The Kinase IKKß Regulates a STING- and NF-κB-Dependent Antiviral Response Pathway in Drosophila.

Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. Here, we showed that two components of the IMD pathway, the kinase dIKKß and the transcription factor Relish, were required to control infection by two picorna-like viruses. We identified a set of genes induced by viral infection and regulated by dIKKß and Relish, which included an ortholog of STING. We showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKß to regulate expression of Nazo, an antiviral factor. Our data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity.

tRNA Maturation Defects Lead to Inhibition of rRNA Processing via Synthesis of pppGpp.

rRNAs and tRNAs universally require processing from longer primary transcripts to become functional for translation. Here, we describe an unsuspected link between tRNA maturation and the 3' processing of 16S rRNA, a key step in preparing the small ribosomal subunit for interaction with the Shine-Dalgarno sequence in prokaryotic translation initiation. We show that an accumulation of either 5' or 3' immature tRNAs triggers RelA-dependent production of the stringent response alarmone (p)ppGpp in the Gram-positive model organism Bacillus subtilis. The accumulation of (p)ppGpp and accompanying decrease in GTP levels specifically inhibit 16S rRNA 3' maturation. We suggest that cells can exploit this mechanism to sense potential slowdowns in tRNA maturation and adjust rRNA processing accordingly to maintain the appropriate functional balance between these two major components of the translation apparatus.

The unique dual targeting of AGO1 by two types of PRMT enzymes promotes phasiRNA loading in Arabidopsis thaliana.

Arginine/R methylation (R-met) of proteins is a widespread post-translational modification (PTM), deposited by a family of protein arginine/R methyl transferase enzymes (PRMT). Regulations by R-met are involved in key biological processes deeply studied in metazoan. Among those, post-transcriptional gene silencing (PTGS) can be regulated by R-met in animals and in plants. It mainly contributes to safeguard processes as protection of genome integrity in germlines through the regulation of piRNA pathway in metazoan, or response to bacterial infection through the control of AGO2 in plants. So far, only PRMT5 has been identified as the AGO/PIWI R-met writer in higher eukaryotes. We uncovered that AGO1, the main PTGS effector regulating plant development, contains unique R-met features among the AGO/PIWI superfamily, and outstanding in eukaryotes. Indeed, AGO1 contains both symmetric (sDMA) and asymmetric (aDMA) R-dimethylations and is dually targeted by PRMT5 and by another type I PRMT in Arabidopsis thaliana. We showed also that loss of sDMA didn't compromise AtAGO1 subcellular trafficking in planta. Interestingly, we underscored that AtPRMT5 specifically promotes the loading of phasiRNA in AtAGO1. All our observations bring to consider this dual regulation of AtAGO1 in plant development and response to environment, and pinpoint the complexity of AGO1 post-translational regulation.

Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector.

Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms.

eIF3 Peripheral Subunits Rearrangement after mRNA Binding and Start-Codon Recognition.

mRNA translation initiation in eukaryotes requires the cooperation of a dozen eukaryotic initiation factors (eIFs) forming several complexes, which leads to mRNA attachment to the small ribosomal 40S subunit, mRNA scanning for start codon, and accommodation of initiator tRNA at the 40S P site. eIF3, composed of 13 subunits, 8 core (a, c, e, f, h, l, k, and m) and 5 peripheral (b, d, g, i, and j), plays a central role during this process. Here we report a cryo-electron microscopy structure of a mammalian 48S initiation complex at 5.8 Å resolution. It shows the relocation of subunits eIF3i and eIF3g to the 40S intersubunit face on the GTPase binding site, at a late stage in initiation. On the basis of a previous study, we demonstrate the relocation of eIF3b to the 40S intersubunit face, binding below the eIF2-Met-tRNAi(Met) ternary complex upon mRNA attachment. Our analysis reveals the deep rearrangement of eIF3 and unravels the molecular mechanism underlying eIF3 function in mRNA scanning and timing of ribosomal subunit joining.

DNA polymerase η is a substrate for calpain: a possible mechanism for pol η retention in UV-induced replication foci.

