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Full Text (PDF) Copyright © 2005 by the Genetics Society of America DOI: 10.1534/genetics.104.034322 Mutations in the Saccharomyces cerevisiae LSM1 Gene That Affect mRNA Decapping and 3؅ End Protection Sundaresan Tharun,*,1 Denise Muhlrad,† Ashis Chowdhury* and Roy Parker† *Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799 and †Department of Molecular and Cellular Biology and Howard Hughes Medical Institute, University of Arizona, Tucson, Arizona 85721 Manuscript received August 6, 2004 Accepted for publication January 20, 2005 ABSTRACT The decapping of eukaryotic mRNAs is a key step in their degradation. The heteroheptameric Lsm1p–7p complex is a general activator of decapping and also functions in protecting the 3Ј ends of deadenylated mRNAs from a 3Ј-trimming reaction. Lsm1p is the unique member of the Lsm1p–7p complex, distinguish- ing that complex from the functionally different Lsm2p–8p complex. To understand the function of Lsm1p, we constructed a series of deletion and point mutations of the LSM1 gene and examined their effects on phenotype. These studies revealed the following: (i) Mutations affecting the predicted RNA- binding and inter-subunit interaction residues of Lsm1p led to impairment of mRNA decay, suggesting that the integrity of the Lsm1p–7p complex and the ability of the Lsm1p–7p complex to interact with mRNA are important for mRNA decay function; (ii) mutations affecting the predicted RNA contact residues did not affect the localization of the Lsm1p–7p complex to the P-bodies; (iii) mRNA 3Ј-end protection could be indicative of the binding of the Lsm1p–7p complex to the mRNA prior to activation of decapping, since all the mutants defective in mRNA 3Ј end protection were also blocked in mRNA decay; and (iv) in addition to the Sm domain, the C-terminal domain of Lsm1p is also important for mRNA decay function. ESSENGER RNA (mRNA) turnover is an impor- 1992; Decker and Parker 1993; Hsu and Stevens 1993; M tant control point in the regulation of gene ex- Muhlrad et al. 1994, 1995; Beelman et al. 1996; Dunckley pression. Numerous examples of genes regulated at the and Parker 1999; Tucker et al. 2001). In the 3Ј to 5Ј level of mRNA stability are known to date (Hentze pathway, deadenylated mRNAs are degraded in a 3Ј to 1991; Peltz et al. 1991; Abler and Green 1996; Guhan- 5Ј exonucleolytic manner by the exosome (Muhlrad iyogi and Brewer 2001; Tharun and Parker 2001b). et al. 1995; Anderson and Parker 1998). Several studies have shown that mRNA decay factors Decapping is a crucial step in the 5Ј to 3Ј decay path- and decay pathways are well conserved in eukaryotes way because it is the site of several regulatory inputs from yeast to humans (Seraphin 1992; Couttet et al. (Coller and Parker 2004). Moreover, a large number 1997; Gao et al. 2001; Wang and Kiledjian 2001; of proteins affect decapping in addition to the decap- Decker and Parker 2002; Liu et al. 2002; Lykke- ping enzyme. The translation initiation machinery and Andersen 2002; Van Dijk et al. 2002, 2003; Wang et the poly(A)-binding protein are antagonistic to decap- al. 2002; Piccirillo et al. 2003; Tseng-Rogenski et al. ping (Caponigro and Parker 1995; Coller et al. 1998; 2003). Schwartz and Parker 1999, 2000; Vilela et al. 2000; In eukaryotes, wild-type mRNAs primarily degrade via Wilusz et al. 2001; Ramirez et al. 2002; Khanna and deadenylation-dependent decay pathways that involve Kiledjian 2004). Conversely, several other factors func- Ј Ј Ј Ј 5 to 3 or 3 to 5 mode of degradation of the mRNA tion as activators of decapping. They include Pat1p, body. In yeast, deadenylation is carried out by the Ccr4p Dhh1p, Lsm1p–7p complex, Edc1p, Edc2p, and Edc3p Ј Ј complex (Tucker et al. 2001). In the 5 to 3 decay (Hatfield et al. 1996; Boeck et al. 1998; Bonnerot et pathway (also known as the “deadenylation-dependent al. 2000; Bouveret et al. 2000; Tharun et al. 2000; decapping pathway”), this is followed by decapping by Wyers et al. 2000; Coller et al. 2001; Dunckley et al. the decapping enzyme (Dcp1p–2p complex) that ex- 2001; Fischer and Weis 2002; Schwartz et al. 2003; Ј Ј poses the body of the message to 5 to 3 exonucleolytic Kshirsagar and Parker 2004). decay by the exonuclease Xrn1p (Muhlrad and Parker The Lsm1p–7p complex is particularly interesting as a decapping activator for several reasons. First, it is a highly conserved component of eukaryotic mRNA decay 1Corresponding author: Department of Biochemistry, Uniformed Ser- vices University of the Health Sciences, 4301 Jones Bridge Rd., machinery. Second, it physically interacts with several Bethesda, MD 20814-4799. E-mail: [email protected] other factors involved in the major mRNA decay path- Genetics 170: 33–46 (May 2005) 34 S. Tharun et