Project description:A massively parallel reporter assay, MPRA, was conducted in mouse embryonic stem cells (mESC). Synthetic cis-regulatory elements comprised of binding sites for pluripotency transcription factors and genomic sequences with comparable binding sites configurations were used in the assay. Transcripts of dsRed were amplified via PCR from the end of the transcript to sequence 3' UTR barcodes.
Project description:The activity of enhancers with dynamic P300 binding or mutagenized nuclear receptor motifs was assessed by massively parallel reporter assay during CM maturation.
Project description:We performed a massively parallel reporter assay for RNA localization in two mouse neuronal cell lines, CAD and Neuro-2a. We transfected a library of around 50,000 different 3'UTR reporters (cloned downstream of GFP) into cells grown on transwell plates and collected soma and neurite fractions separately. RNA was extracted and cDNA synthesized, followed by targeted amplification of the reporter mRNA and Illumina sequencing.
Project description:RNA structural switches are key regulators of gene expression in bacteria, yet their characterization in Metazoa remains limited. Here we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3’UTR of the RORC transcript. In vivo DMS-MaPseq, coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated mRNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally-driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.
Project description:We performed a Massively Parallel Reporter Assay (MPRA) to screen >30,000 human-specific substitutions in ChIP-seq-identified Human Gain Enhancers (HGEs) and Human Accelerated Regions (HARs), highly conserved non-coding regions that show accelerated sequence evolution in humans. After comparing human and chimpanzee reference alleles, we used a second MPRA to deconvolute individual substitutions within differentially active enhancers from substitutions in the same fragment and from other variants (human segregating variants or chimpanzee-specific variants) to isolate their specific effects on enhancer activity.