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Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

ABSTRACT: Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.

SUBMITTER: Horstick EJ

PROVIDER: S-EPMC4402511 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

Horstick Eric J EJ Jordan Diana C DC Bergeron Sadie A SA Tabor Kathryn M KM Serpe Mihaela M Feldman Benjamin B Burgess Harold A HA

Nucleic acids research 20150127 7

Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of gene ...[more]

PMID: 25628360

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Project description:Random or stochastic monoallelic expressed genes (StMA genes) represent a unique form of monoallelic expression where allelic choice is made at random early in development. The consequential clonal diversity provides opportunity for functional heterozygosity in tissues such as the brain, and can impact on both development and disease. We investigate the relationship of StMA expressed genes previously identified in clonal neural stem cells with the neurodevelopmental disorders autism and schizophrenia. We found that StMA genes show an overrepresentation of schizophrenia risk candidates identified by genome wide association studies from the genetic association database. Similar suggestive enrichment was also found for genes from the NHGRI genome-wide association study catalog and a psychiatric genetics consortium schizophrenia dataset although these latter more robust gene lists did not achieve statistical significance. We also examined multiple sources of copy number variation (CNV) datasets from autism and schizophrenia cohorts. After taking into account total gene numbers and CNV size, both autism and schizophrenia associated CNVs appeared to show an enrichment of StMA genes relative to the control CNV datasets. Since the StMA genes were originally identified in neural stem cells, bias due to the neural transcriptome is possible. To address this, we randomly sampled neural stem cell expressed genes and repeated the tests. After a significant number of iterations, neural stem cell expressed genes did not show an overrepresentation in autism or schizophrenia CNV datasets. Therefore, irrespective of the neural derived transcriptome, StMA genes originally identified in neural stem cells show an overrepresentation in CNVs associated with autism and schizophrenia. If this association is functional, then the regulation (or dysregulation) of this form of allelic expression status within tissues such as the brain may be a contributory risk factor for neurodevelopmental disorders and may also influence disease discordance sometimes observed in monozygotic twins.

Dataset Information

Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

Publications

Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

Similar Datasets

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