Project description:To investigate gene specificity at the level of translation in both the human genome and viruses we devised a high-throughput bicistronic assay to quantify cap-independent translation. We uncover thousands of novel cap-independent translation sequences and provide insights on the landscape of translational regulation in both human and viruses. We find extensive translational elements in the 3â untranslated region (3âUTR) of human transcripts and the polyprotein region of un-capped RNA viruses. Through the characterization of regulatory elements underlying cap-independent translation activity we identify potential mechanisms of secondary structure, short sequence motif and base-pairing with the 18S rRNA. Furthermore, we systematically map the 18S rRNA regions for which reverse complementary enhance translation. Thus we provide insights into the mechanisms of translational control in humans and viruses. high-throughput bicistronic assay for obtaining cap-independent translation measurements of 55,000 fully designed sequences in parallel using fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq).
Project description:One of the major challenges in genomics is to build computational models that accurately predict genome-wide gene expression from the sequences of regulatory elements. Promoters play a key role in gene regulation, yet their regulatory logic remains incompletely understood. Here, we present PARM, a cell-type specific deep learning model trained on specially designed massively parallel reporter assays that query human promoter sequences. PARM is computationally light-weight and reliably predicts autonomous promoter activity across the genome from DNA sequence alone, in multiple cell types. PARM can also design purely synthetic strong promoters. We leveraged PARM to systematically identify transcription factor (TF) binding sites that likely to contribute to the activity of each natural human promoter, and to detect the rewiring of these regulatory interactions upon various stimuli to the cells. We also uncovered and experimentally confirmed striking positional preferences of TFs that differ between activating and repressive regulatory functions, as well as a complex grammar of motif-motif interactions. Our approach provides a foundation towards a deeper understanding of the dynamic regulation of human promoters by TFs.
Project description:Combinatorial promoter expression level estimation via cell sorting The purpose of this experiment was to determine the expression level of a library of synthetic promoters. The promoters were cloned in front of a GFP reporter and the resulting library transformed into yeast, sorted by FACS into six fluorescence bins, and the contents of the bins sequenced to determine the distribution of each promoter among each fluorescence bin. This was then used to calculate an expression level for each promoter with enough data.