Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional response of Saccharomyces cerevisiae to the proapoptotic unsaturated fatty aldehyde t-2-hexadecenal by RNAseq

ABSTRACT: Hexadecenal, a trans (-2,3-) unsaturated fatty aldehyde, is an intermediate of the sphingolipid degradation pathway. This pathway, induced during cellular stress, involves the conversion of sphingosine-1-phosphate to hexadecenal by Sphingosine-1-Phosphate-Lyase (SPL) in humans and DPL1 in yeast. Hexadecenal is further metabolized to hexadecenoic acid by Fatty Aldehyde Dehydrogenase (ALDH3A2 in humans and HFD1 in yeast). Trans-2-hexadecenal (t-2-hex), has been found to induce mitochondrial dysfunction in a conserved manner from yeast to humans. However, the specific mechanisms and biological targets underlying this lipid-induced mitochondrial inhibition remain largely unknown. In this study, we employed unbiased transcriptomic approaches using the Saccharomyces cerevisiae yeast model to elucidate the principal mechanisms and biological targets associated with t-2-hex-induced mitochondrial dysfunction. To investigate the effects of t-2-hex, we utilized an hfd1 mutant strain lacking the HFD1 gene. This strain exhibited increased sensitivity to t-2-hex treatment compared to wild-type cells. Exponentially growing hfd1 mutant cells were exposed to 100 μM t-2-hex for a duration of 3 hours, while control cells were incubated with the solvent dimethyl sulfoxide (DMSO), in which hexadecenal is dissolved. Total RNA was extracted from both treated and control cells for high-throughput sequencing analysis. Through transcriptomic profiling, we aimed to identify differentially expressed genes, pathways, and regulatory networks associated with t-2-hex-induced mitochondrial dysfunction in yeast. This study provides a comprehensive analysis of the transcriptional response to t-2-hex treatment, shedding light on the molecular mechanisms and potential biological targets underlying lipid-induced mitochondrial dysfunction.

INSTRUMENT(S): DNBSEQ-G400

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Josep Fita-Torró

PROVIDER: E-MTAB-13188 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Lipid intermediates derived from sphingolipid metabolism are crucial regulators of mitochondrial function from yeast to humans. Among these intermediates, trans-2-hexadecenal (t-2hex) within the sphingolipid degradation pathway exhibits remarkable efficiency in inducing mitochondria-mediated cell death. In yeast cell cultures, the addition of t-2-hex triggers complete disintegration of the mitochondrial network, leading to subsequent cell death. This effect is particularly pronounced in yeast cells lacking the activity of the t-2-hex degrading enzyme, Hfd1. However, the molecular mechanisms of t-2-hex induction of mitochondrial dysfunction are completely unknown. In this project, we want to exploit the unprecedented power of yeast genetics to unveil novel genetic determinants involved in t-2-hex's pro-apoptotic function. To accomplish this, we employed the SATAY method, which combines saturated transposon mutagenesis with high-throughput sequencing to functionally explore the yeast genome. In our screening approach, hfd1 mutant cells harboring a plasmid-encoded inducible MiniDs transposon were induced by galactose, resulting in extensive integration of the transposon throughout the yeast genome. Cells with the plasmid excised and the transposon genomically integrated were pooled together, creating a high-density transposon library comprising approximately 2.3E+06 independent insertion mutants. Subsequently, the pooled mutant library was subjected to treatment with the mitochondria-mediated death inducer, t-2-hexadecenal. As a control, cells were also incubated with the solvent dimethyl sulfoxide (DMSO), in which hexadecenal is dissolved. Following the treatments, cells were collected for genomic DNA extraction and digestion, using restriction enzymes with frequent four-base pair recognition sites. The resulting library fragments were circularized using T4 DNA ligase, and the transposon-genome junctions were selectively amplified through PCR with outward-facing primers specific to the transposon. Finally, the pooled and purified amplicons were subjected to massive sequencing on an Illumina MiSeq platform. The obtained sequences were then aligned to the reference genome of Saccharomyces cerevisiae, allowing for the mapping of transposon insertions and the calculation of transposon counts per gene. This project enabled the identification of genes required for the resistance and toxicity to t-2hex.

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Transcriptional response of Saccharomyces cerevisiae to the proapoptotic unsaturated fatty aldehyde t-2-hexadecenal by RNAseq

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