Xrn1 regulates cAMP-PKA specificity during thermotolerance

ORTOLÁ MARTÍNEZ MARÍA CLARA; GALELLO, FIORELLA; PAUTASSO, CONSTANZA; PORTELA, PAULA; ROSSI, SILVIA

Mendoza

Congreso; LVIII Reunión Anual SAIB; 2022

SAIB

Organisms are constantly challenged by changing environments. Complex signalling circuits sense these changes. Signal transduction pathways play a major role in controlling enzyme cascades that ultimately promote transcriptome and proteome remodelling in multi-stress and stress-specific manners. Several physiological mechanisms help organisms to prepare for recurring stressors. In the “acquired stress resistance” or "adaptive response" cells exposed to a mild stress dose survive a subsequent lethal dose of the same or a different stress condition. One of the most studied signalling pathways is the conserved cAMP-PKA pathway. PKA from S. cerevisiae is composed of two catalytic (Tpk1, Tpk2 or Tpk3) and two regulatory subunits (Bcy1). Although each Tpk isoform controls different biological processes, little is known about the principles that govern PKA signalling specificity. One of the mechanisms involved in signalling specificity is regulation of PKA subunits expression. We have previously demonstrated that all PKA subunits share a negative expression regulation mechanism mediated by PKA activity. However, Tpk1 is the only subunit transcriptionally upregulated during heat shock. To further understand the molecular basis of cAMP-PKA specificity, we evaluated the mechanisms involved in PKA subunits expression during thermal stress adaptation. To this aim, we exposed cells to a scheme of two consecutive heat shocks: 37°C for 30 minutes and 45°C for 10 minutes (thermotolerance). We observed that only TPK1 promoter activity and mRNA levels increase during thermotolerance. Gene expression in eukaryotes does not always follow a linear progression from transcription to translation and mRNA degradation. There is an important crosstalk between transcription and mRNA decay machineries which functions from the nucleus to the cytoplasm and vice versa. The transcription-degradation crosstalk is grounded on the co-transcriptional imprinting of mRNAs with general factors such as RNA Pol II subunits (Rpb4/7) and mRNA decay factors (Xrn1 or Ccr4-NOT). Thus, we evaluated the role of Xrn1 in PKA activity and TPK1 expression. PKA activity was assessed by analysing different physiological readouts of the cAMP-PKA pathway such as heat resistance and glycogen accumulation. The xrn1Δ strain is more resistant to heat shock and shows a higher glycogen accumulation than the WT strain, consistent with a lower PKA activity. These results indicate that Xrn1 could play a role in the regulation of PKA activity. Moreover, TPK1 mRNA half-life increases in a xrn1Δ strain. TPK1 promoter activity and mRNA levels are upregulated upon thermotolerance in a Xrn1-dependent manner. Accordingly, Xrn1 is recruited to the TPK1 promoter upon stress. Although during thermotolerance TPK1 mRNA levels are strikingly upregulated in a xrn1Δ strain, Tpk1 protein levels severely decrease. These results indicate that Xrn1 regulates TPK1 expression at different levels during thermotolerance, affecting transcription, mRNA decay and translational efficiency. However, neither thermotolerance nor Xrn1 have an effect on the regulation of the other PKA subunits. Our results demonstrate that TPK1 expression is specifically regulated during thermotolerance and that Xrn1 has an important role in this process. Thus, we disclosed a tricky mechanism of the expression regulation of PKA subunits that impacts on cAMP-PKA pathway specificity.