Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Kozak sequence libraries for characterizing transgenes across expression levels

ABSTRACT: Typical mammalian overexpression systems test protein sequence variants with little control over expression levels and steady-state protein abundances, hindering interpretations of how protein sequence and expression converge to yield phenotypic outcomes. We explored the translation initiation sequence, commonly referred to as the Kozak sequence, as a means to modulate protein steady-state abundance and cellular function. We performed sort-seq on a randomized library of the 6 nucleotides preceding the start codon, amounting to 4,042 sequences. Calibrating the scores revealed a ~100-fold range of protein steady-state abundances possible through manipulation of the Kozak sequence. We identified human germline variants with predicted expression-reducing Kozak substitutions in disease-associated genes. Modulating the cell surface abundance of the host cell receptor ACE2 controlled the rate at which those cells became infected by SARS-like coronavirus spike pseudotyped particles. We demonstrated the potential of the approach by simultaneously testing Kozak libraries with a small panel of coding variants for ACE2 and STIM1. This approach lays the methodological groundwork for linking the causal relationships between protein sequence, abundance, and functional outcome.

ORGANISM(S): synthetic construct

PROVIDER: GSE295498 | GEO | 2025/05/01

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Analysis of Kozak sequences variants of haploinsufficient genes

Project description:We conceived that the availability of base editors and the possible presence of suboptimal Kozak sequences in the human genome could provide an elegant approach to modulate translational efficiency for the molecular compensation of some haploinsufficient disorders. Moreover, such an approach would be independent of the type of alteration inactivating the defective allele and, therefore, highly suitable for gene therapy protocols. To find base conversions upregulating translational efficiency, we systematically screened 5261 (4621 unique) variants of Kozak sequences for translational strength. We designed these variants in the main AUG context of 230 previously annotated haploinsufficient genes. Analysis of our library of Kozak variants, bearing the conversions that base editors can reproduce, proved that weak Kozak sequences are present at about a 20% frequency in our gene sample. We validated the variants of five genes and base edited one of them in a cell model, providing proof of principle of the approach, which we called BOOST (Base editing cOrrection of haplOinSufficiency by Translational enhancement).

2022-09-13 | GSE210035 | GEO

Rps26 directs mRNA-specific translation by recognition of Kozak sequence elements

Project description:The “Kozak sequence”, immediately upstream of the start codon, regulates the translational efficiency of an mRNA. Nevertheless, how this sequence is recognized remains unexplored. Here, we describe a novel approach to separate two ribosome populations from the same cells and use this method, and RNA-seq, to identify the mRNAs bound to ribosomes with and without Rps26, a protein linked to the pathogenesis of Diamond Blackfan Anemia (DBA). These analyses reveal that Rps26 contributes to recognition of the Kozak sequence in well-translated mRNAs, and that Rps26-deficient ribosomes preferentially translate mRNA from select stress response pathways. Surprisingly, we demonstrate that exposure of cells to these stresses leads to the formation of Rps26-deficient ribosomes and to the translation of their targets. Thus, Rps26-deficient ribosomes play aberrant pathogenic roles in DBA as well as physiological roles in augmenting the well-characterized stress transcriptional response with a heretofore unknown translational stress response, thereby creating a feed forward loop in gene-expression.

2017-07-30 | GSE86203 | GEO

Analysis of Kozak sequences variants of haploinsufficient genes

Project description:Analysis of Kozak sequences variants of haploinsufficient genes

| PRJNA863493 | ENA

Causal signals between codon bias, mRNA structure and the efficiency of translation and elongation

Project description:Ribosome profiling data reports on the distribution of translating ribosomes, at steady-state, with codon-level resolution. We present a robust method to extract codon translation rates and protein synthesis rates from these data, and identify causal features associated with elongation and translation efficiency in physiological conditions in yeast. We show that neither elongation rate nor translational efficiency is improved by experimental manipulation of the abundance or body sequence of the rare AGG tRNA. Deletion of three of the four copies of the heavily used ACA tRNA shows a modest efficiency decrease that could be explained by other rate-reducing signals at gene start. This suggests that correlation between codon bias and efficiency arises as selection for codons to utilize translation machinery efficiently in highly translated genes. We also show a correlation between efficiency and RNA structure calculated both computationally and from recent structure probing data, as well as the Kozak initiation motif, which may comprise a mechanism to regulate initiation. We test whether tRNA abundance affects elongation or translation efficiency by changing the tRNA levels through deletion or over expression and measuring the ribosomal dwell time at each codon using a robust statistical method that accounts for flow conservation.

2015-01-05 | E-GEOD-63789 | biostudies-arrayexpress

HOT3/eIF5B1 confers Kozak motif-dependent translational control of photosynthesis-associated nuclear genes for chloroplast biogenesis

Project description:Photosynthesis requires chloroplasts, where most proteins are nucleus-encoded and produced via cytoplasmic translation. The translation initiation factor eIF5B gates the transition from initiation (I) to elongation (E), and the Kozak motif is associated with translation efficiency, but their relationship is previously unknown. Here, with ribosome profiling, we determined the genome-wide I-E transition efficiencies. We discovered that the most prevalent Kozak motif is associated with high I-E transition efficiency in Arabidopsis, rice, and wheat, thus implicating the Kozak motif in facilitating the I-E transition. Indeed, the effects of most Kozak motifs, including the most effective ones, depend on HOT3/eIF5B1 in Arabidopsis. HOT3 preferentially promotes the translation of photosynthesis-associated nuclear genes in a Kozak motif-dependent manner, which explains the chloroplast defects of hot3 mutants. Our study reveals an evolutionarily-conserved link between the Kozak motif and eIF5B-mediated translational I-E efficiency and elucidates a HOT3-mediated translational control of nuclear photosynthesis genes for chloroplast biogenesis.

2024-10-03 | GSE212856 | GEO

HOT3/eIF5B1 confers Kozak motif-dependent translational control of photosynthesis-associated nuclear genes for chloroplast biogenesis

2024-10-03 | GSE212855 | GEO

Kozak sequence libraries for characterizing transgenes across expression levels

Project description:Kozak sequence libraries for characterizing transgenes across expression levels

| PRJNA1254985 | ENA

Causal signals between codon bias, mRNA structure and the efficiency of translation and elongation

2015-01-05 | GSE63789 | GEO

Multidimensional PTEN missense variant analysis reveals variant subgroups including potential dominant negatives

Project description:Determining the pathogenicity of human genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes will likely require generalizable, scalable assays. We previously developed Variant Abundance by Massively Parallel Sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance in a single experiment. Here, we reapplied VAMP-seq to quantify the abundances of additional PTEN missense variants which were missing from the original experiment.

2021-09-13 | GSE159469 | GEO

Expression data from mouse collecting duct cell, mpkCCD, in response to a peptide hormone vasopressin analog, dDAVP

Project description:Vasopressin is the major hormone that regulates renal water excretion. It does so by binding to a receptor in renal collecting duct cells, triggering signaling pathways that ultimately regulate the abundance, location, and activity of the water channel protein aquaporin 2. We took an advantage of quantitative large scale proteomic technologies and oligonucleotide microarrays to quantify steady state changes in protein and transcript abundances in response to vasopressin in a collecting duct cell line, mpkCCD clone 11 (Yu et al. PNAS 2009, 106:2441-2446). This cell line originally developed by Alan Vandewalle’s group recapitulates vasopressin-mediated AQP2 expression and phosphorylation as seen in native colleting duct cells.

2010-12-09 | GSE19938 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data