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PTRE-seq reveals mechanism and interactions of RNA binding proteins and miRNAs.

ABSTRACT: RNA binding proteins (RBP) and microRNAs (miRNAs) often bind sequences in 3' untranslated regions (UTRs) of mRNAs, and regulate stability and translation efficiency. With the identification of numerous RBPs and miRNAs, there is an urgent need for new technologies to dissect the function of the cis-acting elements of RBPs and miRNAs. We describe post-transcriptional regulatory element sequencing (PTRE-seq), a massively parallel method for assaying the target sequences of miRNAs and RBPs. We use PTRE-seq to dissect sequence preferences and interactions between miRNAs and RBPs. The binding sites for these effector molecules influenced different aspects of the RNA lifecycle: RNA stability, translation efficiency, and translation initiation. In some cases, post-transcriptional control is modular, with different factors acting independently of each other, while in other cases factors show specific epistatic interactions. The throughput, flexibility, and reproducibility of PTRE-seq make it a valuable tool to study post-transcriptional regulation by 3'UTR elements.

SUBMITTER: Cottrell KA

PROVIDER: S-EPMC5775260 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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PTRE-seq reveals mechanism and interactions of RNA binding proteins and miRNAs.

Cottrell Kyle A KA Chaudhari Hemangi G HG Cohen Barak A BA Djuranovic Sergej S

Nature communications 20180119 1

RNA binding proteins (RBP) and microRNAs (miRNAs) often bind sequences in 3' untranslated regions (UTRs) of mRNAs, and regulate stability and translation efficiency. With the identification of numerous RBPs and miRNAs, there is an urgent need for new technologies to dissect the function of the cis-acting elements of RBPs and miRNAs. We describe post-transcriptional regulatory element sequencing (PTRE-seq), a massively parallel method for assaying the target sequences of miRNAs and RBPs. We use P ...[more]

PMID: 29352242

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Project description:BackgroundSarcopenia is defined as an age-related progressive loss of muscle mass and/or strength. Although different factors can contribute to this disease, the underlying mechanisms remain unclear. We assessed transcriptional heterogeneity in skeletal muscles from sarcopenic and control mice at single-cell resolution.MethodsA mouse model was established to study sarcopenic skeletal muscles. Single-cell RNA-seq was performed on tibialis anterior (TA) muscle cells collected from sarcopenic and control mice. A series of bioinformatic analyses were carried out to identify and compare different cell types under different conditions. Immunofluorescence staining and western blotting were used to validate the findings from single-cell experiments. Tube formation assays were conducted to further evaluate the effects of Gbp2 on endothelial cells during angiogenesis.ResultsA murine sarcopenia model was successfully established using a senescence-accelerated mouse strain (SAMP6, n = 5). Sarcopenia phenotype was induced by administration of dexamethasone (20 mg/kg) and reduced physical activity. Senescence-resistant mice strain (SAMR1) and SAMP6 strain with similar activity but injected with PBS were recruited as two control groups. As signs of sarcopenia, body weight, muscle cell counts and cross-sectional fibre area were all significantly decreased in sarcopenic mice (P value = 0.004, 0.03 and 0.035, respectively). After quality control, 13 612 TA muscle single-cell transcriptomes were retained for analysis. Fourteen cell clusters were identified from the profiled cells. Among them, two distinct endothelial subtypes were found to be dominant in the sarcopenia group (42.2% cells) and in the two control groups (59.1% and 47.9% cells), respectively. 191 differentially expressed genes were detected between the two endothelial subtypes. Sarcopenia-specific endothelial cell subtype exhibited a dramatic increase in the interferon family genes and the interferon-inducible guanylate-binding protein (GBP) family gene expressions. For example, Igtp and Gbp2 in sarcopenic endothelial cells were 5.4 and 13.3 times higher than those in the control groups, respectively. We further validated our findings in muscle specimens of sarcopenia patients and observed that GBP2 levels were increased in endothelial cells of a subset of patients (11 of 40 patients, 27.5%), and we identified significantly higher CD31 and GBP2 co-localization (P value = 0.001128). Finally, we overexpressed Gbp2 in human umbilical vein endothelial cells in vitro. The endothelial cells with elevated Gbp2 expression displayed compromised tube formation.ConclusionsOur single-cell-based results suggested that endothelial cells may play critical roles in sarcopenia development through interferon-GBP signalling pathways, highlighting new therapeutic directions to slow down or even reverse age-related sarcopenia.

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PTRE-seq reveals mechanism and interactions of RNA binding proteins and miRNAs.

Publications

PTRE-seq reveals mechanism and interactions of RNA binding proteins and miRNAs.

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