Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless.

RNA unwinding by DExH-type helicases underlies most RNA metabolism and function. It remains unresolved if and how the basic unwinding reaction of helicases is regulated by auxiliary domains. We explored the interplay between the RecA and auxiliary domains of the RNA helicase maleless (MLE) from Drosophila using structural and functional studies. We discovered that MLE exists in a dsRNA-bound open conformation and that the auxiliary dsRBD2 domain aligns the substrate RNA with the accessible helicase tunnel. In an ATP-dependent manner, dsRBD2 associates with the helicase module, leading to tunnel closure around ssRNA. Furthermore, our structures provide a rationale for blunt-ended dsRNA unwinding and 3'-5' translocation by MLE. Structure-based MLE mutations confirm the functional relevance of our model for RNA unwinding. Our findings contribute to our understanding of the fundamental mechanics of auxiliary domains in DExH helicase MLE, which serves as a model for its human ortholog and potential therapeutic target, DHX9/RHA.

Molecular basis of mRNA transport by a kinesin-1-atypical tropomyosin complex.

Kinesin-1 carries cargos including proteins, RNAs, vesicles, and pathogens over long distances within cells. The mechanochemical cycle of kinesins is well described, but how they establish cargo specificity is not fully understood. Transport of oskar mRNA to the posterior pole of the Drosophila oocyte is mediated by Drosophila kinesin-1, also called kinesin heavy chain (Khc), and a putative cargo adaptor, the atypical tropomyosin, aTm1. How the proteins cooperate in mRNA transport is unknown. Here, we present the high-resolution crystal structure of a Khc-aTm1 complex. The proteins form a tripartite coiled coil comprising two in-register Khc chains and one aTm1 chain, in antiparallel orientation. We show that aTm1 binds to an evolutionarily conserved cargo binding site on Khc, and mutational analysis confirms the importance of this interaction for mRNA transport in vivo. Furthermore, we demonstrate that Khc binds RNA directly and that it does so via its alternative cargo binding domain, which forms a positively charged joint surface with aTm1, as well as through its adjacent auxiliary microtubule binding domain. Finally, we show that aTm1 plays a stabilizing role in the interaction of Khc with RNA, which distinguishes aTm1 from classical motor adaptors.

Dynamic, variable oligomerization and the trafficking of variant surface glycoproteins of Trypanosoma brucei.

African trypanosomes cause disease in humans and livestock, avoiding host immunity by changing the expression of variant surface glycoproteins (VSGs); the major glycosylphosphatidylinositol (GPI) anchored antigens coating the surface of the bloodstream stage. Proper trafficking of VSGs is therefore critical to pathogen survival. The valence model argues that GPI anchors regulate progression and fate in the secretory pathway and that, specifically, a valence of two (VSGs are dimers) is critical for stable cell surface association. However, recent reports that the MITat1.3 (M1.3) VSG N-terminal domain (NTD) behaves as a monomer in solution and in a crystal structure challenge this model. We now show that the behavior of intact M1.3 VSG in standard in vivo trafficking assays is consistent with an oligomer. Nevertheless, Blue Native Gel electrophoresis and size exclusion chromatography-multiangle light scattering chromatography of purified full length M1.3 VSG indicates a monomer in vitro. However, studies with additional VSGs show that multiple oligomeric states are possible, and that for some VSGs oligomerization is concentration dependent. These data argue that individual VSG monomers possess different propensities to self-oligomerize, but that when constrained at high density to the cell surface, oligomeric species predominate. These results resolve the apparent conflict between the valence hypothesis and the M1.3 NTD VSG crystal structure.

Structure and dynamics of the quaternary hunchback mRNA translation repression complex.

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.

Extended N-Terminal Acetyltransferase Naa50 in Filamentous Fungi Adds to Naa50 Diversity.

Most eukaryotic proteins are N-terminally acetylated by a set of Nα acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi Chaetomium thermophilum and Neurospora crassa with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that CtNaa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution.

NatB-Mediated N-Terminal Acetylation Affects Growth and Biotic Stress Responses.

