Open Access Research article

Regulation of the cardiomyocyte transcriptome vs translatome by endothelin-1 and insulin: translational regulation of 5' terminal oligopyrimidine tract (TOP) mRNAs by insulin

Thomais Markou, Andrew K Marshall, Timothy E Cullingford, El L Tham, Peter H Sugden and Angela Clerk*

Author Affiliations

National Heart and Lung Institute (Cardiovascular Sciences), Faculty of Medicine, Imperial College London, London, UK

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BMC Genomics 2010, 11:343  doi:10.1186/1471-2164-11-343

Published: 29 May 2010

Additional files

Additional file 1:

Regulation of the cardiomyocyte global transcriptome by ET-1 or insulin (Microsoft Word Table). Cardiomyocytes were unstimulated (control), or exposed to 100 nM ET-1 or 50 mU/ml insulin (1 h). Total RNA expression was determined using microarrays. Transcripts were identified with significant changes in expression (>1.5-fold change induced by ET-1 or insulin relative to controls; FDR < 0.05, * ET-1 vs control; # Insulin vs control; † ET-1 vs insulin, one-way ANOVA with Tukey post-test and Benjamini-Hochberg FDR correction). Mean raw values are given for controls with the mean change relative to controls for ET-1 or insulin (n = 4). For transcripts represented by more than one probeset, the probesets and corresponding raw values are listed with the average of the mean relative change. RNA responses are clustered according to up- or down-regulation, and the effect of ET-1 and/or insulin. Clusters (i) - (viii) correspond to the summarised data in Figure 2A of the associated manuscript. Clusters are colour-coded according to response to ET-1 and insulin (green), ET-1 only (yellow) or insulin only (cyan). AS, Antisense.

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Additional file 2:

Functional classifications of the top 10th percentile RNAs identified in neonatal rat cardiomyocytes (Microsoft Word Table). Total and polysomal RNA from unstimulated neonatal rat cardiomyocytes (serum-starved, 20 h) were analysed using microarrays. Results are the mean raw fluorescence values (n = 4). For transcripts represented by more than one probeset, the probesets and mean corresponding raw values are listed. Differential expression in polysome vs total RNA was analysed by t-test with Benjamini-Hochberg FDR correction (*p < 0.05). RNAs are grouped according to general functional categories (summarised in Figure 3C of the associated manuscript) with detailed functions within the Table.

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Additional file 3:

Differential expression of cardiomyocyte transcripts in polysomal and total RNA (Microsoft Word Table). Total and polysomal RNA from unstimulated neonatal rat cardiomyocytes were analysed using microarrays. The data were normalised to the gene median. Transcripts with differential expression in total and polysomal RNA pools were identified (t-test with Benjamini-Hochberg FDR correction p < 0.05; >3-fold difference). Mean raw fluorescence and normalised values are given (n = 4). For transcripts represented by more than one probeset, the probesets and mean corresponding raw values are listed. RNAs are listed according to relative expression in polysomal or total pools and whether they are protein-coding, non-protein-coding or associated with no known gene. AS = Antisense.

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Additional file 4:

Regulation of polysomal and total RNA expression in cardiomyocytes in response to insulin (Microsoft Word Table). Neonatal rat cardiomyocytes were exposed to insulin (50 mU/ml, 1 h) or left unstimulated (controls). Total and polysomal RNA were analysed using microarrays. The data were normalised to controls. Transcripts with differential expression in insulin-treated cells relative to controls were identified (>1.25-fold difference; * FDR < 0.05 insulin vs controls for polysomal RNA, # FDR < 0.05 insulin vs controls for total RNA, t-test with Benjamini and Hochberg false discovery rate correction). Mean raw fluorescence values are provided for controls, with mean expression relative to controls for insulin-treated cells (n = 4). For transcripts represented by more than one probeset, the probesets and corresponding raw values are listed. RNAs are listed according to translational regulation and in order of functional category then alphabetical order of the gene symbol. AS = Antisense.

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Additional file 5:

Regulation of polysomal and total RNA expression in cardiomyocytes in response to ET-1 (Microsoft Word Table). Neonatal rat cardiomyocytes were exposed to ET-1 (100 nM, 1 h) or left unstimulated (controls). Total and polysomal RNA were analysed using Affymetrix rat genome 230 2.0 microarrays. The data were normalised to controls. Transcripts with differential expression in ET-1-treated cells relative to controls were identified (>1.25-fold difference; * FDR < 0.05 ET-1 vs Controls for polysomal RNA, # FDR < 0.05 ET-1 vs Controls for total RNA, t-test with Benjamini and Hochberg false discovery rate correction). Mean raw fluorescence values are provided for controls, and mean expression relative to controls is provided for ET-1-treated cells (n = 4). For transcripts represented by more than one probeset, the probesets and mean corresponding raw values are listed. RNAs are listed according to translational regulation and in order of functional category then alphabetical order of the gene symbol. AS = Antisense.

