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Pervasive cis-antisense transcription negatively regulates gene expression in E. coli K12 in response to environmental changes

ABSTRACT: cis-antisense RNAs (cis-asRNAs) in prokaryotes are more pervasive than originally thought. However, little is known about their expression patterns and functions. Here we determined transcriptomes and proteomes of E. coli at multiple time points in five culture conditions using directional RNA-seq and tandem mass spectrometry. We found that a highly varying portion (27.2%~90.8%) of transcribed genes had cis-asRNAs dependent on growth phases and culture conditions, and that almost all transcribed genes changed their cis-asRNA levels relative to the mRNA level at different growth phases and culture conditions. Intriguingly, the correlation between the protein and mRNA levels of genes is abolished the increasing cis-asRNA levels relative to the mRNA levels, suggesting that cis-asRNAs might directly or indirectly inhibit translation by forming duplexes with mRNAs. All data sets are available for data mining from a unified resource to support further biological discoveries and insights into cis-asRNA-mediated gene expressional regulation.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Escherichia Coli Str. K-12 Substr. Mg1655

SUBMITTER: Ehsan Tabari

LAB HEAD: Zhengchang Su

PROVIDER: PXD001640 | Pride | 2022-02-28

REPOSITORIES: Pride

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			Action	DRS
	022313_HS1_01.RAW	Raw
	022313_HS1_01.mzid.gz	Mzid
	022313_HS1_01.mzid_022313_HS1_01.MGF	Mzid
	022313_HS1_01.mzid_022313_HS1_01.pride.mgf.gz	Mzid
	022313_HS1_01.pride.mztab.gz	Mztab

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Project description:Lung cancer is one of the most common and fatal cancer worldwide. There are two major types of lung cancer, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Recently, gefitinib, a small molecule, which target tyrosine kinase, has been regarded as the first-line treatment in NSCLC patients. However, several patients have been observed tumor recurrence and eventually developed progressive outcomes after target therapy. Thus, an effective therapeutic approach need to be explored. Here, we found that ectopically expressed ATP synthase on plasma membrane exhibited gefitinib-resistance properties in lung cancer cell lines. Furthermore, we unraveled that citreoviridin, an ectopic ATP synthase inhibitor, suppressed the abilities of both proliferation and colony formation in lung cancer cell lines. To elucidate the comprehensive mechanism regulated by citreoviridin, we performed microarray analysis. The results indicated that not only mRNAs but also long non-coding RNAs (lncRNAs) are involved in citreoviridin-treated cell death. One of the well-known lncRNAs, growth arrest-specific transcript (GAS5), was robustly upregulated after citreoviridin treatment. In order to investigate the upstream modulator of GAS5, we utilized chromatin immunoprecipitation (ChIP) assay and revealed that E2F transcription factor 1 (E2F1) could bind to the promoter of GAS5. Consistently, both microarray and qPCR data showed the expression level of E2F1 was negatively correlated to GAS5 after citreoviridin treatment. The evidence suggests that E2F1 might be a potential repressor of GAS5. Furthermore, combining microarray and Gene Set Enrichment Analysis (GSEA) analysis as well as qPCR demonstrated that p53 pathway was activated. To further realize the GAS5-p53 regulating network, RNA-protein pull-down assay followed by LC-MS/MS will be utilized to dissect the GAS5-interacting proteomic profiling.From the results, we identified 107 GAS5-interacting proteins in common from A549 and H1975 cell lines. Our proteomics experiments identified topoisomerase 2-alpha (TOP2A) as the key protein involved in the citreoviridin-regulated gefitinib-resistance pathway, therefore, we picked out TOP2A for further study. Additionally, we further validated the interaction between TOP2A and GAS5 by western blot and RNA immunoprecipitation (RIP). Taken together, this study suggests targeting E2F1/GAS5/p53 axis is a potential therapeutic strategy for gefitinib-resistant lung cancer.

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Pervasive cis-antisense transcription negatively regulates gene expression in E. coli K12 in response to environmental changes

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