Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-Seq of H1299 cells transfected with different types of bi-directional plasmid to better understand the transcriptional changes occurring with the different design frameworks

ABSTRACT: Our previous work has shown that exogenous DNA impose a burden to the host cells due to the finite amount of transcriptional and translational resources, resulting in a limiting productivity capacity. We thus designed context-aware genetic networks in which miRNAs were successfully exploited as burden mitigators, and observed upregulation of protein expression in the targeted cells. We co-transfected H1299, the testbed of our previous studies with a bi-directional plasmid encoding EGFP and mKate, either in an open loop circuit architecture (OL) that lack miRNA regulation, or in the form of a miRNA-iFFL, placing miR31 target sites, highly expressed in this cell line, in the 3’ (iFFL-3’) or 5’(iFFL-5’) UTR of mKate (Fig. 2a). We next sorted the cells that were either non-transfected (NTr) or transfected (Tr) according to fluorescence expression, to compare the OL that are more sensitive to burden, with iFFL-3’ and iFFL-5’ architectures.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Antonio Rinaldi

PROVIDER: E-MTAB-14355 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Expression changes of competitive endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts that contain miRNA binding sites. Here, we find that although miRNA levels define the extent of repression, they do not affect the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ~20% of that observed for canonical 8-nt sites. Sites with extensive additional complementarity can be even more potent, but this occurs predominantly through miRNA degradation rather than competition. Cooperative binding of closely spaced sites for different miRNAs can also increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNA and target abundance, target-site spacing and affinity requirements for ceRNA-mediated gene regulation and specify the unusual circumstances in which ceRNA-mediated gene regulation might be observed. Sixty-four mRNA profiles were generated of 1) primary hepatocytes of mice expressing variable levels of a recombinant Adenovirus expressing the transcript of AldolaseA (Ad-AldoA), containing sites matching miR-122, let-7, miR-192, miR-194 or a mutated site (no site) or 2) embryonic stem (ES) cells that were transfected with a Tet-inducible dual-color reporter construct expressing enhanced yellow fluorescent protein (eYFP) and mCherry that contains zero (0s) or three (3s) 8 nt miRNA seed matches for miR-293 or miR-92 in the 3â?² UTR. ES cells were sorted with a FACSAria IIIu flow cytometer into three bins based on their eYFP fluorescence intensity. All samples were sequenced in duplicates by an Illumina HiSeq 2500. Library preparation and sequencing were performed by Fasteris SA (Switzerland) or by the Functional Genomics Centre ZÃ¼rich (FGCZ). Three small RNA profiles or either primary hepatocytes or embryonic stem cells were also generated by Solexa sequencing.

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RNA-Seq of H1299 cells transfected with different types of bi-directional plasmid to better understand the transcriptional changes occurring with the different design frameworks

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