The patent's assignee is The Regents of the
News editors obtained the following quote from the background information supplied by the inventors: "Accumulating evidence now reveals that transcription elongation is not a straightforward read-out of the downstream DNA sequence. Co-transcriptional processing events dictate the covalent nature and fate of RNA transcripts (Moore, M. J. &
"Historically, two strategies have been used to provide snapshots of transcriptional activity in vivo. In the first approach, RNAP is crosslinked to DNA and RNAP-bound DNA elements are identified by microarrays or deep sequencing (Kim, T. H. et al. Nature 436, 876-880 (2005); Lefrancois, P. et al. BMC genomics 10, 37 (2009)). While providing a global view of RNAP binding sites, these measurements are of limited spatial and temporal resolution and do not reveal the identity of the transcribed strand or even if RNAP molecules are engaged in transcription. In the second approach, transcription is halted in vivo and then reinitiated in isolated nuclei under conditions that allow labeling of nascent chains thereby enabling them to be distinguished from bulk RNA (Core, L. J., et al. Science 322, 1845-1848 (2008); Rodriguez-Gil, A. et al. The distribution of active RNA polymerase II along the transcribed region is gene-specific and controlled by elongation factors.
As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "Provided herein are methods of identifying a native cellular nascent RNA transcript by (a) arresting transcription in a cell; (b) purifying a cellular nascent RNA transcript; and © sequencing said cellular nascent RNA transcript thereby identifying the cellular nascent RNA transcript.
BRIEF DESCRIPTION OF THE DRAWINGS
"FIG. 1A-1C: NET-seq visualizes active transcription via capture of 3' RNA termini. FIG. 1A, Schematic diagram of NET-Seq protocol. A yeast culture is flash frozen and cryogenically lysed. Nascent RNA is co-purified via an immunoprecipitation (IP) of the RNAPII elongation complex. Conversion of RNA into DNA results in a DNA library with the RNA as an insert between DNA sequencing linkers. The sequencing primer is positioned such that the 3' end of the insert is sequenced. m7G refers to the 7-methylguanosine cap structure at the 5' end of nascent transcripts. FIG. 1B, The 3' end of each sequence is mapped to the yeast genome and the number of reads at each nucleotide is plotted at the RPL30 locus for nascent RNA and lightly fragmented mature RNA. Note for the nascent transcripts, the introns (grey box) and regions after the poly-adenylation site (black arrow) are readily detected. FIG. 1C, Metagene analysis for well-expressed genes (N=471, >1.5 reads/bp in both conditions) of the mean read density in the presence (black) and absence (grey) of transcription inhibitor, .alpha.-amanitin.
"FIG. 2A-2D: Observation of divergent transcripts reveals strong directionality at most promoters. FIG. 2A, Nascent and mature transcripts initiating from URA1 and RPL5 promoters in the sense and antisense directions. Note that there are cryptic unstable transcripts (CUTs) in the antisense direction for URA1 but not RPL5. FIG. 2B, A histogram of the transcription ratio (antisense/sense transcription levels) for 1875 genes. The dark grey left box and light grey right box indicate the subset of genes with a ratio of less than 1:8 and less than 1:3, respectively. FIG. 2C, Antisense transcription levels are plotted versus sense transcription for each tandem gene (Spearman correlation coefficient, r.sub.s=0.34). FIG. 2D, The level of antisense transcription for each promoter is plotted versus the local enrichment for H4 hyperacetylation using available data (Pokholok, D. K. et al., Cell 122:517-527 (2005)) (r.sub.s=0.65).
"FIG. 3A-3C: Rco1 suppresses antisense transcription at divergent promoters. FIG. 3A, Examples of cryptic unstable transcripts (CUTs, light grey data below CUTs) upstream and antisense of DBF2, DRN1 and VAS1 promoters. The fold increase of CUT transcription in the rco1 .DELTA. strain is marked at bottom. FIG. 3B, The transcription ratio (antisense:sense) in the rco1 .DELTA. strain is plotted against the transcription ratio in the wild type strain for each gene. FIG. 3C, A metagene analysis of well-expressed antisense transcription (N=171, >1 read/bp); wild type (black), rco1D (light grey).