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Additional file 6:

Recruitment of TOP mRNAs to polysomes in cardiomyocytes in response to insulin (Microsoft Excel Spreadsheet). Neonatal rat cardiomyocytes were exposed to insulin (50 mU/ml, 1 h) or left unstimulated (controls). Total and polysomal RNA were analysed using microarrays. Probesets corresponding to ribosomal protein mRNAs and other established TOP mRNAs were identified. The mean raw values are provided for controls with the mean fold change (n = 4) induced by insulin relative to controls. *FDR < 0.05 for polysomal RNA from insulin-treated cells vs controls (t-test with Benjamini and Hochberg false discovery rate correction). Mouse and rat 5' UTR sequences up to and including the initiation codon are provided. Sequence identification used FANTOM3 and NCBI databases and the database of transcriptional start sites (DBTSS). The TOP sequence is shown in red. [N.B. For some rat genes, a pseudogene was identified on the NCBI site; the correct gene was identified by comparison with the mouse genome on the basis of intron structure and location relative to adjacent genes (#). For other rat genes, the 5' UTR was short and the genomic sequence was used to identify regions of homology with the mouse genome (†)].

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Additional file 7:

Recruitment of probable non-TOP mRNAs to polysomes in cardiomyocytes in response to insulin (Microsoft Excel Spreadsheet). Neonatal rat cardiomyocytes exposed to insulin (50 mU/ml, 1 h) or left unstimulated (controls). Total and polysomal RNA were analysed using microarrays. Probesets corresponding to mRNAs with >1.25-fold increase in expression in polysomal RNA (but not total RNA) in response to insulin (FDR < 0.05, t-test with Benjamini -Hochberg false discovery rate correction) were identified. Mean raw fluorescence values are given for the controls with the mean fold change (n = 4) induced by insulin relative to controls. Mouse 5' UTR sequences up to and including the initiation codon are provided. Sequence identification used FANTOM3 and NCBI databases in addition to the database of transcriptional start sites (DBTSS). Presence (Y) or absence (N) of polypyrimidine or GC tracts, upstream open reading frames (uORFs), or 5' untranslated exons are indicated, in addition to GC content (%), UTR length and predicted minimum free energy (Mfe).

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Additional file 8:

Response of putative TOP mRNAs to insulin stimulation in cardiomyocytes (Microsoft Excel Spreadsheet). Neonatal rat cardiomyocytes were exposed to insulin (50 mU/ml, 1 h) or ET-1 (100 nM, 1 h) or left unstimulated (controls). Total and polysomal RNA prepared were analysed using Affymetrix rat genome 230 2.0 microarrays. Probesets corresponding to TOP mRNAs identified by Yameshita et al. (Nuclei Acids Res. (2008) 36:3707-3715) were identified and the microarray data mined for the cardiomyocyte response. Results are the mean fold change (n = 4) in response to insulin or ET-1 relative to controls. Mean raw fluorescence values are given for the controls.

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Additional file 9:

Recruitment of probable non-TOP mRNAs to polysomes in cardiomyocytes in response to ET-1 (Microsoft Excel Spreadsheet). Neonatal rat cardiomyocytes exposed to ET-1 (100 nM, 1 h) or left unstimulated (controls). Total and polysomal RNA were analysed using microarrays. Probesets corresponding to mRNAs with >1.25-fold increase in expression in polysomal RNA (but not total RNA) in response to ET-1 (FDR < 0.05, t-test with Benjamini -Hochberg false discovery rate correction) were identified. Mean raw fluorescence values are given for the controls with the mean fold change (n = 4) induced by insulin relative to controls. Mouse 5' UTR sequences up to and including the initiation codon are provided. Sequence identification used FANTOM3 and NCBI databases in addition to the database of transcriptional start sites (DBTSS). Presence (Y) or absence (N) of polypyrimidine or GC tracts, upstream open reading frames (uORFs), or 5' untranslated exons are indicated, in addition to GC content (%), UTR length and predicted minimum free energy (Mfe).

Format: XLS Size: 58KB Download file

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