Glucose-dependent turnover of the mRNAs encoding succinate dehydrogenase peptides in Saccharomyces cerevisiae: sequence elements in the 5' untranslated region of the Ip mRNA play a dominant role.

Abstract

We have demonstrated previously that glucose repression of mitochondrial biogenesis in Saccharomyces cerevisiae involves the control of the turnover of mRNAs for the iron protein (Ip) and flavoprotein (Fp) subunits of succinate dehydrogenase (SDH). Their half-lives are >60 min in the presence of a nonfermentable carbon source (YPG medium) and <5 min in glucose (YPD medium). This is a rare example in yeast in which the half-life of an mRNA can be controlled by manipulating external conditions. In our current studies, a series of Ip transcripts with internal deletions as well as chimeric transcripts with heterologous sequences (internally or at the ends) have been examined, and we established that the 5'-untranslated region (5' UTR) of the Ip mRNA contains a major determinant controlling its differential turnover in YPG and YPD. Furthermore, the 5' exonuclease encoded by the XRN1 gene is required for the rapid degradation of the Ip and Fp mRNAs upon the addition of glucose. In the presence of cycloheximide the nucleolytic degradation of the Ip mRNA can be slowed down by stalled ribosomes to allow the identification of intermediates. Such intermediates have lost their 5' ends but still retain their 3' UTRs. If protein synthesis is inhibited at an early initiation step by the use of a prtl mutation (affecting the initiation factor eIF3), the Ip and Fp mRNAs are very rapidly degraded even in YPG. Significantly, the arrest of translation by the introduction of a stable hairpin loop just upstream of the initiation codon does not alter the differential stability of the transcript in YPG and YPD.These observations suggest that a signaling pathway exists in which the external carbon source can control the turnover of mRNAs of specific mitochondrial proteins. Factors must be present that control either the activity or more likely the access of a nuclease to the select mRNAs. As a result, we propose that a competition between initiation of translation and nuclease action at the 5' end of the transcript determines the half-life of the Ip mRNA.

The AMP-activated Protein Kinase Snf1 Regulates Transcription Factor Binding, RNA Polymerase II Activity, and mRNA Stability of Glucose-repressed Genes in Saccharomyces cerevisiae

“…The data are expressed as binding (ChIP/input) for ADH2tss and ORF relative to ChIP/input at the TEL region used as a reference. as could involve enhanced mRNA decay, analogous to the glucose-induced mRNA decay that has been documented for SDH2 (50), JEN1 (48), GAL (2), and PCK1 (49) mRNAs. The apparent half-life of an mRNA can be estimated by the time required to reach a new steady state level (51).…”

“…Addition of a fermentable carbon source to cells growing oxidatively leads to the rapid decay of transcripts derived from the glucose-repressible genes CYC1 (56), SUC2, SDH1, SDH2 (50,(57)(58)(59)(60), FBP1, PCK1 (49,61), and JEN1 (48). In addition GAL mRNAs respond to a shift from galactose-to-glucose metabolism by a dramatic shortening of their half-lives (2,62).…”

The Target of Rapamycin Signaling Pathway Regulates mRNA Turnover in the YeastSaccharomyces cerevisiae

“…Therefore, the stabilization of mRNAs after rapid glucose depletion is likely to be due to a mechanism that is different from what we have observed. It should also be noted that, unlike the destabilization of mRNAs that we have observed at diauxic shift when glucose becomes limiting and cells switch to respiration, the SDH1 and SDH2 mRNAs are stabilized when cells are grown in carbon sources that require respiration and are destabilized in the presence of glucose (Lombardo et al, 1992;Cereghino et al, 1995). Whether TOR is involved in the regulation of the stability of these mRNAs remains to be determined.…”

Analysis of the 5′ untranslated region (5′UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris

“…P. pastoris RNA was prepared by a standard procedure adapted for total yeast RNA isolation (Cereghino et al, 1995). Samples containing 10 µg of total RNA were loaded into the wells of 1.5% agarose/denaturing formaldehyde gels and electrophoresed for separation.…”