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Integrative transcriptomic and proteomic profiling of the effects of cell confluency on gene expression

ABSTRACT: In this study we examine the impact of cell confluency on gene expression. We focused on Argonaute (AGO) protein dynamics and associated gene and protein expression in HEK293, A375, and SHSY5Y cell lines. As a consequence of cell confluency, AGO2 protein translocates into the nucleus. Therefore, we generated transcriptomic data using RNA sequencing to compare gene expression in subconfluent versus confluent cells, which highlighted significant alterations in gene regulation patterns directly corresponding to changes in cell density. Our study also encompasses miRNA profiling data obtained through small RNA sequencing, revealing miRNA expressional changes dependent on cellular confluency, as well as cellular localization. Finally, we derived proteomic data from mass spectrometry analyses following AGO1-4 immunoprecipitation, providing a comprehensive view of AGO interactome in both nuclear and cytoplasmic compartments under varying confluency. These datasets offer a detailed exploration of the cellular and molecular dynamics, influenced by cell confluency, presenting a valuable resource for further research in cellular biology, particularly in understanding the basic mechanisms of cell density in cancer cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE249290 | GEO | 2024/04/19

REPOSITORIES: GEO

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Project description:Small non-coding RNAs have emerged as key players in modulation of viral infection. A unique example is the critical dependence of hepatitis C virus (HCV) on the liver-specific microRNA (miRNA), miR-122, which has surfaced as therapeutic target. Here, we used crosslinking immunoprecipitation (CLIP) of the Argonaute (AGO) protein to characterize strengths and specificities of miRNA interactions across 15 viral genomes. Intriguingly, replication of pestiviruses, which are major threats to milk and meat industry, critically depends on cellular miR-17 and let-7 interactions with the viral 3âUTR. Like HCV, miRNA binding enhanced translation and prevented viral RNA degradation. On the cellular transcriptome, pestiviral miR-17 sequestration in vitro and ex vivo conferred reduced AGO binding and functional mRNA de-repression for miR-17 targets. These findings generalize the concept of RNA virus dependence on cellular miRNAs, highlight such interactions as therapeutic targets, and connect functional regulation of the transcriptome in primary cells to miRNA sequestration. Several subseries of analyses were performed. For each sample, subseries to which it belongs are indicated. In the âVirus AGO-CLIPâ subseries, AGO-CLIP was performed on cells infected with virus as indicated. Processed reads were aligned to the host genome (hg18 or BosTau7) and to the respective viral genome. The primary data of interest from this subseries concerns AGO binding to viral RNA, and is given as processed data file âTable S1â. In the âVirus AGO-CLIP: BVDV vs. Mockâ subseries, AGO-CLIP was performed on four replicates each of BVDV and mock infected MDBK cells. CLIP reads aligned to BosTau7 were clustered, and differential analysis was performed on binding to each cluster. In addition, differential analysis of AGO bound miRNAs was performed. The associated data is given as processed data file âTable S3 and S4â. In the âAGO-CLIP: tinyLNA-17 vs. Mockâ AGO-CLIP was performed on four replicates each of tinyLNA-17 and mock treated MDBK cells. CLIP reads aligned to BosTau7 were clustered, and differential analysis was performed on binding to each cluster. The associated data is given as processed data file âTable S5â. In the âRNA-seq: BVDV vs. Mockâ subseries, mRNA-seq was performed on two replicates each of MDBK cells infected with different biotypes of BVDV or with miR-17 seed site mutant BVDV. The latter (incl. mock controls) were trans-complemented with the corresponding mutated miR-17. Differential gene expression analysis was performed between selected conditions. The associated data is given as processed data file âTable S6â.

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Integrative transcriptomic and proteomic profiling of the effects of cell confluency on gene expression

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