Function of the Ski4p (Csl4p) and Ski7p Proteins in 3′-to-5′ Degradation of mRNA

Abstract

One of two general pathways of mRNA decay in the yeast Saccharomyces cerevisiae occurs by deadenylation followed by 3-to-5 degradation of the mRNA body. Previous results have shown that this degradation requires components of the exosome and the Ski2p, Ski3p, and Ski8p proteins, which were originally identified due to their superkiller phenotype. In this work, we demonstrate that deletion of the SKI7 gene, which encodes a putative GTPase, also causes a defect in 3-to-5 degradation of mRNA. Deletion of SKI7, like deletion of SKI2, SKI3, or SKI8, does not affect various RNA-processing reactions of the exosome. In addition, we show that a mutation in the SKI4 gene also causes a defect in 3-to-5 mRNA degradation. We show that the SKI4 gene is identical to the CSL4 gene, which encodes a core component of the exosome. Interestingly, the ski4-1 allele contains a point mutation resulting in a mutation in the putative RNA binding domain of the Csl4p protein. This point mutation strongly affects mRNA degradation without affecting exosome function in rRNA or snRNA processing, 5 externally transcribed spacer (ETS) degradation, or viability. In contrast, the csl4-1 allele of the same gene affects rRNA processing but not 3-to-5 mRNA degradation. We identify csl4-1 as resulting from a partial-loss-of-function mutation in the promoter of the CSL4 gene. These data indicate that the distinct functions of the exosome can be separated genetically and suggest that the RNA binding domain of Csl4p may have a specific function in mRNA degradation.

The evolutionarily conserved subunits Rrp4 and Csl4 confer different substrate specificities to the archaeal exosome

“…Fig. 5C and D shows that a G(GU) 16 transcript was well degraded and prolonged. However, the degradation of MCS-RNA was more efficient than the degradation of its polyuridylated form MCS-RNA(U) 20 in competition assays (Fig.…”

“…For in vitro transcription of MCS-RNA 20 nucleotide (nt) and 30 nt variants, the primer 5 0 -TCGAGCAGCTGAAGCTTCCCTATAGTGAGTCGTATTA-3 0 and 5 0 -TCGA GCAGCTGAAGCTTGCATGCCTGCACCCTATAGTGAGTCGTATTA-3 0 were annealed with the T7-oligonucleotide (5 0 -TAATACGACTCACTA-TAGGG-3 0 ), and the MEGAshortscript™ Kit (Ambion) was used. Oligonucleotides with a 3 0 -sequence complementary to the T7-oligonucleotide were also used for the in vitro transcription of the GGG(CA) 15 and G(GU) 16 substrates. Heteropolymeric transcripts were internally labelled using [a-32 P] UTP or [a-32 P] ATP.…”

“…This and the fact that the G-content of naturally occurring RNA tails in S. solfataricus is higher than 30%, suggest that G is efficiently bound by the exosome. This may explain the efficient processing of G(GU) 16 RNA in comparison to polyurydylated RNA. The assumption that the exosome of S. solfataricus prefers purines over pyrimidines is in agreement with its failure to degrade or to polyadenylate a 16S ribosomal RNA (rRNA)-based substrate with a pyrimidine-rich 3 0 -end in vitro [10].…”

“…5. The vast majority of a GGG(CA) 15 -transcript is not processed by the exosome, while a G(GU) 16 -transcript is degraded or polyadenylated. Substrates (0.4 pmol per assay), processing products and protein complexes are indicated.…”

“…1). It is noteworthy that Rrp4 and Csl4 are conserved in the exosomes of Archaea and Eukarya [3,[16][17][18], suggesting important differential roles for these proteins, most probably in substrate selection. The archaeal exosome is a suitable system for analysis of the functions of Rrp4 and Csl4: in contrast to the eukaryotic exosome, active protein complexes with homotrimeric Rrp4 or Csl4 caps bound to the hexameric core can be reconstituted [6,[9][10][11].…”