Genetics: Published Articles Ahead of Print, published on February 16, 2005 as 10.1534/genetics.104.034322

Mutations in the Saccharomyces cerevisiae LSM1 that affect

mRNA decapping and 3’ end protection

Sundaresan Tharun1, Ashis Chowdhury1, Denise Muhlrad2 and Roy Parker2

1Department of Biochemistry

Uniformed Services University of the Health Sciences

Bethesda MD 20814-4799

2Department of Molecular and Cellular Biology &

Howard Hughes Medical Institute

University of Arizona

Tucson, Arizona 85721

1 Running title: Mutagenic analysis of yeast Lsm1p

Corresponding author: Sundaresan Tharun, Department of Biochemistry, Uniformed

Services University of the Health Sciences (USUHS), 4301, Jones Bridge Road,

Bethesda, Maryland 20814-4799. email: [email protected].

Phone: 301-295-9423 Fax: 301-295-3512

2 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 3’ ends of deadenylated mRNAs from a 3’ trimming reaction.

Lsm1p is the unique member of the Lsm1p-7p complex distinguishing that complex from the functionally different Lsm2p-8p complex. To understand the function of Lsm1p, we constructed a series of deletion and point mutants of the LSM1 gene and examined their phenotypes. These studies revealed the following. (i) Integrity of the Lsm1p-7p complex is important for its mRNA decay function since mutations affecting the predicted inter- subunit interaction surfaces of Lsm1p lead to impairment of mRNA decay. (ii)

Interactions between Lsm1p and mRNA are necessary for the activation of decapping by the Lsm1p-7p complex. However, even when these interactions are disrupted, the

Lsm1p-7p complex could localize to the P-bodies. (iii) 3’ 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 3’ end protection were also blocked in mRNA decay. (iv) In addition to the Sm domain the C-terminal domain of Lsm1p is also important for mRNA decay function.

3 INTRODUCTION

Messenger RNA turnover is an important control point in the regulation of . Numerous examples of regulated at the level of mRNA stability are known to date (ABLER and GREEN 1996; GUHANIYOGI and BREWER 2001; HENTZE 1991;

PELTZ et al. 1991; THARUN and PARKER 2001b). Several studies have shown that mRNA decay factors and decay pathways are well conserved in eukaryotes from yeast to humans

(COUTTET et al. 1997; DECKER and PARKER 2002; GAO et al. 2001; LIU et al. 2002;

LYKKE-ANDERSEN 2002; PICCIRILLO et al. 2003; SERAPHIN 1992; TSENG-ROGENSKI et al.

2003; VAN DIJK et al. 2002; VAN DIJK et al. 2003; WANG et al. 2002; WANG and

KILEDJIAN 2001).

In eukaryotes, wild type mRNAs primarily degrade via deadenylation-dependent decay pathways which involve 5’ to 3’ or 3’ to 5’ mode of degradation of the mRNA body. In yeast, deadenylation is carried out by the Ccr4p complex (TUCKER et al. 2001). In the 5’ to 3’ decay pathway (also known as the ‘deadenylation-dependent decapping pathway’), this is followed by decapping by the decapping enzyme (Dcp1p-Dcp2p complex) which exposes the body of the message to 5’ to 3’ exonucleolytic decay by the exonuclease

Xrn1p (BEELMAN et al. 1996; DECKER and PARKER 1993; DUNCKLEY and PARKER 1999;

HSU and STEVENS 1993; MUHLRAD et al. 1994; MUHLRAD et al. 1995; MUHLRAD and

PARKER 1992; TUCKER et al. 2001). In the 3’ to 5’ pathway, deadenylated mRNAs are degraded in a 3’ to 5’ exonucleolytic manner by the exosome (ANDERSON and PARKER

1998; MUHLRAD et al. 1995).

4 Decapping is a crucial step in the 5’ to 3’ decay pathway because it is the site of several regulatory inputs (COLLER and PARKER 2004b). Moreover, a large number of affect decapping in addition to the decapping enzyme. The translation initiation machinery and the poly(A) binding are antagonistic to decapping (CAPONIGRO and PARKER 1995; COLLER et al. 1998; KHANNA and KILEDJIAN 2004; RAMIREZ et al.

