Abstract
BackgroundChanges in cellular phenotype result from underlying changes in mRNA transcription and translation. Endothelin-1 stimulates cardiomyocyte hypertrophy with associated changes in mRNA/protein expression and an increase in the rate of protein synthesis. Insulin also increases the rate of translation but does not promote overt cardiomyocyte hypertrophy. One mechanism of translational regulation is through 5' terminal oligopyrimidine tracts (TOPs) that, in response to growth stimuli, promote mRNA recruitment to polysomes for increased translation. TOP mRNAs include those encoding ribosomal proteins, but the full panoply remains to be established. Here, we used microarrays to compare the effects of endothelin-1 and insulin on the global transcriptome of neonatal rat cardiomyocytes, and on mRNA recruitment to polysomes (i.e. the translatome).ResultsGlobally, endothelin-1 and insulin (1 h) promoted >1.5-fold significant (false discovery rate < 0.05) changes in expression of 341 and 38 RNAs, respectively. For these transcripts with this level of change there was little evidence of translational regulation. However, 1336 and 712 RNAs had >1.25-fold significant changes in expression in total and/or polysomal RNA induced by endothelin-1 or insulin, respectively, of which ~35% of endothelin-1-responsive and ~56% of insulin-responsive transcripts were translationally regulated. Of mRNAs for established proteins recruited to polysomes in response to insulin, 49 were known TOP mRNAs with a further 15 probable/possible TOP mRNAs, but 49 had no identifiable TOP sequences or other consistent features in the 5' untranslated region.ConclusionsEndothelin-1, rather than insulin, substantially affects global transcript expression to promote cardiomyocyte hypertrophy. Effects on RNA recruitment to polysomes are subtle, with differential effects of endothelin-1 and insulin on specific transcripts. Furthermore, although insulin promotes recruitment of TOP mRNAs to cardiomyocyte polysomes, not all recruited mRNAs are TOP mRNAs.
Highlights
Changes in cellular phenotype result from underlying changes in mRNA transcription and translation
Signaling through to protein synthesis by ET-1 and insulin To compare the effects of ET-1 and insulin on activation of intracellular signaling pathways in cardiomyocytes, extracts were immunoblotted with antibodies selective for phosphorylated forms of extracellular signal-regulated kinases 1/2 (ERK1/2), PKB/ Akt, mammalian target of rapamycin (mTOR), p70 S6 kinases (p70S6Ks) and ribosomal subunit protein S6 (Rps6)
Insulin consistently stimulated a greater increase in phospho-mTOR(Ser2448) than ET-1, this was not reflected in the degree of phosphorylation of p70S6K(Thr389) and Rps6(Ser235/236)
Summary
Changes in cellular phenotype result from underlying changes in mRNA transcription and translation. Endothelin-1 stimulates cardiomyocyte hypertrophy with associated changes in mRNA/protein expression and an increase in the rate of protein synthesis. One mechanism of translational regulation is through 5' terminal oligopyrimidine tracts (TOPs) that, in response to growth stimuli, promote mRNA recruitment to polysomes for increased translation. We used microarrays to compare the effects of endothelin-1 and insulin on the global transcriptome of neonatal rat cardiomyocytes, and on mRNA recruitment to polysomes (i.e. the translatome). The global rate of protein synthesis relates to the capacity for and efficiency of translation [1,2]. Capacity is increased by synthesis of ribosomal subunits and other translational components, whereas efficiency is regulated by the rate of translational initiation (assembly of initiation factors, "unwinding" of RNA secondary structures, scanning and recognition of the initiation codon), and the rate of peptide chain elongation. The 5' UTR influences the rate of initiation via 5' terminal oligopyrimidine tracts (TOPs), inclusion of short upstream open reading frames (uORFs), GC content and UTR length [2,3]
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