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.

FIG. 1.

(A) Structure of the Hsp70 protein. Hsp70s consist of an N-terminal ATPase domain and linker and variable C-terminal domains of unknown function. The position of the point mutation (P417L), which makes the SSA1 gene temperature sensitive, is indicated. (B) Structure of the MFA2 mRNA. The MFA2 mRNA consists of the 117-nt coding region and a 169-nt 3′-UTR. The 3′-UTR is divided into a 58-nt domain, domain I (nt 184 to 241), and a 44-nt domain, domain II (nt 245 to 288). The putative AREs are underlined.

FIG. 2.

Stabilization of MFA2 mRNA occurs as a result of a loss-of-function mutation in SSA1. The half-life of MFA2 was assessed by Northern blotting in a wild-type strain (A), an ssa1^ts strain (B), and an ssa1^ts strain overexpressing SSA1 (C).

FIG. 3.

Non-ARE-containing transcripts are not affected by the ssa1^ts mutation. GCN4 (A), HTB (B), and PGK1 (C) transcripts were tested for altered decay in the ssa1^ts strain by Northern blot analysis. None of these transcripts were differentially stabilized, indicating non-ARE-containing transcripts are not affected. (D) A nonsense-containing transcript, mini-PGK1, is also immune to ssa1^ts, with no demonstrable difference in half-life, indicating that NMD substrates are not targeted for regulation by ssa1^ts.

FIG. 4.

Regulation of stability by SSA1 is specific for the MFA2 mRNA and does not affect TIF51A. (A) The ARE-containing TIF51A mRNA is not further stabilized by the ssa1^ts mutation under glucose conditions. (B) The ssa1^ts mutation regulates MFA2 but not TIF51A stability under nonglucose conditions. Northern blot profiles show that TIF51A is unstable in the ssa1^ts mutant, decaying at a rate similar to that observed for the wild type.

FIG. 5.

MFA2 mRNA decay is not affected by the Ssa1p partner proteins Ydj1p and Sis1p. Northern blots showing the decay profile of the MFA2 transcript in wild-type and mutant YDJ1 (A) and SIS1 (B) strains. MFA2 stability is not affected by either of these mutants.

FIG. 6.

The AU-rich 3′-UTR sequence of MFA2 is sufficient to mediate Hsp70-dependent regulation. (A) Map of the GCN4-MFA2 3′-UTR chimera. This chimeric construct contains the functional GCN4 uORF1 fused to the MFA2 3′-UTR. (B) MFA2 3′-UTR can stabilize a heterologous mRNA in the ssa1^ts mutant. Northern blot analysis of the chimeric construct in SSA1 and ssa1^ts strains shows that GCN4, which is normally not affected by ssa1^ts, can now respond to loss of Ssa1p activity, and behaves in the same manner as MFA2.

FIG. 7.

SSA1 regulates MFA2 mRNA stability through the AU-rich domain I of the MFA2 3′-UTR. (A) Shown are half-life measurements of wild-type and mutant (Δ1 or Δ2) MFA2 transcripts analyzed in SSA1 and ssa1^ts strains after shifting to nonpermissive temperature. (B) Northern blots depicting the decay profiles of the transcripts. A domain II deletion (MFA2-Δ2) is stabilized similarly to the endogenous transcript in an ssa1^ts mutant. A domain I deletion (MFA2-Δ1), however, makes the transcript unstable in an ssa1^ts mutant, indicating that the domain I region is critical for the regulation of MFA2 stability by SSA1.

FIG. 8.

Deadenylation of MFA2 mRNA is inhibited in the ssa1^ts strain. (A) Pathway of mRNA decay. In yeast most mRNAs degrade by the deadenylation-dependent decay pathway where deadenylation is followed by decapping and finally by predominantly 5′→3′ exonucleolytic decay. The entire MFA2 gene was fused downstream of the inducible CTR1 promoter in order to dissect which step of the deadenylation-dependent decay pathway was affected by the ssa1^ts mutant. (B) ssa1^ts affects deadenylation rates. The polyacrylamide Northern blot of MFA2 mRNA shows that deadenylation is dramatically reduced in the ssa1^ts mutant. RH denotes samples annealed with oligo(dT) and treated with RNase H prior to loading, in order to indicate the size of the deadenylated transcript.