2002; SCHWARTZ and PARKER 1999; SCHWARTZ and PARKER 2000; VILELA et al. 2000;

WILUSZ et al. 2001). Conversely, several other factors function as activators of decapping. They include Pat1p, Dhh1p, Lsm1p-7p complex, Edc1p, Edc2p and Edc3p

(BOECK et al. 1998; BONNEROT et al. 2000; BOUVERET et al. 2000; COLLER et al. 2001;

DUNCKLEY et al. 2001; FISCHER and WEIS 2002; HATFIELD et al. 1996; KSHIRSAGAR and

PARKER 2004; SCHWARTZ et al. 2003; THARUN et al. 2000; WYERS et al. 2000).

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 machinery. Second, it physically interacts with several other factors involved in the major mRNA decay pathway including the decapping activators Pat1p and Dhh1p and the 5’ to 3’ exonuclease Xrn1p (BONNEROT et al. 2000; BOUVERET et al. 2000; COLLER et al. 2001; THARUN et al. 2000). Third, in vivo, it associates with a pool of deadenylated mRNPs that is bound to the decapping enzyme and targeted for decay but distinct from translating mRNPs (THARUN and PARKER 2001a). Fourth, several studies suggest that in addition to mRNA decapping, this complex is also involved in protecting mRNA 3’ ends from trimming (BOECK et al. 1998; HE and PARKER 2001). Finally, mammalian cells

5 overexpressing Lsm1p show a transformed phenotype, suggesting a possible role for

Lsm1p in growth control (GUMBS et al. 2002; SCHWEINFEST et al. 1997).

The Lsm1p-7p complex, which is conserved in all eukaryotes, is a heptameric complex made of seven proteins, Lsm1p through Lsm7p. The Lsm (‘Like Sm’) proteins are homologous to the Sm proteins and these proteins form a family of protein complexes which includes complexes found in eubacteria, archaea, and eukaryotes (ACHSEL et al.

1999; ACHSEL et al. 2001; COLLINS et al. 2001; KAMBACH et al. 1999; MOLLER et al.

2002; MURA et al. 2001; SCHUMACHER et al. 2002; TORO et al. 2001). Individual members of this protein family are characterized by the presence of a bipartite sequence motif, referred to as the Sm domain. The Sm domain consists of two conserved segments of amino acids (Sm motifs I and II) separated by a nonconserved segment of variable length (COOPER et al. 1995; HERMANN et al. 1995; SERAPHIN 1995).

These Sm and Lsm proteins also show similarity in their tertiary structure and the quarternary structure of the complexes they form. Typically they exist in cells in the form of ring-shaped hexa or heptameric complexes (ACHSEL et al. 1999; ACHSEL et al.

2001; COLLINS et al. 2001; KAMBACH et al. 1999; MOLLER et al. 2002; MURA et al.

2001; SCHUMACHER et al. 2002; TORO et al. 2001). For example, the seven Sm proteins conserved in all eukaryotes associate into a ring shaped heptameric ‘Sm complex’

(KAMBACH et al. 1999) which assembles onto the U1, U2, U4 and U5 snRNAs to form the cores of the corresponding spliceosomal (LERNER and STEITZ 1981;

LUHRMANN et al. 1990). On the other hand, there are eight Lsm proteins (Lsm1p through

6 Lsm8p) conserved in all eukaryotes and they appear to form two heptameric complexes

(BOUVERET et al. 2000; SALGADO-GARRIDO et al. 1999; THARUN et al. 2000). One complex, consisting of Lsm1p through Lsm7p proteins is an activator of mRNA decapping (BOECK et al. 1998; BOUVERET et al. 2000; THARUN et al. 2000), while the other made of Lsm2p through Lsm8p proteins functions in pre-mRNA splicing by forming the core of U6 snRNP (ACHSEL et al. 1999; COOPER et al. 1995; MAYES et al.

