Study / S25OHOMST2018-01-19

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

Kyle A. Cottrell, Hemangi G. Chaudhari, Barak A. Cohen, Sergej Djuranovic

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About this study

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.

Full author list & citation

Kyle A. Cottrell, Hemangi G. Chaudhari, Barak A. Cohen, Sergej Djuranovic. PTRE-seq reveals mechanism and interactions of RNA binding proteins and miRNAs. 2018-01-19. https://doi.org/10.1038/s41467-017-02745-0

Experiments 5

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PTRE-seq RNA expression in HEK293 cells

The shared synthetic 3′UTR reporter library was transfected into T-REx-293 cells, and one RNA output sample was barcode-sequenced against the two-replicate plasmid input library.

3' UTR / RNA Stability MPRA (MPRAu)Human

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HeLa PTRE-seq multi-fraction synthetic 3′UTR library

An episomal CMV/TO-EGFP reporter library containing 642 synthetic 3′UTR identities, each represented by barcoded oligonucleotides, was transfected into HeLa cells. Total RNA, polysome-associated RNA, and 40S-associated RNA were quantified against plasmid input to measure RNA expression, translation efficiency, translation initiation, and 40S association.

3' UTR / RNA Stability MPRA (MPRAu)Human

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PTRE-seq RNA expression in mouse N2A cells

The shared synthetic 3′UTR reporter library was transfected into mouse Neuro-2a neuroblastoma cells, and one RNA output sample was barcode-sequenced against the two-replicate plasmid input library.

3' UTR / RNA Stability MPRA (MPRAu)Mouse

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PTRE-seq under mCherry-Smaug overexpression in HeLa cells

The shared synthetic 3′UTR reporter library was co-transfected with a mCherry-tagged Drosophila Smaug expression plasmid in HeLa cells. One RNA output sample was barcode-sequenced against the shared two-replicate plasmid input library.

Trans-Factor Perturbation MPRAHuman

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PTRE-seq RNA expression in neonatal human dermal fibroblasts

The shared synthetic 3′UTR reporter library was transfected into neonatal human dermal fibroblasts (HDFn), and one RNA output sample was barcode-sequenced against the two-replicate plasmid input library.

3' UTR / RNA Stability MPRA (MPRAu)Human

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Raw source data 26 files

Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.