Expression regulation of yeast PKA subunits, Tpk1 and Bcy1, by uORF

GATTAS, MJ; DAVILA GALESIO, J; GALELLO, F; ROSSI, S

Mar del Plata

Congreso; LI Reunion SAIB; 2015

SAIB

Upstream open reading frames (uORFs) are regulatory elements located in 5? untranslated regions, which can repress the translation of downstream coding sequences. In S. cerevisiae, the catalytic subunit of protein kinase A (PKA) is encoded by three genes TPK1, TPK2 and TPK3; while the regulatory subunit is encoded by the BCY1 gene. In TPK1 an uORF was described in the 5? UTR, containing five codons length, and in BCY1 5´UTR we defined one uORF of 50codons length. In order to study the role of TPK1 and BCY1 uORFs, full-length 5´ sequence (WT-UTR) or a version with the mutated uORF start codon (mut-UTR) of each subunit were cloned into a β-galactosidase (β-Gal) reporter vector.β-Gal activity and mRNA levels were measured. We found that TPK1 and BCY1 uORFs negatively regulate translation and stabilize the mRNAs. Under heat shock stress, β-Gal activity was upregulated for bothTpk1 WT-UTR and mut-UTR while mRNA levels were upregulated only for Tpk1 WT-UTR.Therefore the translational efficiency resulted higher for the mut-UTR construct. In the case of BCY1, heat shock stress resulted in a downregulation of β-Gal activity and mRNA levels for both WT-UTR and mut-UTR. Thus, uORF could contribute to regulate the expression of PKA Tpk1 and Bcy1 subunits at the post-transcriptional level.