The DEAD-box RNA helicase Ded1p affects and accumulates in Saccharomyces cerevisiae P-bodies

Abstract

Recent results suggest that cytoplasmic mRNAs can form translationally repressed messenger ribonucleoprotein particles (mRNPs) capable of decapping and degradation, or accumulation into cytoplasmic processing bodies (P-bodies), which can function as sites of mRNA storage. The proteins that function in transitions between the translationally repressed mRNPs that accumulate in P-bodies and mRNPs engaged in translation are largely unknown. Herein, we demonstrate that the yeast translation initiation factor Ded1p can localize to P-bodies. Moreover, depletion of Ded1p leads to defects in P-body formation. Overexpression of Ded1p results in increased size and number of P-bodies and inhibition of growth in a manner partially suppressed by loss of Pat1p, Dhh1p, or Lsm1p. Mutations that inactivate the ATPase activity of Ded1p increase the overexpression growth inhibition of Ded1p and prevent Ded1p from localizing in P-bodies. Combined with earlier work showing Ded1p can have a positive effect on translation, these results suggest that Ded1p is a bifunctional protein that can affect both translation initiation and P-body formation.

Figure 1.

Ded1p-GFP accumulation in P-bodies is dependent on ATP-hydrolysis. (A) P-body formation of wild-type strain yRP2065 containing Ded1p-GFP (pRP1558) and Dcp2p-RFP (pRP1155) with or without dextrose or at high OD₆₀₀. (B) yRP2065 containing Ded1p-GFP (pRP1558) or Ded1p-DAAD-GFP (pRP1562). Percentage of foci-containing cells is indicated.

Figure 2.

Belle accumulates in RNA granules in Drosophila neurites. Localization of Belle in neurites (A) and cell bodies (B) as determined by indirect immunofluorescence in Drosophila neurons expressing a dFMR-YFP transgene as a marker of neuronal granules. Neuronal culturing, staining, and microscopy were performed as described earlier (Barbee et al., 2006).

Figure 3.

Depletion of Ded1p inhibits translation and P-body formation, but not mRNA decay. (A) ³⁵S incorporation in Tet-off DED1 strain (yRP2433) in the absence or presence 10 μg/ml doxycycline. Cells were assayed with 10 μg/ml cyclohexamide in the presence of 2% dextrose (CHX), the presence of 2% dextrose only (dex), or the absence of dextrose (−dex). Recovery refers to a 10-min incubation after the reintroduction of 2% dextrose to cells starved of dextrose. (B) Polysome analysis of Tet-off DED1 strain (yRP2433) in the presence or absence of 10 μg/ml doxycycline. (C) RNA stability is unaffected by depletion of Ded1p. Tet-off strain (yRP2433) transformed with a plasmid carrying MFA2pG (pRP590). (D) Tet-off DED1 strain (yRP2433) carrying Dcp2p-GFP (pRP1175) in the presence or absence of 10 μg/ml doxycycline. Cells were analyzed in the presence of dextrose, in the absence of dextrose, or in water. Percentage of cells with foci is indicated.

Figure 4.

Overexpression of DED1 or the ATP-hydrolysis mutant inhibits translation, but does not affect mRNA stability. (A) Wild-type strain yRP840 was transformed with either a 2μ vector (pRP247), Gal-DED1 (pRP1559), or mutant Gal-ded1-DAAD (pRP1564). ³⁵S incorporation was measured in the presence of cyclohexamide (C), before galactose induction of overexpression (0), and after 2 h of induction (2). (B) Polysome analysis of the strains as described in A after a 2-h induction in 2% galactose. (C) Decay analysis of PGK1pG mRNA in the strains described in A after a 2-h induction in 2% galactose.

Figure 5.

Overexpression of DED1 can promote the formation of P-bodies. For the Ded1-GFP strain (bottom row), yRP840 was transformed with DED1-GFP (pRP1557) and either 2μ control vector (pRP247) or Gal-DED1 (pRP1559). For all other strains, a strain containing a GFP-tagged RNA metabolism factor (see Table 1) was transformed with either 2μ control vector (pRP247) or Gal-DED1 (pRP1559). All strains were monitored in 2% sucrose (suc) before induction (0 h) and after 2 h (2 h) of induction in selective media containing 2% galactose (gal).

Figure 6.

Overexpression of DED1 confers a growth defect that is partially suppressed by deletion of PAT1, DHH1, or LSM1. Wild-type (yRP2065) or deletion strains (see Table 1) were transformed with either 2μ control vector (pRP247), Gal-DED1 (pRP1559), or Gal-ded1-DAAD (pRP1564) and grown on selective media containing either 2% dextrose or 2% galactose.

Figure 7.

Ded1p fractionates with RNPs and the 40S subunit. A polysome analysis of wild type strain yRP840 transformed with ded1-DAAD-GFP (pRP1562) and a Western blot of collected fractions probed with anti-Ded1p antibody.