Unknown,Transcriptomics,Genomics,Proteomics

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Comparative Dynamic Transcriptome Analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation

ABSTRACT: Data was pre-processed array-wise using expresso (R/Bioconductor) with the RMA background correction method. We created our own probe annotation environment (cdf), which excludes probes in probesets that show cross-hybridization between Sc and Sp. 8708 annotated Sc probes and 13,317 annotated Sp probes out of a total of 120,855 probes showed cross-hybridization when a conservative intensity cut-off of 4.5 (log intensity values after preprocessing) was used. Cross-hybridizing probes were excluded from further analysis. This included 16 whole probe sets. Note that the standard GC-RMA method is not suitable for our purposes, since its bias model cannot handle bimodal intensity distributions, as caused by the simultaneous hybridization of Sc and Sp transcripts with global differences in RNA abundance. Labeling bias estimation and correction was done as described (Miller et al. 2011). Between-array normalization of arrays containing mixed Sc and Sp total RNA was done by proportional rescaling, such that the median Sp gene expression level was 1. Accordingly, between-array normalization of arrays containing mixed Sc and Sp labeled RNA was done by proportionally scaling the array to a median labeled Sp gene expression level of c. The constant c scales the median half-life of all experiments. We calibrated c in a way that the resulting median Sc wild-type mRNA half-life equaled that observed previously (Miller et al. 2011). Now, all Sc RNA levels, no matter if total or labeled, no matter from which experiment, can be compared on an absolute level. Decay rates and synthesis rates were obtained as described (Miller et al. 2011). The whole analysis workflow has been carried out using the open source R/Bioconductor package DTA

ORGANISM(S): Schizosaccharomyces pombe

SUBMITTER: Mai Sun

PROVIDER: E-MTAB-760 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation.

Sun Mai M Schwalb Björn B Schulz Daniel D Pirkl Nicole N Etzold Stefanie S Larivière Laurent L Maier Kerstin C KC Seizl Martin M Tresch Achim A Cramer Patrick P

Genome research 20120330 7

To monitor eukaryotic mRNA metabolism, we developed comparative dynamic transcriptome analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae (Sc) cells with the use of Schizosaccharomyces pombe (Sp) as an internal standard. cDTA uses nonperturbing metabolic labeling that supersedes conventional methods for mRNA turnover analysis. cDTA reveals that Sc and Sp transcripts that encode orthologous proteins have similar synthesis rates, whereas decay rate ...[more]

PMID: 22466169

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Project description:The rates of mRNA synthesis and degradation determine cellular mRNA levels and can be monitored by comparative Dynamic Transcriptome Analysis (cDTA) that uses non-perturbing metabolic RNA labeling. Here we present cDTA data for 46 yeast strains lacking genes involved in mRNA degradation and metabolism. In these strains, changes in mRNA degradation rates are generally compensated by changes in mRNA synthesis rates, resulting in a buffering of mRNA levels. We show that buffering of mRNA levels requires the RNA exonuclease Xrn1. The buffering is rapidly established when mRNA synthesis is impaired, but is delayed when mRNA degradation is impaired, apparently due to Xrn1-dependent transcription repressor induction. Cluster analysis of the data defines the general mRNA degradation machinery, reveals different substrate preferences for the two mRNA deadenylase complexes Ccr4-Not and Pan2-Pan3, and unveils an interwoven cellular mRNA surveillance network. For this purpose, cDTA was carried out as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). S. cerevisiae cultures were grown at 30¡ãC in 50 ml aliquots of YPD medium. S. pombe cultures were grown at 32¡ãC in YES medium. For inhibitor perturbation, BY4741 cells (OD600=0.1) were inoculated from a saturated overnight culture in two 100 ml aliquots of YPD liquid medium, incubated at 30¡ãC and grown to early-log phase (OD600=0.8). Cells were labeled for 6 minutes (sample 0 min). Then cycloheximide was added and cells were labeled for 10 minutes and 60 minutes. Cells were harvested and treated as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). 1,10-Phenanthroline was added to the culture to a final concentration of 25 _g/ml as described (Dori-Bachash et al., 2012), and labeled for 6 minutes after 18 minutes treatment. Cells were treated as above. For the anchor-away analysis, S. cerevisiae cells (OD600=0.1) were cultured in two 200 ml aliquots of YPD containing 1_g/ml rapamycin and incubated at 30¡ãC untill OD600 reached 0.8. Due to the instability of rapamycin, we added 1_g/ml rapamycin every two hours. Then a 20 ml sample was taken and labeled with 4sU. The cells were then treated as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111).

Project description:Genome-wide occupancy analysis of TBX5, NKX2-5 and GATA4 in differentiating WT, Nkx2-5KO (NKO), Tbx5KO (TKO) and Nkx2-5;Tbx5KO (DKO) cells at the cardiac precursor (CP) and cardiomyocyte (CM) differentiation stages. Analysis of TBX5, NKX2-5 and GATA4 occupancy a and gene expression in WT, Tbx5KO, Nkx2-5KO and DoubleKO precursor (CP) and mature (CM) in vitro differentiated cardiomyocytes. We performed ChIP-exo experiments for NKX2-5, TBX5 and GATA4 in differentiating WT, Nkx2-5KO (NKO), Tbx5KO (TKO) and Nkx2-5;Tbx5KO (DKO) cells at the CP and CM stages. ChIP-exo was performed according to methods published previously (Boyer et al., 2005; Serandour et al., 2013; Wamstad et al., 2012) using specific antisera to TBX5 (sc-17866 XS, lot no. B1213), NKX2-5 (sc-8697 XS, lot no. B1213), and GATA4 (sc-1237 XS, lot no. B1213) on chromatin isolated from 10 mill. cells. Re-ChIP-exo was performed from 40 mill cells, combining methods published previously (Serandour et al., 2013; Shankaranarayanan et al., 2011). ChIP-exo Analysis: Barcoded libraries were single-end 50bp sequenced on an Illumina HiSeq 2500 instrument. Reads were trimmed using the fastq-mcf program (Aronesty, 2011), and then aligned to the mm9 mouse genome assembly using bowtie2 (ChIP-seq, ChIP-exo) (Langmead and Salzberg, 2012). Samtools was then used to retain reads that had a mapq score of 30 or greater (Li et al., 2009), thereby ensuring that each mapped uniquely to the genome assembly. The 5' -most position of reads that mapped to the reference strand and the 3Ã¢??-most position of reads that mapped to the non-reference strand were identified for each read as the actual edges of each exonuclease-treated fragment. The genome was divided into 20bp genomic bins and a normalized tag density was calculated for each genomic bin 'i' as follows: tag density(i)=([#tags within 75bp of i] X [total# genomic bins])/(total #of tags) To identify broad regions of binding, bins with tag densities of greater than 100 were merged to generate a peak list for each sample. Within 1kb of each region, strand-specific single-base-resolution tag densities were calculated for each dataset by dividing each region into 1bp bins, then counting the number of tags within 5bp of each bin. For each region of binding, the footprint for each bound region was defined as the span from the peak position of '+' strand binding to the peak position of '-' strand binding as seen from the high-resolution tag densities. For subsequent analyses, any overlapping footprint for replica/factor/stage/genotype was merged to generate an average footprint list. Only average footprints present in at least two replicas of the same factor, stage and genotype were considered (Final Average Footprints).

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Comparative Dynamic Transcriptome Analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation

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Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation.

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