Biology subjects

Bochler, A.

Publications and source records attributed to Bochler, A..

5 recordsLinked to original sources

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↗

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

Structural insights into the Kozak sequence interactions within the mammalian late-stage initiation complexes

In higher eukaryotes, the mRNA sequence in direct vicinity of the start codon, called the Kozak sequence (CRCCaugG, where R is a purine), is known to influence the rate of the initiation process. However, the molecular basis underlying its role remains poorly understood. Here, we present the cryo-electron microscopy (cryo-EM) structures of mammalian late-stage 48S initiation complexes (LS48S IC) in the presence of two different native mRNA sequences, {beta}-globin and histone 4 (H4) at overall resolution of 3[A] and 3.5[A], respectively. Our high-resolution structures unravel key interactions from the mRNA to eukaryotic initiation factors (eIF): 1A, 2, 3, 18S rRNA, and several 40S ribosomal proteins. In addition, we were able to study the structural role of ABCE1 in the formation of native 48S ICs. Our results reveal a comprehensive map of the ribosome/eIFs -mRNA and -tRNA interactions and suggest the impact of mRNA sequence on the structure of the LS48S IC.

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↗