scattering.

Over the past two decades small-angle X-ray scattering (SAXS) has become a popular method to characterize solutions of biomolecules including ribonucleic acid (RNA). In an integrative structural approach, SAXS is complementary to crystallography, NMR, and electron microscopy and provides information about RNA architecture and dynamics. This chapter highlights the practical advantages of combining size-exclusion chromatography and SAXS at synchrotron facilities. It is illustrated by practical case studies of samples ranging from single hairpins and tRNA to a large IRES. The emphasis is also put on sample preparation which is a critical step of SAXS analysis and on optimized protocols for in vitro RNA synthesis ensuring the production of mg amount of pure and homogeneous molecules.

Deinococcus glutaminyl-tRNA synthetase is a chimer between proteins from an ancient and the modern pathways of aminoacyl-tRNA formation.

Glutaminyl-tRNA synthetase from Deinococcus radiodurans possesses a C-terminal extension of 215 residues appending the anticodon-binding domain. This domain constitutes a paralog of the Yqey protein present in various organisms and part of it is present in the C-terminal end of the GatB subunit of GatCAB, a partner of the indirect pathway of Gln-tRNA(Gln) formation. To analyze the peculiarities of the structure-function relationship of this GlnRS related to the Yqey domain, a structure of the protein was solved from crystals diffracting at 2.3 A and a docking model of the synthetase complexed to tRNA(Gln) constructed. The comparison of the modeled complex with the structure of the E. coli complex reveals that all residues of E. coli GlnRS contacting tRNA(Gln) are conserved in D. radiodurans GlnRS, leaving the functional role of the Yqey domain puzzling. Kinetic investigations and tRNA-binding experiments of full length and Yqey-truncated GlnRSs reveal that the Yqey domain is involved in tRNA(Gln) recognition. They demonstrate that Yqey plays the role of an affinity-enhancer of GlnRS for tRNA(Gln) acting only in cis. However, the presence of Yqey in free state in organisms lacking GlnRS, suggests that this domain may exert additional cellular functions.

Post-translational modifications guard yeast from misaspartylation.

Yeast aspartyl-tRNA synthetase (AspRS) is downregulated at the post-transcriptional level. This complex retro-inhibition mechanism causes the cell to equilibrate cellular concentrations of tRNAAsp, AspRS, and its encoding mRNA. This strategy hinders AspRS accumulation to keep misacylation of heterologous tRNAs under control. Here, the AspRS concentration was increased artificially in vivo but did not generate tRNAAsn and/or tRNAGlu misaspartylation or the logical consecutive post-translational stress. This work allowed the detection of an additional subtle cellular lock capable of blocking AspRS toxicity. This study revealed the presence of post-translational modifications in the N-terminal extension of AspRS. We hypothesize that by neutralizing the lysine-rich motif contained in this domain, the cell mobilizes an additional strategy that considerably reduces the probability of the enzyme binding and aspartylating noncognate tRNAs and thus harming its own translation.

Plasmodium apicoplast tyrosyl-tRNA synthetase recognizes an unusual, simplified identity set in cognate tRNATyr.

The life cycle of Plasmodium falciparum, the agent responsible for malaria, depends on both cytosolic and apicoplast translation fidelity. Apicoplast aminoacyl-tRNA synthetases (aaRS) are bacterial-like enzymes devoted to organellar tRNA aminoacylation. They are all encoded by the nuclear genome and are translocated into the apicoplast only after cytosolic biosynthesis. Apicoplast aaRSs contain numerous idiosyncratic sequence insertions: An understanding of the roles of these insertions has remained elusive and they hinder efforts to heterologously overexpress these proteins. Moreover, the A/T rich content of the Plasmodium genome leads to A/U rich apicoplast tRNA substrates that display structural plasticity. Here, we focus on the P. falciparum apicoplast tyrosyl-tRNA synthetase (Pf-apiTyrRS) and its cognate tRNATyr substrate (Pf-apitRNATyr). Cloning and expression strategies used to obtain an active and functional recombinant Pf-apiTyrRS are reported. Functional analyses established that only three weak identity elements in the apitRNATyr promote specific recognition by the cognate Pf-apiTyrRS and that positive identity elements usually found in the tRNATyr acceptor stem are excluded from this set. This finding brings to light an unusual behavior for a tRNATyr aminoacylation system and suggests that Pf-apiTyrRS uses primarily negative recognition elements to direct tyrosylation specificity.