"FIG. 4A-4C: Frequent RNAPII pausing throughout gene bodies. FIG. 4A, NET-seq data at the GPM1 gene for biological replicates. FIG. 4B, A histogram of the mean distance between pauses for each well-expressed gene (N=1006, >2 reads/bp). FIG. 4C, The consensus sequence of the DNA coding strand surrounding pause sites found from all genes.
"FIG. 5A-5D: Dst1 relieves RNAPII pausing after backtracking FIG. 5A, A schematic describing an existing model for how RNAPII pauses at an obstacle (dark grey square), backtracks and is induced to cleave its transcript through binding to Dst1 (Izban, M. G. &
"FIG. 6 Nucleosomes are a major barrier to transcription. Plot of mean pause densities in dst1 .DELTA. data relative to the first four nucleosomes after the transcription start site using available nucleosome positioning data (Weiner, A. et al., Genome Res 20:90-100 (2010)). Error bars represent one standard deviation.
"FIG. 7 Western blot detecting FLAG-labeled Rpb3 of immunoprecipitation samples of input lysate, unbound lysate and eluted protein.
"FIG. 8A-8D: Evidence of co-transcriptional splicing in yeast. FIG. 8A, A schematic showing how co-transcriptional splicing intermediates (e.g. spliced exon and excised lariat (light grey)) would remain bound to RNAP II via the spliceosome (small circle). FIG. 8B, Read densities for two spliced genes, ACT1 and MOB2. Note the high densities at their precise exon-intron junctions indicated by stars. FIG. 8C, Average number of reads per base pair for spliced genes versus the gene's reads at the 3' end of splice junctions and FIG. 8D, one base pair downstream from splice junctions; exon (light grey), lariat (black) for FIG. 8C and FIG. 8D.
"FIG. 9A-9D: Antisense transcription correlations. FIG. 9A-9B) Antisense transcription level versus the width of the promoter's nucleosome free region (NFR) and nucleosome occupancy from available data (Weiner, A. et al., Genome Res. 20:90-100 (2010)). R values are Spearman correlation coefficients. FIG. 9C-9D) Antisense transcription level versus H3 acetylation enrichment from available data (Pokholok, D. K. et al., Cell 122:517-527 (2005)).
"FIG. 10 Comparison between sense transcription in wild type strain and the rco1 .DELTA. strain at divergent promoters. R=0.965, Pearson correlation coefficient. R=0.914, Spearman correlation coefficient.
"FIG. 11A-11D: Comparison of fold increases of antisense transcription (tx) in mutant strains compared to that in wild type.
"FIG. 12 Average pause density across gene bodies for highly expressed genes (N=361, >4 reads/bp).
"FIG. 13A-13C: Pause finding analysis on mRNA data FIG. 13A) Sequence consensus of extracted pauses in mRNA data shows a strong propensity for G's to occur at the base following the 3' end of the fragmented transcript. This bias occurred during the fragmentation of full length mRNA. After removing all pauses that are followed by a G, the average distance between pauses was measured for each gene for nascent RNA (FIG. 13B) and for fragmented mRNA (FIG. 13C).
"FIG. 14 Histogram of the fraction of pauses that are found in both wildtype and dst1 .DELTA. data.
"FIG. 15 Mean pause density of the wild type strain at the first four nucleosomes following transcription start sites. Error bars are placed at one standard deviation.
"FIG. 16 Histogram of the fraction of reads at pause sites for highly-expressed genes (N=256, >10 reads/bp). The reads at all pause sites was summed and then divided by the total number of reads for the gene resulting in the fraction of reads at pause sites."
For additional information on this patent application, see: Weissman, Jonathan; Churchman,
Keywords for this news article include: Antisense Technology, Biotechnology, Genetics, Genomics, Chromatin, Proteomics, Chromosomes, Nucleosomes,
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