Xrn1 and Rpb4 are mRNA coordinators that regulate cAMP-PKA specificity upon heat stress

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

Rosario

Congreso; LIX SAIB Annual Meeting; 2023

SAIB

Signal transduction pathways control transcriptome and proteome remodeling in response to differentenvironmental conditions and stressors. The cAMP-PKA pathway plays a critical role in regulatingseveral cellular processes and is conserved across species. Considering PKA’s multiple functions, an important question is how signaling specificity in response to different stimuli is achieved. In S.cerevisiae, PKA is composed of two catalytic (Tpk1, Tpk2 or Tpk3) and two regulatory subunits (Bcy1).Diverse mechanisms determine specificity of PKA signaling. One of them is the regulation of PKAsubunits expression, which is reflected in changes in the isoform composition of the holoenzyme. Wehave previously demonstrated that the expression of each PKA subunit is different in response to stress.Tpk1 is the only subunit upregulated during mild heat shock. To further understand the molecular basisof cAMP-PKA specificity, we evaluated the mechanisms involved in PKA subunits expression duringthermal stress adaptation or thermotolerance, where cells exposed to a mild heat stress dose (37°C for30 minutes) survive a subsequent severe heat stress dose (45°C for 10 minutes). We observed that onlyTPK1 promoter activity and mRNA levels increase during thermotolerance. Gene expression implies animportant crosstalk between mRNA transcription, decay and translation. This is possible since keyfactors involved in one stage of gene expression may play non-canonical roles in other stages. Thesefactors were referred to as “mRNA coordinators” and act through co-transcriptional imprinting ofmRNAs. Rpb4/7 subunits of RNA Pol II and mRNA decay factors such as the 5’-3’ exonuclease Xrn1 orCcr4-NOT were described as mRNA coordinators. We evaluated the role of Rpb4 and Xrn1 in cAMP-PKApathway specificity. First, we investigated their effect on PKA activity in vivo through the analysis ofphysiological PKA readouts (stress resistance and glycogen accumulation). Both xrn1Δ and rpb4Δmutant strains are more resistant to heat shock than a WT strain. A xrn1Δ strain shows higher glycogenaccumulation than a WT strain, consistent with a lower PKA activity. However, a rpb4Δ strain shows nosignificant differences in comparison with a WT strain. We also evaluated the roles of Xrn1 and Rpb4 inTPK1 gene expression. TPK1 mRNA half-life increases in a xrn1Δ strain under optimal growth conditions.Moreover, TPK1 promoter activity and mRNA levels are upregulated upon thermotolerance in anXrn1-dependent manner. Accordingly, Xrn1 is recruited to the TPK1 promoter upon heat stress, consistentwith its role in TPK1 transcription regulation. Although during thermotolerance TPK1 mRNA levels arestrikingly upregulated in a xrn1Δ strain, Tpk1 protein levels severely decrease, indicating that Xrn1 alsoregulates Tpk1 translation. Noteworthy, Xrn1 does not regulate other PKA subunits expression. Regardingthe role of Rpb4 in TPK1 expression, TPK1 mRNA levels are higher in a rpb4Δ strain than in a WT strain, butdo not change in response to stress. Tpk1 protein levels are also higher in a rpb4Δ than in a WT strain inall conditions. Bcy1 protein levels are highly upregulated in rpb4Δ, explaining the lack of differences inglycogen accumulation in comparison to WT strain. Our results support the idea that Xrn1 and Rpb4 arecentral elements that coordinate TPK1 transcription in the nucleus with posttranscriptional cytoplasmicprocesses upon heat stress.