N∝-terminal acetylation (NTA) is one of the most abundant protein modifications in eukaryotes. In humans, NTA is catalyzed by seven Nα-acetyltransferases (NatA-F and NatH). Remarkably, the plant Nat machinery and its biological relevance remain poorly understood, although NTA has gained recognition as a key regulator of crucial processes such as protein turnover, protein-protein interaction, and protein targeting. In this study, we combined in vitro assays, reverse genetics, quantitative N-terminomics, transcriptomics, and physiological assays to characterize the Arabidopsis (Arabidopsis thaliana) NatB complex. We show that the plant NatB catalytic (NAA20) and auxiliary subunit (NAA25) form a stable heterodimeric complex that accepts canonical NatB-type substrates in vitro. In planta, NatB complex formation was essential for enzymatic activity. Depletion of NatB subunits to 30% of the wild-type level in three Arabidopsis T-DNA insertion mutants (naa20-1, naa20-2, and naa25-1) caused a 50% decrease in plant growth. A complementation approach revealed functional conservation between plant and human catalytic NatB subunits, whereas yeast NAA20 failed to complement naa20-1 Quantitative N-terminomics of approximately 1000 peptides identified 32 bona fide substrates of the plant NatB complex. In vivo, NatB was seen to preferentially acetylate N termini starting with the initiator Met followed by acidic amino acids and contributed 20% of the acetylation marks in the detected plant proteome. Global transcriptome and proteome analyses of NatB-depleted mutants suggested a function of NatB in multiple stress responses. Indeed, loss of NatB function, but not NatA, increased plant sensitivity toward osmotic and high-salt stress, indicating that NatB is required for tolerance of these abiotic stressors.

The Arabidopsis Nα -acetyltransferase NAA60 locates to the plasma membrane and is vital for the high salt stress response.

In humans and plants, N-terminal acetylation plays a central role in protein homeostasis, affects 80% of proteins in the cytoplasm and is catalyzed by five ribosome-associated N-acetyltransferases (NatA-E). Humans also possess a Golgi-associated NatF (HsNAA60) that is essential for Golgi integrity. Remarkably, NAA60 is absent in fungi and has not been identified in plants. Here we identify and characterize the first plasma membrane-anchored post-translationally acting N-acetyltransferase AtNAA60 in the reference plant Arabidopsis thaliana by the combined application of reverse genetics, global proteomics, live-cell imaging, microscale thermophoresis, circular dichroism spectroscopy, nano-differential scanning fluorometry, intrinsic tryptophan fluorescence and X-ray crystallography. We demonstrate that AtNAA60, like HsNAA60, is membrane-localized in vivo by an α-helical membrane anchor at its C-terminus, but in contrast to HsNAA60, AtNAA60 localizes to the plasma membrane. The AtNAA60 crystal structure provides insights into substrate-binding, the broad substrate specificity and the catalytic mechanism probed by structure-based mutagenesis. Characterization of the NAA60 loss-of-function mutants (naa60-1 and naa60-2) uncovers a plasma membrane-localized substrate of AtNAA60 and the importance of NAA60 during high salt stress. Our findings provide evidence for the plant-specific evolution of a plasma membrane-anchored N-acetyltransferase that is vital for adaptation to stress.

Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction.

Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 protein-binding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SRP S domain (SRP RNA, SRP19 and SRP68) detailing all interactions of SRP72 within SRP. The SRP72-PBD is a tetratricopeptide repeat, which binds an extended linear motif of SRP68 with high affinity. The SRP72-RBD is a flexible peptide crawling along the 5e- and 5f-loops of SRP RNA. A conserved tryptophan inserts into the 5e-loop forming a novel type of RNA kink-turn stabilized by a potassium ion, which we define as K+-turn. In addition, SRP72-RBD remodels the 5f-loop involved in ribosome binding and visualizes SRP RNA plasticity. Docking of the S domain structure into cryo-electron microscopy density maps reveals multiple contact sites between SRP68/72 and the ribosome, and explains the role of SRP72 in the SRP pathway.

