Biology subjects

Soufari, H.

Publications and source records attributed to Soufari, H..

9 recordsLinked to original sources

bioRxiv2025

Missing steps of mitochondrial translation initiation identified in plants

Translation initiation, the first step of the translation cycle, involves initiation factors (mtIF) 2 and 3 in mitochondria. mtIF3 release from the ribosomal small subunit was believed to be a prerequisite for the recruitment of the initiator tRNA. Here we use cryogenic electron microscopy to characterize plant mitochondria pre-initiation complexes and to reveal how plant mtIF3 binds the initiator tRNA and facilitates its accommodation in the decoding center.

molecular biology↗

bioRxiv2021

Crystal structures and functional analysis of the ZnF5-WWE1-WWE2 region of PARP13/ZAP define a new mode of engaging poly(ADP-ribose)

PARP13/ZAP acts against multiple viruses through recognizing and promoting degradation of cytoplasmic viral mRNA. PARP13 has four N-terminal Zn-finger motifs that bind CG-rich nucleotide sequences, and a C-terminal ADP ribosyltransferase fold similar to other PARPs. A central region predicted to contain a fifth Zn-finger and two tandem WWE domains is implicated in binding poly(ADP-ribose); however, there are limited insights into the structure and function of this PARP13 region (ZnF5-WWE1-WWE2). Here, we present crystal structures of ZnF5-WWE1-WWE2 from mouse PARP13 in complex with ADP-ribose and with ATP. ZnF5-WWE1-WWE2 crystallized as a dimer with major contacts formed between WWE1 and WWE2 originating from different monomers, indicative of a more compact monomeric arrangement of the tandem WWE domains. Solution scattering experiments and biophysical analysis indicated a monomer in solution, suggesting that the crystal dimer represents domain swapping that could potentially represent a PARP13 conformation assumed when signaling viral RNA detection. The crystal structure and binding studies demonstrate that WWE2 interacts with ADP-ribose and ATP, whereas WWE1 does not have a functional binding site. The shape of the WWE2 binding pocket disfavors interaction with the ribose-ribose linkage of poly(ADP-ribose). Binding studies with poly(ADP-ribose) ligands indicate that WWE2 serves as an anchor for preferential binding to the terminal end of poly(ADP-ribose), and the composite structure of ZnF5-WWE1-WWE2 forms an extended surface to engage polymer chains of ADP-ribose. This model represents a novel mode of poly(ADP-ribose) recognition and provides a structural framework for investigating poly(ADP-ribose) impact on PARP13 function.

biochemistry↗

bioRxiv2021

Regulation and mechanistic basis of macrolide resistance by the ABC-F ATPase MsrD

Antibiotic resistance ABC-Fs (ARE ABC-Fs) are translation factors currently proliferating among human pathogens that provide resistance against clinically important ribosome-targeting antibiotics. Here, we combine genetic and structural approaches to determine the regulation of streptococcal ARE ABC-F gene msrD in response to macrolide exposure and also demonstrate that MsrD twin-ATPase sites work asymmetrically to mediate the dynamic of MsrD interaction with the ribosome. We show that cladinose-containing macrolides lead to insertion of MsrDL leader peptide into an undocumented conserved crevice of the ribosomal exit tunnel concomitantly with 23S rRNA rearrangements that prevent peptide bond formation and preclude accommodation of release factors. The stalled ribosome obstructs formation of a Rho-independent terminator which prevents msrD transcriptional attenuation. This stalled ribosome is rescued by MsrD, but not by MsrD mutants which do not provide antibiotic resistance, showing evidence of equivalence between MsrD function in antibiotic resistance and its action on this complex.

microbiology↗

bioRxiv2021

The Chlamydomonas mitochondrial ribosome: how to build a ribosome from RNA fragments

Mitochondria are the powerhouse of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few essential messenger RNAs still encoded by mitochondrial genomes. Here, we present a biochemical and structural characterization of the mitoribosome in the model green alga Chlamydomonas reinhardtii, as well as a functional study of some of its specific components. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene pieces. Novel proteins, mainly helical repeat proteins, including OPR, PPR and mTERF proteins are found in Chlamydomonas mitoribosome, revealing the first structure of an OPR protein in complex with its RNA target. Targeted amiRNA silencing indicated that the novel ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the mitochondrial inner membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of the mitoribosome diversity and the various strategies they adopt for membrane tethering. Highlights* Structure of the Chlamydomonas reinhardtii mitoribosome * Fragmented ribosomal RNAs are stabilized by highly intertwined interactions with Chlamydomonas-specific proteins * Specific r-proteins are essential for rRNA homeostasis and respiratory fitness * Cryo-ET reveals the mitoribosome association to the inner mitochondrial membrane

molecular biology↗

bioRxiv2020

The Halastavi arva virus intergenic region IRES promotes translation by the simplest possible initiation mechanism

Dicistrovirus intergenic region internal ribosomal entry sites (IGR IRES) do not require initiator tRNA, an AUG codon or initiation factors, and jumpstart translation from the middle of the elongation cycle via formation of IRES/80S complexes resembling the pre-translocation state. eEF2 then translocates the [codon-anticodon]-mimicking pseudoknot I (PKI) from ribosomal A to P sites, bringing the first sense codon into the decoding center. Halastavi arva virus (HalV) contains an IGR that is related to previously described IGR IRESs, but lacks domain 2, which enables these IRESs to bind to individual 40S ribosomal subunits. By employing in vitro reconstitution and cryo-electron microscopy, we now report that the HalV IGR IRES functions by the simplest initiation mechanism that involves binding to 80S ribosomes such that PKI is placed in the P site, so that the A site contains the first codon that is directly accessible for decoding without prior eEF2-mediated translocation of PKI.