DNA polymerase η (pol η) is specifically required for translesion DNA synthesis across UV-induced DNA lesions. Recruitment of this error-prone DNA polymerase is tightly regulated during replication to avoid mutagenesis and perturbation of fork progression. Here, we report that pol η interacts with the calpain small subunit-1 (CAPNS1) in a yeast two-hybrid screening. This interaction is functional, as demonstrated by the ability of endogenous calpain to mediate calcium-dependent cleavage of pol η in cell-free extracts and in living cells treated with a calcium ionophore. The proteolysis of pol η was found to occur at position 465, leading to a catalytically active truncated protein containing the PCNA-interacting motif PIP1. Unexpectedly, cell treatment with the specific calpain inhibitor calpeptin resulted in a decreased extent of pol η foci after UV irradiation, indicating that calpain positively regulates pol η accumulation in replication foci.

Nucleos'ID: A New Search Engine Enabling the Untargeted Identification of RNA Post-transcriptional Modifications from Tandem Mass Spectrometry Analyses of Nucleosides.

As RNA post-transcriptional modifications are of growing interest, several methods were developed for their characterization. One of them established for their identification, at the nucleosidic level, is the hyphenation of separation methods, such as liquid chromatography or capillary electrophoresis, to tandem mass spectrometry. However, to our knowledge, no software is yet available for the untargeted identification of RNA post-transcriptional modifications from MS/MS data-dependent acquisitions. Thus, very long and tedious manual data interpretations are required. To meet the need of easier and faster data interpretation, a new user-friendly search engine, called Nucleos'ID, was developed for CE-MS/MS and LC-MS/MS users. Performances of this new software were evaluated on CE-MS/MS data from nucleoside analyses of already well-described Saccharomyces cerevisiae transfer RNA and Bos taurus total tRNA extract. All samples showed great true positive, true negative, and false discovery rates considering the database size containing all modified and unmodified nucleosides referenced in the literature. The true positive and true negative rates obtained were above 0.94, while the false discovery rates were between 0.09 and 0.17. To increase the level of sample complexity, untargeted identification of several RNA modifications from Pseudomonas aeruginosa 70S ribosome was achieved by the Nucleos'ID search following CE-MS/MS analysis.

A NYN domain protein directly interacts with DECAPPING1 and is required for phyllotactic pattern.

In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DECAPPING2 (DCP2), and its interaction with decapping enhancers, including its main partner DECAPPING1 (DCP1). Here, we report that in Arabidopsis thaliana, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we found DNE1 predominantly associated with DCP1, but not with DCP2, and reciprocally, suggesting the existence of two distinct protein complexes. We also showed that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines led to growth defects and a similar gene deregulation signature than inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness.

Arabidopsis mTERF9 protein promotes chloroplast ribosomal assembly and translation by establishing ribonucleoprotein interactions in vivo.

The mitochondrial transcription termination factor proteins are nuclear-encoded nucleic acid binders defined by degenerate tandem helical-repeats of ∼30 amino acids. They are found in metazoans and plants where they localize in organelles. In higher plants, the mTERF family comprises ∼30 members and several of these have been linked to plant development and response to abiotic stress. However, knowledge of the molecular basis underlying these physiological effects is scarce. We show that the Arabidopsis mTERF9 protein promotes the accumulation of the 16S and 23S rRNAs in chloroplasts, and interacts predominantly with the 16S rRNA in vivo and in vitro. Furthermore, mTERF9 is found in large complexes containing ribosomes and polysomes in chloroplasts. The comprehensive analysis of mTERF9 in vivo protein interactome identified many subunits of the 70S ribosome whose assembly is compromised in the null mterf9 mutant, putative ribosome biogenesis factors and CPN60 chaperonins. Protein interaction assays in yeast revealed that mTERF9 directly interact with these proteins. Our data demonstrate that mTERF9 integrates protein-protein and protein-RNA interactions to promote chloroplast ribosomal assembly and translation. Besides extending our knowledge of mTERF functional repertoire in plants, these findings provide an important insight into the chloroplast ribosome biogenesis.

Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis.

Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed in Trypanosoma brucei already highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoans Leishmania tarentolae and Trypanosoma cruzi mitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.

Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity.

The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.

Opportunistic use of catecholamine neurotransmitters as siderophores to access iron by Pseudomonas aeruginosa.

