The Two Proteins Pat1p (Mrt1p) and Spb8p Interact In Vivo, Are Required for mRNA Decay, and Are Functionally Linked to Pab1p

Abstract

We report here the characterization of a bypass suppressor of pab1⌬ which leads to a fourfold stabilization of the unstable MFA2 mRNA. Cloning of the wild-type gene for that suppressor reveals that it is identical to PAT1 (YCR077c), a gene whose product was reported to interact with Top2p. PAT1 is not an essential gene, but its deletion leads to a thermosensitive phenotype. Further analysis has shown that PAT1 is allelic with mrt1-3, a mutation previously reported to affect decapping and to bypass suppress pab1⌬, as is also the case for dcp1, spb8, and mrt3. Coimmunoprecipitation experiments show that Pat1p is associated with Spb8p. On sucrose gradients, the two proteins cosediment with fractions containing the polysomes. In the absence of Pat1p, however, Spb8p no longer cofractionates with the polysomes, while the removal of Spb8p leads to a sharp decrease in the level of Pat1p. Our results suggest that some of the factors involved in mRNA degradation could be associated with the mRNA that is still being translated, awaiting a specific signal to commit the mRNA to the degradation pathway.A general pathway of mRNA degradation in the yeast Saccharomyces cerevisiae has been described in which mRNAs are deadenylated prior to decapping and then degraded in the 5Ј-to-3Ј direction by the exonuclease Xrn1p (4, 12). The decapping enzyme has been identified in yeast and named Dcp1p (5). In most yeast strains, depletion of Dcp1p leads to a slowgrowth phenotype and to stabilization of mRNAs that are accumulated in a capped and oligoadenylated form (5). However, to become functional, Dcp1p requires the activity of Dcp2p, a putative pyrophosphatase (14). Recently, a polypeptide (M r 70,000) copurifying with Dcp1p has been identified as Ssa1p or Ssa2p, Hsp70 family members (50). Interestingly, two mutations that inhibit decapping, vps16 and mrt1, enhance the interaction of Ssa1p or Ssa2p with Dcp1p. This observation suggests that Ssa1p or Ssa2p could be involved in regulating the activity of Dcp1p.In addition to Dcp1p and Dcp2p, other factors might regulate the decapping activity, since several mutations have been isolated that lead to stabilization of capped, oligoadenylated mRNAs. The best characterized of these factors is Spb8p (6), a protein containing an Sm-like domain that has also been referred to as Lsm1p (40). The Sm motif is found in a set of proteins that interact with the small nuclear RNAs involved in mRNA splicing. However, Spb8p is distinguished from other Sm or Lsm proteins by not being associated with any known small nuclear RNA (29, 38). Mutations within three other loci named mrt1, mrt3, and vps16 lead to an accumulation of capped, oligoadenylated mRNAs (21, 50). While VPS16 has been characterized (22, 50), the genes for mrt1 and mrt3 have not yet been cloned.Several lines of evidence indicate the existence of a link between mRNA translation and degradation (23). It is now well established that the two structures present on the ends of an mRNA, the cap at the 5Ј end and the poly(A) tail at the 3Ј end, act s...

Fission Yeast Hsk1 (Cdc7) Kinase Is Required After Replication Initiation for Induced Mutagenesis and Proper Response to DNA Alkylation Damage

“…MRN is required for end resection and homologous recombination (reviewed in Williams et al 2007). We also isolated snf22 (two alleles), a SNF2-related ATPase characterized for its role in chromatin remodeling in meiosis (Yamada et al 2004); swi9/rad16, the ScRAD1 ortholog required for excision repair and some forms of recombination (Carr et al 1994;Farah et al 2005); and a mutation in SPBC19G7.10c, encoding an uncharacterized homolog of the topoisomerase-interacting S. cerevisiae Pat1 protein required for mRNA decapping (Wang et al 1996;Bonnerot et al 2000). We were unable to identify the genes corresponding to two mutations, rad37 and rad39.…”

The DEAD box helicase, Dhh1p, functions in mRNA decapping and interacts with both the decapping and deadenylase complexes

“…The process and regulation of mRNA turnover is a fundamental aspect of gene expression+ A major pathway of mRNA turnover for both stable and unstable transcripts has been identified in Saccharomyces cerevisiae+ In this pathway, degradation is initiated by deadenylation of the 39 poly(A) tail (Muhlrad & Parker, 1992;Decker & Parker, 1993)+ In yeast, deadenylation is followed by removal of the 59 m7 GpppN cap structure, and exonucleolytic digestion in a 59-39 direction (Decker & Parker, 1993;Hsu & Stevens, 1993;Beelman et al+, 1996)+ Poly(A) shortening requires two proteins, Ccr4p and Pop2p, which have recently been shown to be components of the yeast deadenylase )+ Following deadenylation, the Dcp1p/Dcp2p decapping complex decaps mRNAs at their 59 end Dunckley & Parker 1999)+ Although Dcp1p and Dcp2p are the only proteins known to be required for decapping (Dunckley & Parker, 1999, the rate of decapping is affected by several trans-acting factors+ These accessory factors include Pat1p/Mrt1p, the Lsm complex (consisting of Lsm1p-Lsm7p), Vps16p, Edc1p, and Edc2p Zhang et al+, 1999;Bonnerot et al+, 2000;Tharun et al+, 2000;Wyers et al+, 2000;Dunckley et al+, 2001)+ Pat1p/Mrt1p was originally identified as a genetic lesion that slows mRNA decapping, and was later shown to also be required for efficient translational initiation Wyers et al+, 2000)+ There are nine Lsm proteins in yeast, which form two distinct seven-member ring structures of differing function (Salgado-Garrido et al+, 1999)+ Lsm2p-Lsm8p associate in the nucleus and are involved in U6 snRNA metabolism, whereas Lsm1p-Lsm7p associate in the cytoplasm and affect decapping of mRNAs (Mayes et al+, 1999;Bouveret et al+, 2000;Tharun et al+, 2000)+ Vps16p was identified as a mutant that affected decapping rates in vitro, and Edc1p and Edc2p have been shown to be in a complex with Dcp1p and Dcp2p (Zhang et al+, 1999;Dunckley et al+, 2001)+ Although the mechanism of how these proteins en-hance decapping is unclear, it is possible a class of decapping enhancers may do so by directly affecting the translatability of the mRNA+ Indeed, translational repression may be a first step required for efficient decapping, as recent studies have shown the cap-binding protein, eIF-4E, can inhibit decapping activity both in vivo and in vitro (Schwartz & Parker, 1999+ Given this, a critical issue to resolve in understanding mRNA decay is to determine the nature of transitions between translating and nontranslating states of mRNAs+…”

mRNA Decay Is Rapidly Induced after Spore Germination ofSaccharomyces cerevisiae

“…It is followed by the removal of the 5Ј-cap structure by the Dcp1p decapping enzyme, which exposes the body of the transcript to Xrn1p,23). A complex of seven Lsm proteins (including Spb8p/Lsm1p) and Pat1p has been shown to interact with mRNA and to be required for mRNA decapping (20,(23)(24)(25)(26).…”