Ilya Vainberg Slutskin, Shira Weingarten-Gabbay, Ronit Nir, Adina Weinberger, Eran Segal
Despite extensive research, the sequence features affecting microRNA-mediated regulation are not well understood, limiting our ability to predict gene expression levels in both native and synthetic sequences. Here we employed a massively parallel reporter assay to investigate the effect of over 14,000 rationally designed 3′ UTR sequences on reporter construct repression. We found that multiple factors, including microRNA identity, hybridization energy, target accessibility, and target multiplicity, can be manipulated to achieve a predictable, up to 57-fold, change in protein repression. Moreover, we predict protein repression and RNA levels with high accuracy (R = 0.84 and R = 0.80, respectively) using only 3′ UTR sequence, as well as the effect of mutation in native 3′ UTRs on protein repression (R = 0.63). Taken together, our results elucidate the effect of different sequence features on miRNA-mediated regulation and demonstrate the predictability of their effect on gene expression with applications in regulatory genomics and synthetic biology.
Ilya Vainberg Slutskin, Shira Weingarten-Gabbay, Ronit Nir, Adina Weinberger, Eran Segal. Unraveling the determinants of microRNA mediated regulation using a massively parallel reporter assay. 2018-02-06. https://doi.org/10.1038/s41467-018-02980-z
The 14,151-element rationally designed 3′ UTR reporter library was transiently transfected into MCF7 cells and harvested 24 h later for RNA measurement. Two RNA replicates were normalized to the shared transient-plasmid DNA library sequencing run.
The 14,151-element rationally designed 3′ UTR reporter library was transiently transfected into HEK293 cells and harvested 24 h later for RNA measurement. Two RNA replicates were normalized to the shared transient-plasmid DNA library sequencing run.
The reporter library was integrated as approximately single-copy constructs at the AAVS1 locus with ZFN-mediated targeting, and K562 cells were sorted into 16 mNeonGreen/mCherry expression bins. The public pooled sequencing run preserves 48 bin-primer barcodes (three per bin) and construct barcodes; this package provides the corrected construct-by-bin count matrix.
The 14,151-element rationally designed 3′ UTR reporter library was transiently transfected into K562 cells and harvested 24 h later for RNA measurement. Two RNA replicates were normalized to the shared transient-plasmid DNA library sequencing run.
The 14,151-element rationally designed 3′ UTR reporter library was transiently transfected into HepG2 cells and harvested 24 h later for RNA measurement. Two RNA replicates were normalized to the shared transient-plasmid DNA library sequencing run.
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.
Download all 27 files (ZIP)bins_processing_summary.jsonREADME.txtSRP128656_runinfo.csvSRR6456893_forward_barcode_counts.tsvSRR6456893_forward_barcode_stats.txtSRR6456894_HEK293_R1_barcode_counts.tsvSRR6456894_HEK293_R1_barcode_stats.txtSRR6456895_forward_barcode_counts.tsvSRR6456895_forward_barcode_stats.txtSRR6456896_forward_barcode_counts.tsvSRR6456896_forward_barcode_stats.txtSRR6456897_forward_barcode_counts.tsvSRR6456897_forward_barcode_stats.txtSRR6456898_forward_barcode_counts.tsvSRR6456898_forward_barcode_stats.txtSRR6456900_Lib1Bins_bin_variant_counts.tsvSRR6456900_Lib1Bins_bin_variant_stats.txtSRR6456903_Lib1TransientPlasmid_forward_barcode_counts.tsvSRR6456903_Lib1TransientPlasmid_forward_barcode_stats.txtSRR6456905_forward_barcode_counts.tsvSRR6456905_forward_barcode_stats.txtSRR6456906_forward_barcode_counts.tsvSRR6456906_forward_barcode_stats.txtSRX3547500_Lib1Bins_metadata.xmlsupplementary_files_archive.zipsupplementary_information.pdftransient_processing_summary.json