ABSTRACT: Primary human foreskin fibroblasts (HFF) were infected with wild-type simplex virus 1 (HSV-1) strain 17 at a multiplicity of infection (MOI) of 10. Ribosome profiling was performed at various times during infection with minor modification to the protocol described in Stern-Ginossar N et al., Science 2012. Translation start site profiling was performed by culturing cells in presence of Harringtonin or Lactimidomycin for 30 min prior to cell harvest. Overall design: Ribosome profiling was performed a 0, 1, 2, 4, 6 and 8 h post infection. Translation start site profiling was performed using Harringtonin (2µ/ml for for 30 min) or Lactimidomycin (50µM for 30 min) prior to cell lysis. Two biological replicates were analysed.
Project description:Translation can initiate at alternate, non-canonical start codons in response to stressful stimuli in mammalian cells. Recent studies suggest that viral infection and anti-viral responses alter sites of translation initiation, and in some cases, lead to production of novel immune epitopes. Here we systematically investigate the extent and impact of alternate translation initiation in cells infected with influenza virus. We perform evolutionary analyses that suggest selection against non-canonical initiation at CUG codons in influenza virus lineages that have adapted to mammalian hosts. We then use ribosome profiling with the initiation inhibitor lactimidomycin to experimentally delineate translation initiation sites in a human lung epithelial cell line infected with influenza virus. We identify several candidate sites of alternate initiation in influenza mRNAs, all of which occur at AUG codons that are downstream of canonical initiation codons. One of these candidate downstream start sites truncates 14 amino acids from the N-terminus of the N1 neuraminidase protein, resulting in loss of its cytoplasmic tail and a portion of the transmembrane domain. This truncated neuraminidase protein is expressed on the cell surface during influenza virus infection, is enzymatically active, and is conserved in most N1 viral lineages. We do not detect globally higher levels of alternate translation initiation on host transcripts upon influenza infection or during the anti-viral response, but the subset of host transcripts induced by the anti-viral response is enriched for alternate initiation sites. Together, our results systematically map the landscape of translation initiation during influenza virus infection, and shed light on the evolutionary forces shaping this landscape.
Project description:Ribosome profiling (RiboSeq) is a high-throughput sequencing technique for globally mapping the positions of translating ribosomes on the transcriptome. We infected Caco2 cells with human astrovirus 1 (HAstV1). Cells were harvested at 12 hpi and either flash frozen with no pre-treatment (NT), or pre-treated with lactimidomycin for 30 minutes followed by flash freezing (LTM). These samples where then used for ribosome profiling.
Project description:Understanding translational control in gene expression relies on precise and comprehensive determination of translation initiation sites (TIS) across the entire transcriptome. The recently developed ribosome-profiling technique enables global translation analysis, providing a wealth of information about both the position and the density of ribosomes on mRNAs. Here we present an approach, global translation initiation sequencing, applying in parallel the ribosome E-site translation inhibitors lactimidomycin and cycloheximide to achieve simultaneous detection of both initiation and elongation events on a genome-wide scale. This approach provides a view of alternative translation initiation in mammalian cells with single-nucleotide resolution. Systemic analysis of TIS positions supports the ribosome linear-scanning mechanism in TIS selection. The alternative TIS positions and the associated ORFs identified by global translation initiation sequencing are conserved between human and mouse cells, implying physiological significance of alternative translation. Our study establishes a practical platform for uncovering the hidden coding potential of the transcriptome and offers a greater understanding of the complexity of translation initiation.
Project description:Translation initiation site (TIS) profiling allows for the genome-wide identification of TISs <i>in vivo</i> by exclusively capturing mRNA fragments within ribosomes that have just completed translation initiation. It leverages translation inhibitors, such as harringtonine and lactimidomycin (LTM), that preferentially capture ribosomes at start codon positions, protecting TIS-derived mRNA fragments from nuclease digestion. Here, we describe a step-by-step protocol for TIS profiling in LTM-treated budding yeast that we developed to identify TISs and open reading frames in vegetative and meiotic cells. For complete details on the use and execution of this protocol, please refer to Eisenberg et al. (2020).
