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

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.

G Protein-Coupled Receptors As Regulators of Localized Translation: The Forgotten Pathway?

“…Hence, most of the mRNA transcribed from IEG appeared also translated in response to ET1. However, 17% of mRNA were increased to a greater extent in the polysome-associated pool than in the total transcriptome whereas some others were excluded, which is indicative of regulations of the translational machinery by ET1R-induced signaling to promote mRNA-selective translation, as the authors confirmed later ( 23 ).…”

Using Polysome Isolation with Mechanism Alteration to Uncover Transcriptional and Translational Dynamics in Key Genes

“…The translating ribosome affinity purification (TRAP) approach described in vivo by Heiman et.al [4] and Doyle et.al [5] allows for freshly transcribed gene products, contributing directly to protein production and precipitated straight from the polysome, to be recovered from murine CNS cells. Other studies have used similar methods to examine TLT in biological contexts ranging from cardiomyocytes [6] to stress response in yeast [7] and surveys of discrete cellular populations in Arabidopsis [8]. In Markou et.al [6], 5' terminal oligopyrimidine tracts (TOPs) are used to indirectly measure translation levels and comparatively assess the activity of TLT and TST among cardiomyocyte populations.…”

“…Other studies have used similar methods to examine TLT in biological contexts ranging from cardiomyocytes [6] to stress response in yeast [7] and surveys of discrete cellular populations in Arabidopsis [8]. In Markou et.al [6], 5' terminal oligopyrimidine tracts (TOPs) are used to indirectly measure translation levels and comparatively assess the activity of TLT and TST among cardiomyocyte populations. Halbeisen and Gerber [7] observe that TLT of yeast, harvested using polyribosome precipitation and bulk purification of the associated mRNA, acts to coordinate messages observed in TST during stress response.…”

Regulation of Gene Expression

“…It is known that most eukaryotes regulate growth through the TOR-S6K signal transduction pathway, selectively stimulating mobilization of the (r)-protein mRNAs into polysomes for (r)-protein synthesis (Grolleau et al 2002, Hamilton et al 2006, Markou et al 2010). This pathway is activated either by insulin or by members of the insulin-like growth factor family in all organisms, from yeast to mammals (Fingar andBlenis 2004, Leevers andHafen 2004), including plants (Reyes de la Cruz et al 2004, Robaglia et al 2004).…”