A truncated form of KlLsm4p and the absence of factors involved in mRNA decapping trigger apoptosis in yeast

Abstract

The LSM4 gene of Saccharomyces cerevisiae codes for an essential protein involved in pre-mRNA splicing and also in mRNA decapping, a crucial step for mRNA degradation. We previously demonstrated that the first 72 amino acids of the Kluyveromyces lactis Lsm4p (KlLsm4p), which contain the Sm-like domains, can restore cell viability in both K. lactis and S. cerevisiae cells not expressing the endogenous protein. However, the absence of the carboxy-terminal region resulted in a remarkable loss of viability in stationary phase cells (). Herein, we demonstrate that S. cerevisiae cells expressing the truncated LSM4 protein of K. lactis showed the phenotypic markers of yeast apoptosis such as chromatin condensation, DNA fragmentation, and accumulation of reactive oxygen species. The study of deletion mutants revealed that apoptotic markers were clearly evident also in strains lacking genes involved in mRNA decapping, such as LSM1, DCP1, and DCP2, whereas a slight effect was observed in strains lacking the genes DHH1 and PAT1. This is the first time that a connection between mRNA stability and apoptosis is reported in yeast, pointing to mRNA decapping as the crucial step responsible of the observed apoptotic phenotypes.

Figure 1

Chromatin fragmentation. DAPI staining and phase contrast of S. cerevisiae strain MCY4 containing plasmids pRS416/KlLSM4 (a and b), pRS416/Kllsm4Δ1 (c–f), and the corresponding [rho°] derivatives (KlLSM4, g and h; Kllsm4Δ1, i–l) grown on YPD.

Figure 2

Electron micrographs of strain MCY4-containing plasmids pRS416/KlLSM4 (a) and pRS416/Kllsm4Δ1 (b–d) grown on YPD. The arrows indicate the condensed and marginated chromatin. N, nucleus; M, mitochondria; V, vacuole. Bar, 1 μm.

Figure 3

DNA strand breakage detection. To express the endogenous LSM4p, MCY4 cells were grown exponentially on galactose (a), whereas MCY4 cells containing plasmids pRS416/KlLSM4 (b) and pRS416/KllsmΔ1 (c) were grown on YPD. After cell fixing and digestion of cell walls, strands breaks in DNA were detected according to the TUNEL protocol.

Figure 4

MCY4 expressing Kllsm4Δ1 accumulates ROS. Cell fluorescence (a–d) and phase contrast displays of cells (e–h) after 2 h incubation with dihydrorhodamine 123 are shown. Wild-type control (a–e), MCY4/Kllsm4Δ1 (b, c, f, and g) grown in glucose and after 2 h incubation with 4 mM H₂O₂ (d and h).

Figure 5

MCY4 expressing Kllsm4Δ1 shows an increased mRNA stability. Strain MCY4 was grown on YP-galactose to induce the GAL1-LSM4 gene (lanes 1–6), whereas MCY4/KlLSM4 (lanes 7–12) and MCY4/Kllsm4Δ1 (lanes 13–18) were grown on YPD media. (A) Total RNAs were prepared from cells grown overnight at 24°C. After 15 h of incubation at 45°C, cultures were backshifted at 24°C and RNA samples were prepared after 15 min, 45 min, 60 min, and 120 min and probed with the SSA4 gene. (B) Total RNAs prepared from overnight cultures and probed with the SNR17 gene (see MATERIALS AND METHODS). M, RNA molecular weight marker.

Figure 6

DAPI staining (a and b) and EM analysis (c and d) of S. cerevisiae mutant strain lsm1. N, nucleus; M, mitochondria; V, vacuole. Bar, 1 μm.

Figure 7

DAPI staining and its phase contrast, TUNEL test, DHR staining of S. cerevisiae mutants dcp1, dcp2, dhh1, and pat1 (see text and MATERIALS AND METHODS for details).

Figure 8

Electron micrographs of strains dcp1 (a), dcp2 (b), dhh1 (c), and pat1 (d). The arrows indicate the condensed and marginated chromatin. N, nucleus; M, mitochondria; V, vacuole; ER, endoplasmic reticulum. Bar, 1 μm.