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Comparative analysis between single-cell RNA-seq and single-molecule RNA FISH indicates that the pyrimidine nucleobase idoxuridine (IdU) globally amplifies transcriptional noise

ABSTRACT: Stochastic fluctuations (noise) in transcription generate substantial cell-to-cell variability, but the physiological roles of noise have remained difficult to determine in the absence of generalized noise-modulation approaches. Previous single-cell RNA-sequencing (scRNA-seq) suggested that the pyrimidine-base analog (5′-iodo-2′-deoxyuridine, IdU) could generally amplify noise without substantially altering mean-expression levels but scRNA-seq technical drawbacks potentially obscured the penetrance of IdU-induced transcriptional noise amplification. Here we quantify global-vs.-partial penetrance of IdU-induced noise amplification by assessing scRNA-seq data using numerous normalization algorithms and complement our previous results with data from Human Jurkat T Lymphocytes, including two biological replicates to allow rigorous statistical analysis. Collectively, this analysis indicates which scRNA-seq algorithms are appropriate for quantifying noise and argues that IdU is a globally penetrant noise-enhancer molecule that could enable investigations of the physiological impacts of transcriptional noise in various cell types and organisms.

ORGANISM(S): Homo sapiens

PROVIDER: GSE263194 | GEO | 2024/08/28

REPOSITORIES: GEO

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Project description:Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy and understanding disease progression. Redox factor-1 (Ref-1), a redox signaling protein, regulates the DNA binding activity of several transcription factors, including HIF-1. The conversion of HIF-1 from an oxidized to reduced state leads to enhancement of its DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia.Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions. Experimental Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model. Matched samples were also collected for bulk proteomic analysis of the four conditions. scRNA-seq data was validated using proteomics and qRT-PCR. Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays and qRT-PCR. Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions.

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Comparative analysis between single-cell RNA-seq and single-molecule RNA FISH indicates that the pyrimidine nucleobase idoxuridine (IdU) globally amplifies transcriptional noise

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