Project description:An increasing amount of studies integrate mRNA sequencing data into MS-based proteomics to complement the translation product search space. We present the generation of a protein synthesis-based database from deep sequencing of ribosome-protected mRNA fragments. This approach increases the overall protein identification rates with 3% and 11% (improved and new identifications) for human and mouse respectively and enables proteome-wide detection of 5’-extended proteoforms, uORF translation and near-cognate translation start sites. Overall design: Ribosome profiling of lactimidomycin and cycloheximide treated HCT116 cells
Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first study on protein turnover using positional proteomics and ribosome profiling to distinguish between N-terminal proteoforms of individual genes. Overall, we monitored the stability of 1,941 human N-terminal proteoforms, including 147 N-terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. Ribosome profiling of lactimidomycin and cycloheximide treated human Jurkat T-lymphocytes
Project description:Tetracycline-inhibited ribosome profiling (TetRP) provides a powerful new experimental tool for comprehensive genome-wide identification of translation initiation sites in bacteria. We validated TetRP by confirming the translation start sites of protein-coding genes in accordance with the 2006 version of Escherichia coli K-12 annotation record (GenBank U000962) and found ?150 new start sites within 60 nucleotides of the annotated site. This analysis revealed 72 per cent of the genes whose initiation site annotations were changed from the 2006 GenBank record to the newer 2014 annotation record (GenBank U000963), indicating a high sensitivity. Also, results from reporter fusion and proteomics of N-terminally enriched peptides showed high specificity of the TetRP results. In addition, we discovered over 300 translation start sites within non-coding, intergenic regions of the genome, using a threshold that retains ?2,000 known coding genes. While some appear to correspond to pseudogenes, others may encode small peptides or have previously unforeseen roles. In summary, we showed that ribosome profiling upon translation inhibition by tetracycline offers a simple, reliable and comprehensive experimental tool for precise annotation of translation start sites of expressed genes in bacteria.
Project description:Translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate codons. We show that queuing of scanning ribosomes behind a paused elongating ribosome promotes initiation at upstream weak start sites. Ribosomal profiling reveals polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in an inhibitory non-AUG-initiated upstream conserved coding region (uCC) of the antizyme inhibitor 1 (AZIN1) mRNA, encoding a regulator of cellular polyamine synthesis. Mutation of the PPW motif impairs initiation at the uCC's upstream near-cognate AUU start site and derepresses AZIN1 synthesis, whereas substitution of alternate elongation pause sequences restores uCC translation. Impairing ribosome loading reduces uCC translation and paradoxically derepresses AZIN1 synthesis. Finally, we identify the translation factor eIF5A as a sensor and effector for polyamine control of uCC translation. We propose that stalling of elongating ribosomes triggers queuing of scanning ribosomes and promotes initiation by positioning a ribosome near the start codon.
Project description:BACKGROUND:The retrovirus murine leukemia virus (MuLV) has an 8.3 kb RNA genome with a simple 5'-gag-pol-env-3' architecture. Translation of the pol gene is dependent upon readthrough of the gag UAG stop codon; whereas the env gene is translated from spliced mRNA transcripts. Here, we report the first high resolution analysis of retrovirus gene expression through tandem ribosome profiling (RiboSeq) and RNA sequencing (RNASeq) of MuLV-infected cells. RESULTS:Ribosome profiling of MuLV-infected cells was performed, using the translational inhibitors harringtonine and cycloheximide to distinguish initiating and elongating ribosomes, respectively. Meta-analyses of host cell gene expression demonstrated that the RiboSeq datasets specifically captured the footprints of translating ribosomes at high resolution. Direct measurement of ribosomal occupancy of the MuLV genomic RNA indicated that ~ 7% of ribosomes undergo gag stop codon readthrough to access the pol gene. Initiation of translation was found to occur at several additional sites within the 5' leaders of the gag and env transcripts, upstream of their respective annotated start codons. CONCLUSIONS:These experiments reveal the existence of a number of previously uncharacterised, ribosomally occupied open reading frames within the MuLV genome, with possible regulatory consequences. In addition, we provide the first direct measurements of stop codon readthrough efficiency during cellular infection.
Project description:Translation of mRNA sequences into proteins typically starts at an AUG triplet. In rare cases, translation may also start at alternative non-AUG codons located in the annotated 5' UTR which leads to an increased regulatory complexity. Since ribosome profiling detects translational start sites at the nucleotide level, the properties of these start sites can then be used for the statistical evaluation of functional open reading frames. We developed a linear regression approach to predict in-frame and out-of-frame translational start sites within the 5' UTR from mRNA sequence information together with their translation initiation confidence. Predicted start codons comprise AUG as well as near-cognate codons. The underlying datasets are based on published translational start sites for human HEK293 and mouse embryonic stem cells that were derived by the original authors from ribosome profiling data. The average prediction accuracy of true vs. false start sites for HEK293 cells was 80%. When applied to mouse mRNA sequences, the same model predicted translation initiation sites observed in mouse ES cells with an accuracy of 76%. Moreover, we illustrate the effect of in silico mutations in the flanking sequence context of a start site on the predicted initiation confidence. Our new webservice PreTIS visualizes alternative start sites and their respective ORFs and predicts their ability to initiate translation. Solely, the mRNA sequence is required as input. PreTIS is accessible at http://service.bioinformatik.uni-saarland.de/pretis.