molecular biology↗

bioRxiv2020

Structure of the full kinetoplastids mitoribosome and insight on its large subunit maturation

Kinetoplastids are unicellular eukaryotic parasites responsible for human pathologies such as Chagas disease, sleeping sickness or Leishmaniasis1. They possess a single large mitochondrion, essential for the parasite survival2. In kinetoplastids mitochondrion, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes3. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes4,5. Structural studies performed in Trypanosoma brucei already highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit3,6. However, several important aspects mainly related to the large subunit remain elusive, such as the structure and maturation of its ribosomal RNA3. 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 large subunit as well as previously unidentified ribosomal proteins. Most importantly, we introduce the structure of an LSU assembly intermediate in presence of 16 identified maturation factors. These maturation factors act both on the intersubunit and solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

Specific features and assembly of the plant mitochondrial complex I revealed by cryo-EM

Mitochondria are the powerhouses of eukaryotic cells and the site of essential metabolic reactions. Their main purpose is to maintain the high ATP/ADP ratio that is required to fuel the countless biochemical reactions taking place in eukaryotic cells1. This high ATP/ADP ratio is maintained through oxidative phosphorylation (OXPHOS). Complex I or NADH:ubiquinone oxidoreductase is the main entry site for electrons into the mitochondrial respiratory chain and constitutes the largest of the respiratory complexes2. Its structure and composition varies across eukaryotes species. However, high resolution structures are available only for one group of eukaryotes, opisthokonts3-6. In plants, only biochemical studies were carried out, already hinting the peculiar composition of complex I in the green lineage. Here, we report several cryo-electron microscopy structures of the plant mitochondrial complex I at near-atomic resolution. We describe the structure and composition of the plant complex I including the plant-specific additional domain composed by carbonic anhydrase proteins. We show that the carbonic anhydrase is an heterotrimeric complex with only one conserved active site. This domain is crucial for the overall stability of complex I as well as a peculiar lipid complex composed cardiolipin and phosphatidylinositols. Moreover we also describe the structure of one of the plant-specific complex I assembly intermediate, lacking the whole PD module, in presence of the maturation factor GLDH. GLDH prevents the binding of the plant specific P1 protein, responsible for the linkage of the PP to the PD module. Finally, as the carbonic anhydrase domain is likely to be associated with complex I from numerous other known eukaryotes, we propose that our structure unveils an ancestral-like organization of mitochondrial complex I.

molecular biology↗

bioRxiv2019

Unique features of mRNA translation initiation in trypanosomatids

Canonical mRNA translation in eukaryotes begins with the formation of the 43S pre-initiation complex (PIC). Its assembly requires the binding of several eukaryotic initiation factors (eIF 1, 1A, 2, 3 and 5), Met-tRNAiMet and the small ribosomal subunit (40S). Compared to their mammalian hosts, trypanosomatids present significant structural differences in their 40S suggesting substantial variability in translation initiation. Here we determined the structure of the 43S PIC from Trypanosoma cruzi, the parasite causing the Chagas disease. Our structure shows numerous specific features, such as the variant eIF3 structure and its unique interactions with the large rRNA ESs 9S, 7S and 6S, and the association of a kinetoplastid-specific ~245 kDa DDX60-like helicase. It also revealed the so-far-elusive 40S-binding site of the eIF5 C-terminal domain and the structures of key terminal tails of several conserved eIFs underlying their activities within the PIC. Our results are corroborated by GST-pulldown assays in both human and T. cruzi and mass-spectrometry data.

molecular biology↗

bioRxiv2019

Cryo-EM structure of the RNA-rich plant mitochondrial ribosome

The vast majority of eukaryotic cells contain mitochondria, essential powerhouses and metabolic hubs1. These organelles have a bacterial origin and were acquired during an early endosymbiosis event2. Mitochondria possess specialized gene expression systems composed of various molecular machines including the mitochondrial ribosomes (mitoribosomes). Mitoribosomes are in charge of translating the few essential mRNAs still encoded by mitochondrial genomes3. While chloroplast ribosomes strongly resemble those of bacteria4,5, mitoribosomes have diverged significantly during evolution and present strikingly different structures across eukaryotic species6-10. In contrast to animals and trypanosomatides, plants mitoribosomes have unusually expanded ribosomal RNAs and conserved the short 5S rRNA, which is usually missing in mitoribosomes11. We have previously characterized the composition of the plant mitoribosome6 revealing a dozen plant-specific proteins, in addition to the common conserved mitoribosomal proteins. In spite of the tremendous recent advances in the field, plant mitoribosomes remained elusive to high-resolution structural investigations, and the plant-specific ribosomal features of unknown structures. Here, we present a cryo-electron microscopy study of the plant 78S mitoribosome from cauliflower at near-atomic resolution. We show that most of the plant-specific ribosomal proteins are pentatricopeptide repeat proteins (PPR) that deeply interact with the plant-specific rRNA expansion segments. These additional rRNA segments and proteins reshape the overall structure of the plant mitochondrial ribosome, and we discuss their involvement in the membrane association and mRNA recruitment prior to translation initiation. Finally, our structure unveils an rRNA-constructive phase of mitoribosome evolution across eukaryotes.

molecular biology↗