Ski7p G protein interacts with the exosome and the Ski complex for 3'-to-5' mRNA decay in yeast

Abstract

Two cytoplasmic mRNA-decay pathways have been characterized in yeast, and both are initiated by shortening of the 3'-poly(A) tail. In the major 5'-to-3' decay pathway, the deadenylation triggers removal of the 5'-cap, exposing the transcript body for 5'-to-3' degradation. An alternative 3'-to-5' decay pathway also follows the deadenylation and requires two multi-complexes: the exosome containing various 3'-exonucleases and the Ski complex consisting of the RNA helicase Ski2p, Ski3p and Ski8p. In addition, Ski7p, which has an N-terminal domain and a C-terminal elongation factor 1alpha-like GTP-binding domain, is involved in the 3'-to-5' decay. However, physical interaction between the exosome and the Ski complex, together with the function of Ski7p, has remained unknown. Here we report that the N domain of Ski7p is required and sufficient for the 3'-to-5' decay. Furthermore, the exosome and the Ski complex interact with the different regions of Ski7p N domain, and both interactions are required for the 3'-to-5' decay. Thus, Ski7p G protein appears to function as a signal-coupling factor between the two multi-complexes operating in the 3'-to-5' mRNA-decay pathway.

Fig. 1. Intracellular localization of Ski7p in yeast. A yeast strain expressing Myc-tagged SKI7 (MC028, top panels) and the wild type (W303a, bottom panels) were analyzed by confocal microscopy as described in Materials and methods. (A) Nomarski images of the cells. (B) DNA of the cells was stained with PicoGreen. (C) Ski7p-Myc was stained with anti-Myc (primary) and anti-mouse IgG coupled to Alexa-568 (secondary) antibodies. (D) Overlay of the two signals shown in (B) and (C).

Fig. 2. The N domain of Ski7p is necessary and sufficient for the 3′-to-5′ degradation of mRNA. Yeast strains MC046 (vector), MC047 (Ski7p/N), MC048 (Ski7p/C) and MC049 (Ski7p/Full), carrying the GAL1:MFA2pG reporter, were grown in a galactose-containing medium and harvested at the mid-log phase (OD₆₀₀ = 0.4–0.5). RNA extracted from the cells was subjected to polyacrylamide gel northern blot analysis as described in Materials and methods. Positions of the full-length and poly(G) (pG) to 3′-end fragments of MFA2pG mRNA are indicated on the left. ORF, open reading frame.

Fig. 3. The N domain of Ski7p interacts with the components of the Ski complex and the exosome. Cell extracts were obtained from the indicated yeast strains expressing Ski2p-Myc and Ski8p-HA (A–E) or Prp4p-Myc and Ski6p-HA (F–J). The yeast strains also expressed the indicated forms of Flag-tagged Ski7p (A–E, lanes 1–4 for MC146–MC149; F–J, lanes 1–4 for MC151–MC154). The cell extracts were immunoprecipitated (IP) with an anti-Flag antibody. The precipitated proteins (B, D, E, G, I and J), together with the whole-cell extracts (A, C, F and H), were immunoblotted (IB) with anti-Myc (9E10), anti-HA (12CA5), and anti-Flag (M2) antibodies to detect Ski2p (or Rrp4p), Ski8p (or Ski6p) and Ski7p. The asterisks in (E) and (J) indicate Flag-tagged Ski7p mutants expressed in the various strains. The arrowheads indicate the positions of IgG bands.

Fig. 4. Interaction of the N domain of Ski7p with Rrp4p is maintained in the Ski complex-disrupted yeast strains. Cell extracts were prepared from wild-type SKI8 or ski8Δ strains carrying Ski2p-Myc (A–C, lanes 1–6 for MC052, MC053, MC055, MC130, MC131 and MC133) or Rrp4p-Myc (D–F, lanes 1–6 for MC126, MC127, MC129, MC134, MC135 and MC137). The yeast strains also expressed the indicated forms of Flag-tagged Ski7p. The cell extracts were immunoprecipitated (IP) with the anti-Flag antibody. The precipitated proteins (B, C, E and F), together with the whole-cell extracts (A and D), were immunoblotted (IB) with anti-Myc (9E10) and anti-Flag (M2) antibodies to detect Ski2p (or Rrp4p) and Ski7p, respectively. The asterisks in (C) and (F) indicate Flag-tagged Ski7p mutants expressed in the various strains. The arrowheads indicate the positions of IgG bands.

Fig. 5. The Ski complex and the exosome interact with the different regions of the Ski7p N domain. Cell extracts were obtained from the indicated yeast strains expressing Skip2-Myc and Ski6p-HA (A–E) or Prp4p-Myc and Ski2p-HA (F–J). The yeast strains (MC146, MC147, MC151, MC152, MC155–164) also expressed the various deletion mutants of Ski7p N domain with a Flag tag (lanes 1–7). The cell extracts were immunoprecipitated (IP) with the anti-Flag antibody. The precipitated proteins (B, D, E, G, I and J), together with the whole-cell extracts (A, C, F and H), were immuno blotted (IB) with anti-Myc (9E10), anti-HA (12CA5) and anti-Flag (M2) antibodies to detect Ski2p (or Rrp4p), Ski8p (or Ski6p) and Ski7p. The asterisks in (E) and (J) indicate Flag-tagged Ski7p mutants expressed in the various strains. The arrowheads indicate the positions of IgG bands.

Fig. 6. Both the Ski complex- and the exosome-binding regions of Ski7p N domain are required for the 3′-to-5′ degradation of mRNA. The ski7Δ strain was transformed with plasmids carrying the indicated deletion mutants of Ski7p N domain (lanes 1–7 for MC046, MC047 and MC138–MC142). The transfected cells were grown in the galactose-containing medium and harvested at mid-log phase. RNA extracted from the cells was subjected to polyacrylamide gel northern blot analysis as described in Figure 2. Positions of the full-length and poly(G) to 3′-end fragments of MFA2pG mRNA are indicated on the left.

Fig. 7. Inhibition of 3′-to-5′ mRNA degradation by overexpressing either the Ski complex- or exosome-binding region of Ski7p N domain. The yeast strain MC035 (SKI7; wild type) was transformed with plasmids carrying the indicated deletion mutants of Ski7p N domain (lanes 1–8 for MC046, MC043, MC118–MC122 and MC042). The transfected cells were grown in the galactose-containing medium and harvested at mid-log phase. RNA extracted from the cells was subjected to polyacrylamide gel northern blot analysis as described in Figure 2. Positions of the full-length and poly(G) to 3′-end fragments of MFA2pG mRNA are indicated on the left.

Fig. 8. The N domain of Ski7p interacting with the Ski complex and the exosome. (A) Deletion mutants of Ski7p used in this study and the summary of the present results obtained in Figures 5, 6 and 7. (B) A proposed model for the Ski complex- and the exosome-interacting sites of the Ski7p N domain.