al. way, including the decapping activators Pat1p and Dhh1p subunit that is either Lsm1p or Lsm8p. However, it is and the 5Ј to 3Ј exonuclease Xrn1p (Bonnerot et al. not known what features of Lsm1p and Lsm8p dictate 2000; Bouveret et al. 2000; Tharun et al. 2000; Coller their respective roles. et al. 2001). Third, in vivo, it associates with a pool of The mechanism by which Lsm1p–7p complex acti- deadenylated mRNPs that is bound to the decapping en- vates decapping is not clear. It is also not known how the zyme and targeted for decay but distinct from translating decapping activation function is related to the mRNA 3Ј mRNPs (Tharun and Parker 2001a). Fourth, several end protection function. Given the structural similarity studies suggest that in addition to mRNA decapping, this of the Lsm1p–7p complex to the Lsm2p–8p and the complex is also involved in protecting mRNA 3Ј ends Sm complexes, a reasonable model would be that simi- from trimming (Boeck et al. 1998; He and Parker 2001). larity also exists at a mechanistic level in the manner in Finally, mammalian cells overexpressing Lsm1p show a which these complexes function. Importantly, given that transformed phenotype, suggesting a possible role for the Sm complex and several Sm-like protein complexes Lsm1p in growth control (Schweinfest et al. 1997; (including the Lsm2p–8p complex) have been shown Gumbs et al. 2002). to directly bind to their RNA substrates to execute their The Lsm1p–7p complex, which is conserved in all functions (Achsel et al. 1999, 2001; Raker et al. 1999; eukaryotes, is a heptameric complex made of seven pro- Toro et al. 2001; Moller et al. 2002; Schumacher et teins, Lsm1p through Lsm7p. The Lsm (“Like Sm”) al. 2002), it is likely that the Lsm1p–7p complex also proteins are homologous to the Sm proteins and these directly binds to mRNAs to protect their 3Ј ends and proteins form a family of protein complexes that in- to activate their decapping. This is consistent with the cludes complexes found in eubacteria, archaea, and earlier co-immunoprecipitation analyses, which re- eukaryotes (Achsel et al. 1999, 2001; Kambach et al. vealed that the Lsm1p–7p complex associates with dead- 1999; Collins et al. 2001; Mura et al. 2001; Toro et al. enylated mRNAs in vivo (Tharun et al. 2000; Tharun 2001; Moller et al. 2002; Schumacher et al. 2002). and Parker 2001a). If this is true, then, disrupting the Individual members of this protein family are character- interaction of the Lsm1p–7p complex with RNA should ized by the presence of a bipartite sequence motif, re- impair both mRNA 3Ј end protection and decapping ferred to as the Sm domain. The Sm domain consists activation functions. of two conserved segments of amino acids (Sm motifs Recent studies have shown that the decapping and I and II) separated by a nonconserved segment of vari- 5Ј to 3Ј exonucleolysis of mRNAs occur in discrete cyto- able length (Cooper et al. 1995; Hermann et al. 1995; plasmic structures called processing bodies (P-bodies), Seraphin 1995). where Xrn1p, the various decapping factors including These Sm and Lsm proteins also show similarity in the Lsm1p through Lsm7p proteins, and mRNA mole- their tertiary structure and the quarternary structure of cules targeted for decay are localized (Ingelfinger et the complexes they form. Typically, they exist in cells al. 2002; Van Dijk et al. 2002; Sheth and Parker 2003; in the form of ring-shaped hexa or heptameric com- Cougot et al. 2004). However, it is not known how the plexes (Achsel et al. 1999, 2001; Kambach et al. 1999; Lsm1p–7p complex and the other decay factors are Collins et al. 2001; Mura et al. 2001; Toro et al. 2001; recruited to the P-bodies. Moller et al. 2002; Schumacher et al. 2002; Zaric et To identify the critical regions of the Lsm1p protein al. 2005). For example, the seven Sm proteins conserved and to determine how they are related to the functions in all eukaryotes associate into a ring-shaped heptameric of Lsm1p, we undertook a mutagenic analysis of Lsm1p. “Sm complex” (Kambach et al. 1999) that assembles Structural and biochemical studies in the past have iden- onto the U1, U2, U4, and U5 snRNAs to form the cores tified residues in the Sm domain of Sm proteins and of the corresponding spliceosomal snRNPs (Lerner archaeal Sm-like proteins that are involved in RNA and and Steitz 1981; Luhrmann et al. 1990). On the other inter-subunit interactions (Kambach et al. 1999; Mura hand, there are eight Lsm proteins (Lsm1p through et al. 2001, 2003; Toro et al. 2001; Urlaub et al. 2001). Lsm8p) conserved in all eukaryotes and they appear to Therefore, we predicted that the homologous residues form two heptameric complexes (Salgado-Garrido et in Lsm1p could have similar roles, mutated such resi- al.
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