Iron is an essential nutrient for bacterial growth and the cause of a fierce battle between the pathogen and host during infection. Bacteria have developed several strategies to access iron from the host, the most common being the production of siderophores, small iron-chelating molecules secreted into the bacterial environment. The opportunist pathogen Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, and is also able to use a wide panoply of xenosiderophores, siderophores produced by other microorganisms. Here, we demonstrate that catecholamine neurotransmitters (dopamine, l-DOPA, epinephrine and norepinephrine) are able to chelate iron and efficiently bring iron into P. aeruginosa cells via TonB-dependent transporters (TBDTs). Bacterial growth assays under strong iron-restricted conditions and with numerous mutants showed that the TBDTs involved are PiuA and PirA. PiuA exhibited more pronounced specificity for dopamine uptake than for norepinephrine, epinephrine and l-DOPA, whereas PirA specificity appeared to be higher for l-DOPA and norepinephrine. Proteomic and qRT-PCR approaches showed pirA transcription and expression to be induced in the presence of all four catecholamines. Finally, the oxidative properties of catecholamines enable them to reduce iron, and we observed ferrous iron uptake via the FeoABC system in the presence of l-DOPA.

A role for PchHI as the ABC transporter in iron acquisition by the siderophore pyochelin in Pseudomonas aeruginosa.

Iron is an essential nutrient for bacterial growth but poorly bioavailable. Bacteria scavenge ferric iron by synthesizing and secreting siderophores, small compounds with a high affinity for iron. Pyochelin (PCH) is one of the two siderophores produced by the opportunistic pathogen Pseudomonas aeruginosa. After capturing a ferric iron molecule, PCH-Fe is imported back into bacteria first by the outer membrane transporter FptA and then by the inner membrane permease FptX. Here, using molecular biology, 55 Fe uptake assays, and LC-MS/MS quantification, we first find a role for PchHI as the heterodimeric ABC transporter involved in the siderophore-free iron uptake into the bacterial cytoplasm. We also provide the first evidence that PCH is able to reach the bacterial periplasm and cytoplasm when both FptA and FptX are expressed. Finally, we detected an interaction between PchH and FptX, linking the ABC transporter PchHI with the inner permease FptX in the PCH-Fe uptake pathway. These results pave the way for a better understanding of the PCH siderophore pathway, giving future directions to tackle P. aeruginosa infections.

The viral protein NSP1 acts as a ribosome gatekeeper for shutting down host translation and fostering SARS-CoV-2 translation.

SARS-CoV-2 coronavirus is responsible for Covid-19 pandemic. In the early phase of infection, the single-strand positive RNA genome is translated into non-structural proteins (NSP). One of the first proteins produced during viral infection, NSP1, binds to the host ribosome and blocks the mRNA entry channel. This triggers translation inhibition of cellular translation. In spite of the presence of NSP1 on the ribosome, viral translation proceeds however. The molecular mechanism of the so-called viral evasion to NSP1 inhibition remains elusive. Here, we confirm that viral translation is maintained in the presence of NSP1. The evasion to NSP1-inhibition is mediated by the cis-acting RNA hairpin SL1 in the 5'UTR of SARS-CoV-2. NSP1-evasion can be transferred on a reporter transcript by SL1 transplantation. The apical part of SL1 is only required for viral translation. We show that NSP1 remains bound on the ribosome during viral translation. We suggest that the interaction between NSP1 and SL1 frees the mRNA accommodation channel while maintaining NSP1 bound to the ribosome. Thus, NSP1 acts as a ribosome gatekeeper, shutting down host translation or fostering SARS-CoV-2 translation depending on the presence of the SL1 5'UTR hairpin. SL1 is also present and necessary for translation of sub-genomic RNAs in the late phase of the infectious program. Consequently, therapeutic strategies targeting SL1 should affect viral translation at early and late stages of infection. Therefore, SL1 might be seen as a genuine 'Achille heel' of the virus.

Phenotypic Adaption of Pseudomonas aeruginosa by Hacking Siderophores Produced by Other Microorganisms.

