Biology subjects

Karousis, E. D.

Publications and source records attributed to Karousis, E. D..

8 recordsLinked to original sources

bioRxiv2025

A human cell-free translation screen identifies the NT-2 mycotoxin as a ribosomal peptidyl transferase inhibitor

Translation inhibitors are invaluable for probing ribosome function and therapeutic applications, but systematic discovery in human systems is limited by the lack of scalable, screening-compatible cell-free platforms. Here, we establish a robust high-throughput screening using human lysates that bypasses cellular cytotoxic effects. After screening [~]28,000 small molecules, we identified known and a novel translation inhibitor, including NT-2, a trichothecene mycotoxin produced by the pathogenic Fusarium sporotrichioides. NT-2 suppressed protein synthesis in human cells and yeast lysates, while sparing translation in bacteria and intact yeast cells. Cryo-EM at 1.76 [A] revealed NT-2 bound at the peptidyl transferase center of the human 60S ribosome, confirming NT-2 as a ribosomal inhibitor associated with ribosomes in an inactive eEF2/SERBP1-associated dormant state. Together, these results expose NT-2 as a previously unrecognized environmental inhibitor of mammalian protein synthesis and demonstrate the power of cell-free translation screening to reveal new inhibitors with unexpected ribosome fates.

biochemistry↗

bioRxiv2024

Efficient cell-free translation from diverse human cell types

MotivationThe difficulties in producing cell-free translation systems from different cell types limit the ability to study regulatory mechanisms that depend on different biological contexts. Developing systems tailored to diverse cell types would be instrumental in investigating cell-type-specific translational control, co- and post-translational modifications, and viral manipulation strategies. Our method addresses this gap by providing a scalable and adaptable solution for producing high-quality lysates that reflect the specific needs of different cell types. SummaryCell-free translation systems are indispensable for studying protein synthesis, enabling researchers to explore translational regulation across different cell types. Here, we present a scalable method for preparing translation-competent lysates from a range of frequently used human cell lines using dual centrifugation. We optimized lysis conditions for adherent and suspension cells, producing high-quality lysates from HEK-293 (adherent and in suspension), HeLa, SH-SY5Y, and U2OS cells. Our results demonstrate that cell-specific factors influence translation efficiency, with adherent HeLa cells showing the highest activity. We also observed that sensitivity to lysis conditions varies between cell lines, underscoring the importance of fine-tuning parameters for efficient protein production. Our method provides a robust and adaptable approach for generating cell-type-specific lysates, broadening the application of in vitro translation systems in studying translational mechanisms.

biochemistry↗

bioRxiv2024

Uncoupling the functional roles of Coronavirus Nsp1

When host cells are infected with coronaviruses, the first viral protein produced is Nsp1. This protein inhibits host protein synthesis and induces host mRNA degradation to enhance viral proliferation. Despite its critical role, the mechanism by which Nsp1 mediates cellular mRNA degradation remains unclear. In this study, we use cell-free translation to address how the host mRNA stability is regulated by Nsp1. We reveal that SARS-CoV-2 Nsp1 binding to the ribosome is enough to trigger mRNA degradation independently of ribosome collisions or active translation. MERS-CoV Nsp1 inhibits translation without triggering degradation, highlighting mechanistic differences between the two Nsp1 counterparts. Nsp1 and viral mRNAs appear to co-evolve, rendering viral mRNAs immune to Nsp1-mediated degradation in SARS-CoV-2, MERS-CoV and Bat-Hp viruses. By providing new insights into the mode of action of Nsp1, our study helps to understand the biology of Nsp1 better and find new strategies for therapeutic targeting against coronaviral infections. SignificanceO_LICell-free assays allow the decoupling of Nsp1-mediated translation inhibition from RNA degradation. C_LIO_LINsp1 interaction with the ribosome is crucial for mRNA degradation, but active translation is not required. C_LIO_LISARS-CoV-2 Nsp1 induces mRNA degradation, while MERS-CoV Nsp1 inhibits translation without triggering degradation. C_LIO_LI5UTR-specific protection of viral mRNAs from Nsp1 indicates a co-evolutionary adaptation between the two features C_LI

molecular biology↗

bioRxiv2023

Universal features of Nsp1-mediated translational shutdown by coronaviruses

Nonstructural protein 1 (Nsp1) produced by coronaviruses shuts down host protein synthesis in infected cells. The C-terminal domain of SARS-CoV-2 Nsp1 was shown to bind to the small ribosomal subunit to inhibit translation, but it is not clear whether this mechanism is broadly used by coronaviruses, whether the N-terminal domain of Nsp1 binds the ribosome, or how Nsp1 specifically permits translation of viral mRNAs. Here, we investigated Nsp1 from three representative Betacoronaviruses - SARS-CoV-2, MERS-CoV, and Bat-Hp-CoV - using structural, biophysical, and biochemical assays. We revealed a conserved mechanism of host translational shutdown across the three coronaviruses. We further demonstrated that the N-terminal domain of Bat-Hp-CoV Nsp1 binds to the decoding center of the 40S subunit, where it would prevent mRNA and eIF1A binding. Structure-based biochemical experiments identified a conserved role of these inhibitory interactions in all three coronaviruses and showed that the same regions of Nsp1 are responsible for the preferential translation of viral mRNAs. Our results provide a mechanistic framework to understand how Betacoronaviruses overcome translational inhibition to produce viral proteins.

biochemistry↗

bioRxiv2021

Production of human translation-competent lysates using dual centrifugation

Protein synthesis is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions. Here we present a robust and fast method based on dual centrifugation that allows for detergent-free cell lysis under controlled mechanical forces. We optimized the lysate preparation to yield cytoplasm-enriched extracts from human cells that efficiently translate mRNAs in a cap-dependent as well as in an IRES-mediated way. Reduction of the phosphorylation state of eIF2 using recombinant GADD34 and 2-aminopurine considerably boosts the protein output, reinforcing the potential of this method to produce recombinant proteins from human lysates.

molecular biology↗

bioRxiv2021

Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells

BackgroundNonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge. ResultsTo identify and analyze endogenous targets of NMD, we applied cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identified, most derive from alternative exon usage. The isoform-aware analysis revealed many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3'UTR and, for those mRNAs with a termination codon in the last exon, the length of the 3'UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, though the main function of NMD seems to be ridding the transcriptome of isoforms resulting from spurious splicing events. ConclusionsLong-read sequencing enabled the identification of many novel NMD-sensitive mRNAs and revealed both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.

molecular biology↗

bioRxiv2020

SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation

The non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, is the first viral protein that is synthesized in SARS-CoV-2 infected human cells to suppress host innate immune functions1,2. By combining cryo-electron microscopy and biochemical experiments, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes including the 43S pre-initiation complex. The protein inserts its C-terminal domain at the entrance to the mRNA channel where it interferes with mRNA binding. We observe potent translation inhibition in the presence of Nsp1 in lysates from human cells. Based on the high-resolution structure of the 40S-Nsp1 complex, we identify residues of Nsp1 crucial for mediating translation inhibition. We further show that the full-length 5’ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines inhibition of translation by Nsp1 with efficient translation of the viral mRNA to achieve expression of viral genes3.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

Human NMD ensues independently of stable ribosome stalling

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that is important for the elimination of faulty and the regulation of normal mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system revealed similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs. Moreover, ribosome profiling revealed a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.

molecular biology↗