Oxygen-dependent regulation of c-di-GMP synthesis by SadC controls alginate production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles. Furthermore, we found that the gene products of PA4330 and PA4331, located in a predicted operon with sadC, have a major impact on alginate production: deletion of PA4330 (odaA, for oxygen-dependent alginate synthesis activator) caused an alginate production defect under anaerobic conditions, whereas a PA4331 (odaI, for oxygen-dependent alginate synthesis inhibitor) deletion mutant produced alginate also in the presence of oxygen, which would normally inhibit alginate synthesis. Based on their sequence, OdaA and OdaI have predicted hydratase and dioxygenase reductase activities, respectively. Enzymatic assays using purified protein showed that unlike OdaA, which did not significantly affect DGC activity of SadC, OdaI inhibited c-di-GMP production by SadC. Our data indicate that SadC, OdaA and OdaI are components of a novel response pathway of P. aeruginosa that regulates alginate synthesis in an oxygen-dependent manner.

A WXW motif is required for the anticancer activity of the TAT-RasGAP317-326 peptide.

TAT-RasGAP317-326, a cell-permeable 10-amino acid-long peptide derived from the N2 fragment of p120 Ras GTPase-activating protein (RasGAP), sensitizes tumor cells to apoptosis induced by various anticancer therapies. This RasGAP-derived peptide, by targeting the deleted in liver cancer-1 (DLC1) tumor suppressor, also hampers cell migration and invasion by promoting cell adherence and by inhibiting cell movement. Here, we systematically investigated the role of each amino acid within the RasGAP317-326 sequence for the anticancer activities of TAT-RasGAP317-326. We report here that the first three amino acids of this sequence, tryptophan, methionine, and tryptophan (WMW), are necessary and sufficient to sensitize cancer cells to cisplatin-induced apoptosis and to reduce cell migration. The WMW motif was found to be critical for the binding of fragment N2 to DLC1. These results define the interaction mode between the active anticancer sequence of RasGAP and DLC1. This knowledge will facilitate the design of small molecules bearing the tumor-sensitizing and antimetastatic activities of TAT-RasGAP317-326.

NrsZ: a novel, processed, nitrogen-dependent, small non-coding RNA that regulates Pseudomonas aeruginosa PAO1 virulence.

The opportunistic pathogen Pseudomonas aeruginosa PAO1 has a remarkable capacity to adapt to various environments and to survive with limited nutrients. Here, we report the discovery and characterization of a novel small non-coding RNA: NrsZ (nitrogen-regulated sRNA). We show that under nitrogen limitation, NrsZ is induced by the NtrB/C two component system, an important regulator of nitrogen assimilation and P. aeruginosa's swarming motility, in concert with the alternative sigma factor RpoN. Furthermore, we demonstrate that NrsZ modulates P. aeruginosa motility by controlling the production of rhamnolipid surfactants, virulence factors notably needed for swarming motility. This regulation takes place through the post-transcriptional control of rhlA, a gene essential for rhamnolipids synthesis. Interestingly, we also observed that NrsZ is processed in three similar short modules, and that the first short module encompassing the first 60 nucleotides is sufficient for NrsZ regulatory functions.

Hierarchical management of carbon sources is regulated similarly by the CbrA/B systems in Pseudomonas aeruginosa and Pseudomonas putida.

The CbrA/B system in pseudomonads is involved in the utilization of carbon sources and carbon catabolite repression (CCR) through the activation of the small RNAs crcZ in Pseudomonas aeruginosa, and crcZ and crcY in Pseudomonas putida. Interestingly, previous works reported that the CbrA/B system activity in P. aeruginosa PAO1 and P. putida KT2442 responded differently to the presence of different carbon sources, thus raising the question of the exact nature of the signal(s) detected by CbrA. Here, we demonstrated that the CbrA/B/CrcZ(Y) signal transduction pathway is similarly activated in the two Pseudomonas species. We show that the CbrA sensor kinase is fully interchangeable between the two species and, moreover, responds similarly to the presence of different carbon sources. In addition, a metabolomics analysis supported the hypothesis that CCR responds to the internal energy status of the cell, as the internal carbon/nitrogen ratio seems to determine CCR and non-CCR conditions. The strong difference found in the 2-oxoglutarate/glutamine ratio between CCR and non-CCR conditions points to the close relationship between carbon and nitrogen availability, or the relationship between the CbrA/B and NtrB/C systems, suggesting that both regulatory systems sense the same sort or interrelated signal.

