Expression regulation of protein kinase A catalytic subunit, Tpk1, from Saccharomyces cerevisiae

DÁVILA GALLESIO, JIMENA; GALELLO, FIORELLA; ROSSI, SILVIA

Congreso; Molecular mechanisms in cell signaling and gene expression" Reunión Anual SAIB; 2013

SAIB

Upstream open reading frames (uORFs) are translational regulatory elements located in 5´ untranslated regions. They can repress the translation of downstream coding sequences (CDS). S. cerevisiae protein kinase A (PKA) is composed of a regulatory subunit Bcy1 and three catalytic subunits, Tpk1, Tpk2 and Tpk3, a TPK1 uORF was described in the 5´UTR (five codons length). We cloned the full-length UTR sequence into a β-galactosidase (β-Gal) reporter. β-Gal activity and mRNA level, quantified by qRT-PCR, were compared between the WT UTR construct and a construct where the uORF start codon was mutated. The results indicated that the uORF regulates negatively TPK1 translation but does not destabilize the mRNA. The analysis was also made under heat shock stress, as our previous results had shown that Tpk1 expression is upregulated. Both β-Gal activity and Tpk1 mRNA level were upregulated, β- Gal activity increased more than mRNA level. A ncRNA antisense (AS) was also identified overlaping 600 pb on CDS 3´end. The AS level was measured in log and stationary growth phases and order heat shock stress. The AS level went with TPK1 mRNA levels, indicating a possible role in activation, but not repression of TPK1 expression. Thus, both mechanisms, uORF and antisense ncRNA could be contributing to regulate the expression of PKA Tpk1 subunit at transcriptional and post-transcriptional levels respectively