EXPRESSION REGULATION OF PROTEIN KINASE A CATALYTIC SUBUNIT, Tpk1, FROM Saccharomyces cerevisiae, by uORF y ncRNA

DAVILA GALLESIO J, ROSSI S

Rosario, Santa Fe

Congreso; L reunion SAIB 2014; 2014

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. In TPK1 an uORF was described in the 5? UTR (five codons length). In order to study the role of TPK1 uORF, the full-length 5´ sequence was cloned into a β-galactosidase (β-Gal) reporter (WT-UTR) and a version with the uORF start codon mutated (mut-UTR) was constructed. β-Gal activity and mRNA levels were measured. The uORF regulates negatively TPK1 translation and stabilized the mRNA. Under heat shock stress, both β-Gal activity and Tpk1 mRNA level were upregulated for WT-UTR and β-Gal activity but not mRNA level was upregulated for mut-UTR. Therefore the translational efficiency resulted higher for the mut-UTR construct. In other hand, a ncRNA antisense (AS) was also identified overlapping 600pb on CDS 3? end. The AS level was measured in log and stationary growth phases and under heat shock stress. The AS level showed an inverse correlation with TPK1 mRNA, during the stress conditions, indicating a possible role in repression of TPK1 expression. Thus, uORF and antisense ncRNA could contribute to regulate the expression of PKA Tpk1 subunit at transcriptional and post-transcriptional levels respectively.