Project description:Our ability to understand the control logic embedded in the human genome is limited by a lack of accurate information of the promoter sequences for most genes. Promoters are a unique class of control sequences, serving as the binding sites for both sequence-specific factors and the general transcription machinery during transcription initiation. In order to obtain a comprehensive map of promoters in the human genome, we have determined the location of the RNA polymerase II preinitiation complex throughout the non-repeat sequences of the human genome in primary fibroblast cells. The resulting map defines 10,571 active promoters corresponding to 6,763 known genes and at least 1,199 un-annotated transcriptional units. The map indicates extensive usage of multiple promoters by the human genes and widespread clustering of active promoters in the genome. Further examination of the genome-wide expression profile reveals four general classes of promoters that define the transcriptome of the cell. Our results provide a global view of the functional relationship among the transcriptional machinery, chromatin structure, and gene expression in human cells. Keywords = GALA Keywords = gene expression Keywords = nimblegen Keywords = promoter profile Keywords = LICR renlab Keywords: other
Project description:SHAPE-MaP structure probing experiment was performed on SARS-CoV-2 infected Vero cells at 4 days post infection with two biological replicates. For each replciate, SHAPE-MaP includes a sample treated with 2-methylnicotinic acid imidazolide acid (modified) or a minue reagent (unmodified). NAI preferentially reacts with unpaired bases in RNA, forming acylated bases. These modifications are encoded as mutation during reverse transcripatse and library preparation. After sequencing and alignment, the reactivity profiles of 'modified' and 'unmodified' samples are used to calculate SHAPE reactivity of each base
Project description:Our ability to understand the control logic embedded in the human genome is limited by a lack of accurate information of the promoter sequences for most genes. Promoters are a unique class of control sequences, serving as the binding sites for both sequence-specific factors and the general transcription machinery during transcription initiation. In order to obtain a comprehensive map of promoters in the human genome, we have determined the location of the RNA polymerase II preinitiation complex throughout the non-repeat sequences of the human genome in primary fibroblast cells. The resulting map defines 10,571 active promoters corresponding to 6,763 known genes and at least 1,199 un-annotated transcriptional units. The map indicates extensive usage of multiple promoters by the human genes and widespread clustering of active promoters in the genome. Further examination of the genome-wide expression profile reveals four general classes of promoters that define the transcriptome of the cell. Our results provide a global view of the functional relationship among the transcriptional machinery, chromatin structure, and gene expression in human cells. Keywords = GALA Keywords = gene expression Keywords = nimblegen Keywords = promoter profile Keywords = LICR renlab Keywords: other
Project description:Structure probing experiments were performed on in vitro transcripts and E. coli and human cell cultures under natively extracted (cell-free) and in-cell conditions to benchmark the performance of the newly introduced PAIR-MaP correlated chemical probing strategy for detecting RNA duplexes. Multiple-hit dimethyl sulfate (DMS) probing was done using new buffer conditions that facilitate DMS modification of all four nucleotides.
Project description:MicroRNA (miRNA) maturation is critically dependent on structural features of primary transcripts (pri-miRNAs). However, the scarcity of determined pri-miRNA structures has limited our understanding of miRNA maturation. Here we employed SHAPE-MaP, a high-throughput RNA structure probing method, to unravel the secondary structures of 476 high-confidence human pri-miRNAs. Our SHAPE-based structures diverge substantially from those inferred solely from computation, particularly in the apical loop and basal segments, underlining the need for experimental data in RNA structure prediction. By comparing the structures with high-throughput processing data, we determined the optimal structural features of pri-miRNAs. The sequence determinants are influenced substantially by their structural contexts. Moreover, we identified an element termed the bulged GWG motif (bGWG) with a 3′ bulge in the lower stem, which promotes processing. Our structure-function mapping better annotates the determinants of pri-miRNA processing and offers practical implications for designing small hairpin RNAs and predicting the impacts of miRNA mutations.
Project description:We show that DANCE-MaP permits measurement of state-specific per-nucleotide reactivities, direct secondary structure PAIRs, and tertiary RINGs for RNA structural ensembles. Here, we demonstrate DANCE-MaP on the V. vulnificus add riboswitch.