Biology subjects

Weingarten-Gabbay, S.

Publications and source records attributed to Weingarten-Gabbay, S..

6 recordsLinked to original sources

bioRxiv2026

Amino Acid Stress Induces Non-AUG Initiation of c-myc Translation

Initiation of translation at non-AUG start codons can generate novel isoforms of important cellular regulators, with activities or localization distinct from their AUG-initiated counterparts. Ribosomes scanning the 5UTR upstream of canonical AUG starts recognize near-cognate starts inefficiently, however, and the mechanisms by which non-canonical starts might be regulated physiologically are poorly understood. We show here that the restriction of utilization of individual amino acid induces a switch in expression of c-myc from its AUG-initiated isoform, c-myc-2, to an upstream CUG-initiated isoform, c-myc-1. This switch is enhanced in cells lacking GCN2, the protein kinase linking amino acid stress to phosphorylation and inhibition of eif2. Induction of upstream CUG start site usage in c-myc by restriction of proline utilization depends on the proximity of proline codons downstream of the canonical AUG start site. These observations support a model in which the stalling of an elongating ribosome near a canonical start leads to the accumulation of a ribosome queue that facilitates initiation at an upstream non-canonical start site. We find similar effects associated with deprivation of amino acids other than proline, and isoform changes with ATF-3 as well as c-myc. These findings suggest a broad mechanism by which amino acid stress can regulate translation initiation site use to modify the N-terminal proteome.

cell biology↗

bioRxiv2023

Pan-viral ORFs discovery using Massively Parallel Ribosome Profiling

Unveiling the complete proteome of viruses is crucial to our understanding of the viral life cycle and interaction with the host. We developed Massively Parallel Ribosome Profiling (MPRP) to experimentally determine open reading frames (ORFs) in 20,170 designed oligonucleotides across 679 human-associated viral genomes. We identified 5,381 ORFs, including 4,208 non-canonical ORFs, and show successful detection of both annotated coding sequences (CDSs) and reported non-canonical ORFs. By examining immunopeptidome datasets of infected cells, we found class I human leukocyte antigen (HLA-I) peptides originating from non-canonical ORFs identified through MPRP. By inspecting ribosome occupancies on the 5UTR and CDS regions of annotated viral genes, we identified hundreds of upstream ORFs (uORFs) that negatively regulate the synthesis of canonical viral proteins. The unprecedented source of viral ORFs across a wide range of viral families, including highly pathogenic viruses, expands the repertoire of vaccine targets and exposes new cis-regulatory sequences in viral genomes.

systems biology↗

bioRxiv2023

The HLA-II immunopeptidome of SARS-CoV-2

Targeted synthetic vaccines have the potential to transform our response to viral outbreaks; yet the design of these vaccines requires a comprehensive knowledge of viral immunogens, including T-cell epitopes. Having previously mapped the SARS-CoV-2 HLA-I landscape, here we report viral peptides that are naturally processed and loaded onto HLA-II complexes in infected cells. We identified over 500 unique viral peptides from canonical proteins, as well as from overlapping internal open reading frames (ORFs), revealing, for the first time, the contribution of internal ORFs to the HLA-II peptide repertoire. Most HLA-II peptides co-localized with the known CD4+ T cell epitopes in COVID-19 patients. We also observed that two reported immunodominant regions in the SARS-CoV-2 membrane protein are formed at the level of HLA-II presentation. Overall, our analyses show that HLA-I and HLA-II pathways target distinct viral proteins, with the structural proteins accounting for most of the HLA-II peptidome and non-structural and non-canonical proteins accounting for the majority of the HLA-I peptidome. These findings highlight the need for a vaccine design that incorporates multiple viral elements harboring CD4+ and CD8+ T cell epitopes to maximize the vaccine effectiveness.

microbiology↗

bioRxiv2020

SARS-CoV-2 infected cells present HLA-I peptides from canonical and out-of-frame ORFs

T cell-mediated immunity may play a critical role in controlling and establishing protective immunity against SARS-CoV-2 infection; yet the repertoire of viral epitopes responsible for T cell response activation remains mostly unknown. Identification of viral peptides presented on class I human leukocyte antigen (HLA-I) can reveal epitopes for recognition by cytotoxic T cells and potential incorporation into vaccines. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two human cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleoprotein not captured by current vaccines. Proteomics analyses of infected cells revealed that SARS-CoV-2 may interfere with antigen processing and immune signaling pathways. Based on the endogenously processed and presented viral peptides that we identified, we estimate that a pool of 24 peptides would provide one or more peptides for presentation by at least one HLA allele in 99% of the human population. These biological insights and the list of naturally presented SARS-CoV-2 peptides will facilitate data-driven selection of peptides for immune monitoring and vaccine development.

immunology↗

bioRxiv2020

The coding capacity of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing Coronavirus disease 19 (COVID-19) pandemic 1,2. In order to understand SARS-CoV-2 pathogenicity and antigenic potential, and to develop diagnostic and therapeutic tools, it is essential to portray the full repertoire of its expressed proteins. The SARS-CoV-2 coding capacity map is currently based on computational predictions and relies on homology to other coronaviruses. Since coronaviruses differ in their protein array, especially in the variety of accessory proteins, it is crucial to characterize the specific collection of SARS-CoV-2 proteins in an unbiased and open-ended manner. Utilizing a suite of ribosome profiling techniques 3-8, we present a high-resolution map of the SARS-CoV-2 coding regions, allowing us to accurately quantify the expression of canonical viral open reading frames (ORF)s and to identify 23 novel unannotated viral translated ORFs. These ORFs include upstream ORFs (uORFs) that are likely playing a regulatory role, several in-frame internal ORFs lying within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. We further show that viral mRNAs are not translated more efficiently than host mRNAs; rather, virus translation dominates host translation due to high levels of viral transcripts. Overall, our work reveals the full coding capacity of SARS-CoV-2 genome, providing a rich resource, which will form the basis of future functional studies and diagnostic efforts.

microbiology↗

bioRxiv2017

Deciphering Transcriptional Regulation of Human Core Promoters

Despite its pivotal role in regulating transcription, our understanding of core promoter function, architecture, and cis-regulatory elements is lacking. Here, we devised a highthroughput assay to quantify the activity of [~]15,000 fully designed core promoters that we integrated and expressed from a fixed location within the human genome. We find that core promoters drive transcription unidirectionally, and that sequences originating from promoters exhibit stronger activity than sequences originating from enhancers. Testing multiple combinations and distances of core promoter elements, we observe a positive effect of TATA and Initiator, a negative effect of BREu and BREd, and a 10bp periodicity in the optimal distance between the TATA and the Initiator. By comprehensively screening TF binding-sites, we show that site orientation has little effect, that the effect of binding site number on expression is factor-specific, and that there is a striking agreement between the effect of binding site multiplicity in our assay and the tendency of the TF to appear in homotypic clusters throughout the genome. Overall, our results systematically assay the elements that drive expression in core- and proximal-promoter regions and shed light on organization principles of regulatory regions in the human genome.

genomics↗