Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human HEK cells transfected with miRNA to investigate the contribution of sequence and structure to mRNA-binding of Argonaute/miRNA complexes and degradation of miRNA targets

ABSTRACT: Relative contribution of sequence and structural features to the mRNA-binding of Argonaute/miRNA complexes and the degradation of miRNA targets; How miRNAs recognize their target sites is a puzzle that many experimental and computational studies aimed to solve. Several features such as perfect pairing of the miRNA seed, additional pairing in the 3â region of the miRNA, relative position in the 3â UTR and the A/U content of the environment of the putative site have been found to be relevant. Here we have used a large number of previously published data sets to assess the power that various sequence and structure features have in distinguishing between putative sites that do and those that do not appear to be functional. We found that although different data sets can give widely different answers when it comes to ranking the relative importance of these features, the sites inferred from most transcriptomics experiments as well as from comparative genomics appear similar at this level. This suggests that miRNA target sites have been selected in evolution on their ability to trigger mRNA degradation. To understand at what step in the miRNA induced response individual features play a role, we further transfected human HEK293 cells with miRNAs and analyzed the association of Argonaute/miRNA complexes with target mRNAs and the degradation of these messages. We found that structural features are only important for Argonaute binding, while sequence features such as the A/U content of the 3â UTR are important for mRNA degradation. Experiment Overall Design: Ago2 IPs of mock, miRNA-7, and miR-124 transfected cells were analyzed for 2 biological replicates. IPs of the biological replicates were analyzed with 2 technical replicates.

ORGANISM(S): Homo sapiens

SUBMITTER: Markus Landthaler

PROVIDER: E-GEOD-14537 | 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.

Dataset Information

Transcription profiling of human HEK cells transfected with miRNA to investigate the contribution of sequence and structure to mRNA-binding of Argonaute/miRNA complexes and degradation of miRNA targets

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