Bacteria secrete siderophores to access iron, a key nutrient poorly bioavailable and the source of strong competition between microorganisms in most biotopes. Many bacteria also use siderophores produced by other microorganisms (exosiderophores) in a piracy strategy. Pseudomonas aeruginosa, an opportunistic pathogen, produces two siderophores, pyoverdine and pyochelin, and is also able to use a panel of exosiderophores. We first investigated expression of the various iron-uptake pathways of P. aeruginosa in three different growth media using proteomic and RT-qPCR approaches and observed three different phenotypic patterns, indicating complex phenotypic plasticity in the expression of the various iron-uptake pathways. We then investigated the phenotypic plasticity of iron-uptake pathway expression in the presence of various exosiderophores (present individually or as a mixture) under planktonic growth conditions, as well as in an epithelial cell infection assay. In all growth conditions tested, catechol-type exosiderophores were clearly more efficient in inducing the expression of their corresponding transporters than the others, showing that bacteria opt for the use of catechol siderophores to access iron when they are present in the environment. In parallel, expression of the proteins of the pyochelin pathway was significantly repressed under most conditions tested, as well as that of proteins of the pyoverdine pathway, but to a lesser extent. There was no effect on the expression of the heme and ferrous uptake pathways. Overall, these data provide precise insights on how P. aeruginosa adjusts the expression of its various iron-uptake pathways (phenotypic plasticity and switching) to match varying levels of iron and competition.

SERRATE interacts with the nuclear exosome targeting (NEXT) complex to degrade primary miRNA precursors in Arabidopsis.

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.

YBEY is an essential biogenesis factor for mitochondrial ribosomes.

Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3'-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter helps to properly incorporate uS11m into the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.

Chemical Modifications Induced by Phthalic Anhydride, a Respiratory Sensitizer, in Reconstructed Human Epidermis: A Combined HRMAS NMR and LC-MS/MS Proteomic Approach.

Chemical skin and respiratory allergies are becoming a major health problem. To date our knowledge on the process of protein haptenation is still limited and mainly derived from studies performed in solution using model nucleophiles. In order to better understand chemical interactions between chemical allergens and the skin, we have investigated the reactivity of phthalic anhydride 1 (PA), a chemical respiratory sensitizer, toward reconstructed human epidermis (RHE). This study was performed using a new approach combining HRMAS NMR to investigate the in situ chemical reactivity and LC-MS/MS to identify modified epidermal proteins. In RHE, the reaction of PA appeared to be quite fast and the major product formed was phthalic acid. Two amide type adducts on lysine residues were observed and after 8h of incubation, we also observed the formation of an imide type cyclized adducts with lysine. In parallel, RHE samples topically exposed to phthalic anhydride (13C)-1 were analyzed using the shotgun proteomics method. Thus, 948 different proteins were extracted and identified, 135 of which being modified by PA, i.e., 14.2% of the extracted proteome. A total of 211 amino acids were modified by PA and validated by fragmentation spectra. We thus identified 154 modified lysines, 22 modified histidines, 30 modified tyrosines, and 5 modified arginines. The rate of modified residues, as a proportion of the total number of modifiable nucleophilic residues in RHE, was rather low (1%). At the protein level, modified proteins were mainly type I and type II keratins and other proteins which are abundant in the epidermis such as protein S100A, Caspase 14, annexin A2, serpin B3, fatty-acid binding protein 5, histone H2, H3, H4, etc. However, the most modified protein, mainly on histidine residues, was filaggrin, a protein that is of low abundance (0.0266 mol %) and rich in histidine.

Structure of mammalian eIF3 in the context of the 43S preinitiation complex.

During eukaryotic translation initiation, 43S complexes, comprising a 40S ribosomal subunit, initiator transfer RNA and initiation factors (eIF) 2, 3, 1 and 1A, attach to the 5'-terminal region of messenger RNA and scan along it to the initiation codon. Scanning on structured mRNAs also requires the DExH-box protein DHX29. Mammalian eIF3 contains 13 subunits and participates in nearly all steps of translation initiation. Eight subunits having PCI (proteasome, COP9 signalosome, eIF3) or MPN (Mpr1, Pad1, amino-terminal) domains constitute the structural core of eIF3, to which five peripheral subunits are flexibly linked. Here we present a cryo-electron microscopy structure of eIF3 in the context of the DHX29-bound 43S complex, showing the PCI/MPN core at ∼6 Šresolution. It reveals the organization of the individual subunits and their interactions with components of the 43S complex. We were able to build near-complete polyalanine-level models of the eIF3 PCI/MPN core and of two peripheral subunits. The implications for understanding mRNA ribosomal attachment and scanning are discussed.