The Use of Baculovirus-Mediated Gene Expression in Mammalian Cells for Recombinant Protein Production.

Baculovirus-mediated gene expression in mammalian cells, BacMam, is a useful alternative to transient transfection for recombinant protein production in various types of mammalian cell lines. We decided to establish BacMam in our lab in order to streamline our workflows for gene expression in insect and mammalian cells, as it is straightforward to parallelize the baculovirus generation for both types of eukaryotic cells. This chapter provides a step-by-step description of the protocols we use for the generation of the recombinant BacMam viruses, the transduction of mammalian cell cultures, and optimization of the protein production conditions through small-scale expression and purification tests.

Tropomyosin 1-I/C coordinates kinesin-1 and dynein motors during oskar mRNA transport.

Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo bidirectional transport by binding to both motors simultaneously. However, it is not known how motor activities are coordinated in such circumstances. In the Drosophila female germline, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliver oskar messenger RNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. We show through in vitro reconstitution that Tm1-I/C, a tropomyosin-1 isoform, links kinesin-1 in a strongly inhibited state to DDBE-associated oskar mRNA. Nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and structural modeling indicate that Tm1-I/C suppresses kinesin-1 activity by stabilizing its autoinhibited conformation, thus preventing competition with dynein until kinesin-1 is activated in the oocyte. Our work reveals a new strategy for ensuring sequential activity of microtubule motors.

Trim66's paternal deficiency causes intrauterine overgrowth.

The tripartite motif-containing protein 66 (TRIM66, also known as TIF1-delta) is a PHD-Bromo-containing protein primarily expressed in post-meiotic male germ cells known as spermatids. Biophysical assays showed that the TRIM66 PHD-Bromodomain binds to H3 N-terminus only when lysine 4 is unmethylated. We addressed TRIM66's role in reproduction by loss-of-function genetics in the mouse. Males homozygous for Trim66-null mutations produced functional spermatozoa. Round spermatids lacking TRIM66 up-regulated a network of genes involved in histone acetylation and H3K4 methylation. Profiling of H3K4me3 patterns in the sperm produced by the Trim66-null mutant showed minor alterations below statistical significance. Unexpectedly, Trim66-null males, but not females, sired pups overweight at birth, hence revealing that Trim66 mutations cause a paternal effect phenotype.

Catabolite repression in Pseudomonas aeruginosa†PAO1 regulates the uptake of C4 -dicarboxylates depending on succinate concentration.

In Pseudomonas aeruginosa carbon catabolite repression (CCR) is exerted by the CbrA/B-CrcZ-Crc global regulatory system. Crc is a translational repressor that, in the presence of preferred carbon sources, such as C4 -dicarboxylates, impairs the utilization of less preferred substrates. When non-preferred substrates are present, the CrcZ sRNA levels increase leading to Crc capture, thereby allowing growth of the bacterium at the expense of the non-preferred substrates. The C4 -dicarboxylate transport (Dct) system in P. aeruginosa is composed of two main transporters: DctA, more efficient at mM succinate concentrations, and DctPQM, more important at µM. In this study, we demonstrate that the Dct transporters are differentially regulated by Crc, depending on the concentration of succinate. At high concentrations, Crc positively regulates the expression of the dctA transporter gene and negatively regulates dctPQM post-transcriptionally. The activation of dctA is explained by a Crc-mediated repression of dctR, encoding a transcriptional repressor of dctA. At low succinate concentrations, Crc regulation is impaired. In this condition, CrcZ levels are higher and therefore more Crc proteins are sequestered, decreasing the amount of Crc available to perform CCR on dctR and dctPQM. As a result, expression of dctA is reduced and that of dctPQM is increased.

RNA pentaloop structures as effective targets of regulators belonging to the RsmA/CsrA protein family.

