Role of cytoplasmic deadenylation and mRNA decay factors in yeast apoptosis.

Strains of Saccharomyces cerevisiae lacking factors involved in 5' to 3' mRNA decay pathway (DCP1, DCP2, DHH1, PAT1, LSM1 and LSM4) exhibit caspase-dependent apoptosis and accelerated chronological aging. In the present study, yeast strains lacking mRNA decapping activation factors (DCP2 and LSM1), cytoplasmic exosome function (SKI2) or cytoplasmic deadenylases (double deletion of CCR4 and PAN2) showed typical markers of eukaryotic apoptosis such as increased cellular reactive oxygen species levels, externalization of phosphatidyl serine, chromatin fragmentation, enhanced caspase gene (YCA1) expression and protein activity in mid-log phase cultures. The transcript levels of negative regulators of mRNA decapping (eIF4E and Pab1) were considerably elevated in strains defective in cytoplasmic deadenylation and reduced in strains lacking cytoplasmic 3' to 5' exosome function or decapping activators. Among the yeast strains studied, lsm1Δ and ccr4Δpan2Δ mutants displayed strongest apoptotic phenotype followed by mutants lacking DCP2 or SKI2. Among yeast strains exhibiting deadenylation defects, slight apoptotic phenotype was observed in ccr4Δ mutants and cell death markers imperceptible in pan2Δ mutants.

Both Sm-domain and C-terminal extension of Lsm1 are important for the RNA-binding activity of the Lsm1-7-Pat1 complex.

Lsm proteins are a ubiquitous family of proteins characterized by the Sm-domain. They exist as hexa- or heptameric RNA-binding complexes and carry out RNA-related functions. The Sm-domain is thought to be sufficient for the RNA-binding activity of these proteins. The highly conserved eukaryotic Lsm1 through Lsm7 proteins are part of the cytoplasmic Lsm1-7-Pat1 complex, which is an activator of decapping in the conserved 5'-3' mRNA decay pathway. This complex also protects mRNA 3'-ends from trimming in vivo. Purified Lsm1-7-Pat1 complex is able to bind RNA in vitro and exhibits a unique binding preference for oligoadenylated RNA (over polyadenylated and unadenylated RNA). Lsm1 is a key subunit that determines the RNA-binding properties of this complex. The normal RNA-binding activity of this complex is crucial for mRNA decay and 3'-end protection in vivo and requires the intact Sm-domain of Lsm1. Here, we show that though necessary, the Sm-domain of Lsm1 is not sufficient for the normal RNA-binding ability of the Lsm1-7-Pat1 complex. Deletion of the C-terminal domain (CTD) of Lsm1 (while keeping the Sm-domain intact) impairs mRNA decay in vivo and results in Lsm1-7-Pat1 complexes that are severely impaired in RNA binding in vitro. Interestingly, the mRNA decay and 3'-end protection defects of such CTD-truncated lsm1 mutants could be suppressed in trans by overexpression of the CTD polypeptide. Thus, unlike most Sm-like proteins, Lsm1 uniquely requires both its Sm-domain and CTD for its normal RNA-binding function.