A Yeast Homologue of Hsp70, Ssa1p, Regulates Turnover of the MFA2 Transcript through Its AU-Rich 3′ Untranslated Region

Abstract

Many eukaryotic mRNAs exhibit regulated decay in response to cellular signals. AU-rich elements (AREs) identified in the 3 untranslated region (3-UTR) of several such mRNAs play a critical role in controlling the half-lives of these transcripts. The yeast ARE-containing mRNA, MFA2, has been studied extensively and is degraded by a deadenylation-dependent mechanism. However, the trans-acting factors that promote the rapid decay of MFA2 have not been identified. Our results suggest that the chaperone protein Hsp70, encoded by the SSA family of genes, is involved in modulating MFA2 mRNA decay. MFA2 is specifically stabilized in a strain bearing a temperature-sensitive mutation in the SSA1 gene. Furthermore, an AU-rich region within the 3-UTR of the message is both necessary and sufficient to confer this regulation. Stabilization occurs as a result of slower deadenylation in the ssa1 ts strain, suggesting that Hsp70 is required for activation of the turnover pathway.Gene expression is a highly controlled process involving regulation at both transcriptional and posttranscriptional levels. In recent years mRNA turnover has emerged as an important target for the regulation of gene expression (33,54). A large number of mRNAs encoding cytokines, growth factors, and proto-oncogenes display regulated decay in response to external signals (44, 50). Hence, understanding the pathways regulating transcript stability is of critical importance. Selective mRNA degradation is mediated by a number of different cisacting sequences, including the most-investigated class, called AU-rich elements (AREs), present in the 3Ј untranslated region (3Ј-UTR) of a variety of mammalian and yeast mRNAs (44,49,54). Recent experiments have shown that the pathways and factors involved in ARE-mediated mRNA decay are conserved between yeast and higher eukaryotes, making yeast an ideal system for the study of this phenomenon (49).The ARE is a stability determinant whose sequence is loosely defined and ranges in size from 50 to 150 nucleotides. These elements are typically found in the 3Ј-UTR and contain one or more copies of the pentameric sequence AUUUA flanked by a high content of U's and A's (8, 47). An important feature of many AREs is that they modulate the stability of transcripts in response to cellular stimuli. They can cause instability under some conditions by enhancing the rate of removal of the poly(A) tail and the subsequent degradation of the body of the transcript (7, 38, 48). In contrast, under stabilizing conditions AREs can inhibit the decay process (see references 49 and 54 and references therein). In yeast, at least two classes of ARE-containing mRNAs have been identified, represented by the MFA2 and TIF51A/HYP2 transcripts (49). In both cases decay proceeds through poly(A) tail shortening followed by decapping and 5Ј33Ј exonucleolytic decay (36, 49). The stability of the TIF51A transcript is modulated in response to changes in carbon source (11,49). The mRNA is stable in cells grown in glucose and unstable in cells grown under nong...

The human c‐fos and TNFα AU‐rich elements show different effects on mRNA abundance and protein expression depending on the reporter in the yeast Pichia pastoris

“…Although the level of expression varies slightly, there is no significant difference between the constructs ( Figure 4A). This finding supports the assumption that flanking sequences can in fact influence ARE-mediated effects (Duttagupta et al, 2003;Fialcowitz et al, 2005;Wilson et al, 2001). .…”

“…It is known that the local ARE structure can be involved in ARE-mediated mRNA turnover and it is hypothesized that ARE-flanking sequences might be involved in binding protein selectivity (Duttagupta et al, 2003;Fialcowitz et al, 2005;Wilson et al, 2001). RNA molecules can form different structures of high complexity.…”

“…In the TNFα ARE, the overlapping UUAUUUA(U/A)(U/A) nonamers are underlined. The core sequence, which has been used in mammalian reporter systems (Xu et al, 1997) and in the yeast S. cerevisiae (Vasudevan and Peltz, 2001), is used in this study and is also underlined suggest that the surrounding context or secondary structure is important for recognition of the ARE of MFA2 by regulatory factors (Duttagupta et al, 2003). Stem-loop structures downstream (Barreau et al, 2006) or within an ARE (Chabanon et al, 2005;Fialcowitz et al, 2005) have already been identified and shown to be important for ARE regulation.…”

Negative Transcriptional Regulation of Multidrug Resistance Gene Expression by an Hsp70 Protein

“…Ssa2p is 96% identical to Ssa1p, whereas Ssa3p and Ssa4p share ϳ80% identity with Ssa1p (40). Furthermore, Pdr3p repression would represent only one of the vast array of functions influenced by these Ssa proteins, including protein translation (41), protein insertion into the endoplasmic reticulum (42), prion production (43), and even mRNA turnover (44). Loss of either SSA1 or SSA2 individually has little effect on the cell as the remaining gene appears to compensate for the absence of its homologue (35).…”

A three-hybrid screen identifies mRNAs controlled by a regulatory protein

“…RNA polymerase inhibition was performed as in Duttagupta et al (2003). Cultures were grown in 50 mL YPAD to an OD 600 z0.8.…”