In the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens CHA0, the dimeric RNA-binding proteins RsmA and RsmE, which belong to the vast bacterial RsmA/CsrA family, effectively repress translation of target mRNAs containing a typical recognition sequence near the translation start site. Three small RNAs (RsmX, RsmY, RsmZ) with clustered recognition sequences can sequester RsmA and RsmE and thereby relieve translational repression. According to a previously established structural model, the RsmE protein makes optimal contacts with an RNA sequence 5'- (A)/(U)CANGGANG(U)/(A)-3', in which the central ribonucleotides form a hexaloop. Here, we questioned the relevance of the hexaloop structure in target RNAs. We found that two predicted pentaloop structures, AGGGA (in pltA mRNA encoding a pyoluteorin biosynthetic enzyme) and AAGGA (in mutated pltA mRNA), allowed effective interaction with the RsmE protein in vivo. By contrast, ACGGA and AUGGA were poor targets. Isothermal titration calorimetry measurements confirmed the strong binding of RsmE to the AGGGA pentaloop structure in an RNA oligomer. Modeling studies highlighted the crucial role of the second ribonucleotide in the loop structure. In conclusion, a refined structural model of RsmE-RNA interaction accommodates certain pentaloop RNAs among the preferred hexaloop RNAs.

Structural inventory of cotranslational protein folding by the eukaryotic RAC complex.

The challenge of nascent chain folding at the ribosome is met by the conserved ribosome-associated complex (RAC), which forms a chaperone triad with the Hsp70 protein Ssb in fungi, and consists of the non-canonical Hsp70 Ssz1 and the J domain protein Zuotin (Zuo1). Here we determine cryo-EM structures of Chaetomium thermophilum RAC bound to 80S ribosomes. RAC adopts two distinct conformations accommodating continuous ribosomal rotation by a flexible lever arm. It is held together by a tight interaction between the Ssz1 substrate-binding domain and the Zuo1 N terminus, and additional contacts between the Ssz1 nucleotide-binding domain and Zuo1 J- and Zuo1 homology domains, which form a rigid unit. The Zuo1 HPD motif conserved in J-proteins is masked in a non-canonical interaction by the Ssz1 nucleotide-binding domain, and allows the positioning of Ssb for activation by Zuo1. Overall, we provide the basis for understanding how RAC cooperates with Ssb in a dynamic nascent chain interaction and protein folding.

In vitro and ex vivo proteomics of Mycobacterium marinum biofilms and the development of biofilm-binding synthetic nanobodies.

The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416, respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm surface-binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection. IMPORTANCE With the currently available antibiotics, the treatment of TB takes months. The slow response to treatment is caused by antibiotic tolerance, which is especially common among bacteria that form biofilms. Such biofilms are composed of bacterial cells surrounded by the extracellular matrix. Both the matrix and the dormant lifestyle of the bacterial cells are thought to hinder the efficacy of antibiotics. To be able to develop faster-acting treatments against TB, the biofilm forms of mycobacteria deserve specific attention. In this work, we characterize the protein composition of Mmr biofilms in bacterial cultures and in mycobacteria extracted from infected adult zebrafish. We identify abundant surface-exposed targets and develop the first sybodies that bind to mycobacterial biofilms. As nanobodies can be linked to other therapeutic compounds, in the future, they can provide means to target therapies to biofilms.

Molecular basis of messenger RNA recognition by the specific bacterial repressing clamp RsmA/CsrA.

Proteins of the RsmA/CsrA family are global translational regulators in many bacterial species. We have determined the solution structure of a complex formed between the RsmE protein, a member of this family from Pseudomonas fluorescens, and a target RNA encompassing the ribosome-binding site of the hcnA gene. The RsmE homodimer with its two RNA-binding sites makes optimal contact with an 5'-A/UCANGGANGU/A-3' sequence in the mRNA. When tightly gripped by RsmE, the ANGGAN core folds into a loop, favoring the formation of a 3-base-pair stem by flanking nucleotides. We validated these findings by in vivo and in vitro mutational analyses. The structure of the complex explains well how, by sequestering the Shine-Dalgarno sequence, the RsmA/CsrA proteins repress translation.