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Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance.

ABSTRACT: Recent studies have reported that competitive endogenous RNAs (ceRNAs) can act as sponges for a microRNA (miRNA) through their binding sites and that changes in ceRNA abundances from individual genes can modulate the activity of miRNAs. Consideration of this hypothesis would benefit from knowing the quantitative relationship between a miRNA and its endogenous target sites. Here, we altered intracellular target site abundance through expression of an miR-122 target in hepatocytes and livers and analyzed the effects on miR-122 target genes. Target repression was released in a threshold-like manner at high target site abundance (?1.5 × 10(5) added target sites per cell), and this threshold was insensitive to the effective levels of the miRNA. Furthermore, in response to extreme metabolic liver disease models, global target site abundance of hepatocytes did not change sufficiently to affect miRNA-mediated repression. Thus, modulation of miRNA target abundance is unlikely to cause significant effects on gene expression and metabolism through a ceRNA effect.

SUBMITTER: Denzler R

PROVIDER: S-EPMC4267251 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance.

Denzler Rémy R Agarwal Vikram V Stefano Joanna J Bartel David P DP Stoffel Markus M

Molecular cell 20140501 5

Recent studies have reported that competitive endogenous RNAs (ceRNAs) can act as sponges for a microRNA (miRNA) through their binding sites and that changes in ceRNA abundances from individual genes can modulate the activity of miRNAs. Consideration of this hypothesis would benefit from knowing the quantitative relationship between a miRNA and its endogenous target sites. Here, we altered intracellular target site abundance through expression of an miR-122 target in hepatocytes and livers and a ...[more]

PMID: 24793693

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Project description:BackgroundIn the competing endogenous RNA (ceRNA) hypothesis, different transcripts communicate through a competition for their common targeting microRNAs (miRNAs). Individual examples have clearly shown the functional importance of ceRNA in gene regulation and cancer biology. It remains unclear to what extent gene expression levels are regulated by ceRNA in general. One major hurdle to studying this problem is the intertwined connections in miRNA-target networks, which makes it difficult to isolate the effects of individual miRNAs.ResultsHere we propose computational methods for decomposing a complex miRNA-target network into largely autonomous modules called microRNA-target biclusters (MTBs). Each MTB contains a relatively small number of densely connected miRNAs and mRNAs with few connections to other miRNAs and mRNAs. Each MTB can thus be individually analyzed with minimal crosstalk with other MTBs. Our approach differs from previous methods for finding modules in miRNA-target networks by not making any pre-assumptions about expression patterns, thereby providing objective information for testing the ceRNA hypothesis. We show that the expression levels of miRNAs and mRNAs in an MTB are significantly more anti-correlated than random miRNA-mRNA pairs and other validated and predicted miRNA-target pairs, demonstrating the biological relevance of MTBs. We further show that there is widespread correlation of expression between mRNAs in same MTBs under a wide variety of parameter settings, and the correlation remains even when co-regulatory effects are controlled for, which suggests potential widespread expression buffering between these mRNAs, which is consistent with the ceRNA hypothesis. Lastly, we also propose a potential use of MTBs in functional annotation of miRNAs.ConclusionsMTBs can be used to help identify autonomous miRNA-target modules for testing the generality of the ceRNA hypothesis experimentally. The identified modules can also be used to test other properties of miRNA-target networks in general.

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.

Project description:BACKGROUND: RNA-seq is now widely used to quantitatively assess gene expression, expression differences and isoform switching, and promises to deliver results for the entire transcriptome. However, whether the transcriptional state of a gene can be captured accurately depends critically on library preparation, read alignment, expression estimation and the tests for differential expression and isoform switching. There are comparisons available for the individual steps but there is not yet a systematic investigation which specific genes are impacted by biases throughout the entire analysis workflow. It is especially unclear whether for a given gene, with current methods and protocols, expression changes and isoform switches can be detected. RESULTS: For the human genes, we report their detectability under various conditions using different approaches. Overall, we find that the input material has the biggest influence and may, depending on the protocol and RNA degradation, exhibit already strong length-dependent over- and underrepresentation of transcripts. The alignment step aligns for 50% of the isoforms up to 99% of the reads correctly; only in the presence of transcript modifications mainly short isoforms will have a low alignment rate. In our dataset, we found that, depending on the aligner and the input material used, the expression estimation of up to 93% of the genes being accurate within a factor of two; with the deviations being due to ambiguous alignments. Detection of differential expression using a negative-binomial count model works reliably for our simulated data but is dependent on the count accuracy. Interestingly, using the fold-change instead of the p-value as a score for differential expression yields the same performance in the situation of three replicates and the true change being two-fold. Isoform switching is harder to detect and for at least 109 genes the isoform differences evade detection independent of the method used. CONCLUSIONS: RNA-seq is a reliable tool but the repetitive nature of the human genome makes the origin of the reads ambiguous and limits the detectability for certain genes. RNA-seq does not equally well represent isoforms independent of their size which may range from ~200nt to ~100'000nt. Researchers are advised to verify that their target genes do not have extreme properties with respect to repeated regions, GC content, and isoform length and complexity.

Dataset Information

Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance.

Publications

Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance.

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