Expression regulation of Protein Kinase A catalytic subunit, Tpk1, from Saccharomyces cerevisiae

DAVILA GALLESIO J, GALELLO F, ROSSI S

Buenos Aires

Congreso; XLVIIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2013

SAIB

Upstream open reading frames (uORFs) are translational regulatory elements located in 5´ untranslated regions. They can repress the translation of the downstream coding sequences (CDS), and participate in the spatio-temporal regulations of protein translation. Protein kinase A (PKA) is composed by a regulatory subunit Bcy1 and three catalytic subunits, Tpk1, Tpk2 and Tpk3. Herein, we describe that TPK1 5´UTR contains an uORF (five codons length). We cloned the full-length UTR sequence into a β-galactosidase (β-Gal) reporter system. 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 not destabilizes the mRNA. The analysis was also made under heat shock stress, conditions in which our previous results had shown that Tpk1 expression is upregulated. Both β-Gal activity and Tpk1 mRNA level were upregulated, but the β-Gal activity increased more that mRNA level. A ncRNA antisense (AS) was also identified that overlaps 600 pb on CDS 3´end. The AS levels were measured in log and stationary growth phases and under heat shock. The AS levels went with TPK1 mRNA levels, indicating a possible role in activation, but not repression of TPK1 expression. In summary, the results shown that both mechanisms, uORF and antisense ncRNA could be contributing to regulate the expression of PKA Tpk1 subunit at transcriptional and post-transcriptional levels respectively