Biology subjects

Brito Querido, J.

Publications and source records attributed to Brito Querido, J..

4 recordsLinked to original sources

bioRxiv2026

Structural basis for repeat-associated non-AUG translation initiation on C9orf72 mRNA

A GGGGCC (G4C2) hexanucleotide repeat expansion in the first intron of C9orf72 is the most common monogenic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). This element serves as a template for repeat-associated non-AUG (RAN) translation, which generates neurotoxic dipeptide repeat (DPR) proteins that aggregate in patient brains and contribute to neurodegeneration in model systems. To understand the mechanisms underlying RAN translation initiation, we used cryo-electron microscopy to determine the structure of an in vitro assembled human late-stage C9orf72 translation initiation complex at 3.2 A resolution and a near-native complex purified from human cell lysate at 3.5 A resolution. RAN translation on C9orf72 mRNA initiates predominantly at a near-cognate CUG codon that is located 24 nucleotides upstream of the G4C2 repeat and is flanked by a strong endogenous Kozak sequence. The structures further reveal a direct interaction between the G4C2 repeat and 18S ribosomal RNA expansion segment 9 (ES9S) on the 40S small ribosomal subunit through a kissing-loop interaction. Preventing this RNA-RNA interaction using complementary antisense oligonucleotides significantly reduces RAN translation in vitro and in patient-derived iNeurons, indicating that this structural interaction is functionally important. These findings provide mechanistic insights into RAN translation initiation and highlight a novel potential therapeutic approach to mitigate toxic DPR production in C9orf72-associated neurodegenerative disease.

molecular biology↗

bioRxiv2026

Structural basis for emetine inhibition of ribosome translocation in Toxoplasma gondii

Apicomplexan parasites, including Toxoplasma gondii and Plasmodium falciparum, are major human pathogens that cause toxoplasmosis and malaria, respectively. The existing structures of T. gondii translational machinery are from empty ribosomes that lack several key components, including ribosomal protein RACK1 (Receptor for Activated C Kinase 1). Here, we used cryo-electron microscopy (cryoEM) to determine high-resolution structures of T. gondii ribosomal complexes, including a translating 80S ribosome bound to mRNA and tRNA. These structures reveal that RACK1 occupies the conserved binding site on the 40S subunit observed in other eukaryotic ribosomes. We also determined the architecture of the ribosomal P-stalk and identified the ribosomal proteins uL10 and uL11, which were not observed in previous T. gondii ribosome structures. In addition, we determined structures of the 80S ribosome bound to mRNA, tRNA, and the translation inhibitor emetine in two distinct conformational states. These snapshots reveal two mechanisms by which emetine inhibits the translocation step of mRNA translation: either by dislodging the mRNA from the E-site of the ribosome or by acting as a molecular glue within the E-site, thereby stalling translocation. Together, these findings provide new insights into the molecular basis of protein synthesis in apicomplexan parasites and establish a structural framework for the development of future antiparasitic therapeutics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/740346v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@18b69d5org.highwire.dtl.DTLVardef@1d125e5org.highwire.dtl.DTLVardef@efc11org.highwire.dtl.DTLVardef@1c6d088_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Human tumor suppressor protein Pdcd4 binds at the mRNA entry channel in 40S small ribosomal subunit

Translation is regulated mainly in the initiation step, and its dysregulation is implicated in many human diseases. Several proteins have been found to regulate translational initiation, including Pdcd4 (programmed cell death gene 4). Pdcd4 is a tumor suppressor protein that prevents cell growth, invasion, and metastasis. It is downregulated in most tumor cells, while global translation in the cell is upregulated. To understand the mechanisms underlying translational control by Pdcd4, we used single-particle cryo-electron microscopy to determine the structure of human Pdcd4 bound to 40S small ribosomal subunit, including Pdcd4-40S and Pdcd4-40S-eIF4A-eIF3-eIF1 complexes. The structures reveal the binding site of Pdcd4 at the mRNA entry site in the 40S, where the C-terminal domain (CTD) interacts with eIF4A at the mRNA entry site, while the N-terminal domain (NTD) is inserted into the mRNA channel and decoding site. The structures, together with quantitative binding and in vitro translation assays, shed light on the critical role of the NTD for the recruitment of Pdcd4 to the ribosomal complex and suggest a model whereby Pdcd4 blocks the eIF4F-independent role of eIF4A during recruitment and scanning of the 5' UTR of mRNA.

biochemistry↗

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↗