1999; PANNONE et al. 2001; PANNONE et al. 1998; SALGADO-GARRIDO et al. 1999).

Thus, although involved in very different functions, the Lsm1p-7p and Lsm2p-8p complexes share six out of their seven subunits and differ in only one subunit which is either Lsm1p or Lsm8p. However, it is not known what features of Lsm1p and Lsm8p dictate their respective roles.

The mechanism by which Lsm1p-7p complex activates decapping is not clear. It is also not known how the decapping activation function is related to the mRNA 3’ end protection function. Given the structural similarity of the Lsm1p-7p complex to the

Lsm2p-8p and the Sm complexes, a reasonable model would be that similarity also exists at a mechanistic level in the manner in which these complexes function. Importantly, given that the Sm complex and several Sm-like protein complexes (including the Lsm2p-

8p complex) have been shown to directly bind to their RNA substrates to execute their functions (ACHSEL et al. 1999; ACHSEL et al. 2001; MOLLER et al. 2002; RAKER et al.

1999; SCHUMACHER et al. 2002; TORO et al. 2001), it is likely that the Lsm1p-7p complex also directly binds to mRNAs to protect their 3’ ends and to activate their decapping. This is consistent with the earlier co-immunoprecipitation analyses which

7 revealed that the Lsm1p-7p complex associates with deadenylated mRNAs in vivo

(THARUN et al. 2000; THARUN and PARKER 2001a). If this is true, then, disrupting the interaction of the Lsm1p-7p complex with RNA should impair both mRNA 3’ end protection and decapping activation functions.

Recent studies have shown that the decapping and 5’ to 3’ exonucleolysis of mRNAs occur in discrete cytoplasmic structures called processing bodies (P-bodies) where

Xrn1p, the various decapping factors including the Lsm1p through Lsm7p proteins and mRNA molecules targeted for decay are localized (COUGOT et al. 2004; INGELFINGER et al. 2002; SHETH and PARKER 2003; VAN DIJK et al. 2002). However, it is not known how the Lsm1p-7p complex and the other decay factors are recruited to the P-bodies.

In order to identify the critical regions of the Lsm1p protein and to determine how they are related to the functions of Lsm1p, we undertook a mutagenic analysis of Lsm1p.

Structural and biochemical studies in the past have identified residues in the Sm domain of Sm proteins and archaeal Sm-like proteins that are involved in RNA and inter-subunit interactions (KAMBACH et al. 1999; MURA et al. 2001; MURA et al. 2003; TORO et al.

2001; URLAUB et al. 2001). Therefore, we predicted that the homologous residues in

Lsm1p could have similar roles, mutated such residues, and examined the effect of such mutations on the functions of Lsm1p. In addition, we also examined the roles of other residues within the Lsm1p including those in the conserved C-terminal domain following the Sm domain.

8 These studies revealed the following. (i) Mutations affecting the predicted RNA-binding surface of Lsm1p lead to impairment of mRNA decay without significantly affecting the localization of the Lsm1p-7p complex to the P-bodies. Thus, interactions with mRNA are critical for activation of decapping but not for recruitment of the Lsm1p-7p complex to P-bodies. (ii) Lesions in the predicted inter-subunit interaction domains also lead to significant impairment of mRNA decay indicating the importance of the Lsm1p-7p complex integrity for mRNA decay. (iii) All the mutants that are defective in mRNA 3’ end protection are also defective in mRNA decay suggesting that 3’ end protection could be indicative of the Lsm1p-7p complex-mRNA interaction which leads to decapping activation. (iv) In addition to the Sm domain, the extended C-terminal domain of Lsm1p is also important for the function of the Lsm1p-7p complex.

9 RESULTS

Mutagenesis of LSM1

Structural and biochemical studies have been conducted on the human Sm sub complexes and several archaebacterial Sm-like protein complexes in the past (KAMBACH et al. 1999;

MURA et al. 2001; MURA et al. 2003; TORO et al. 2001; URLAUB et al. 2001). Such studies revealed the crucial amino acid residues -- involved in RNA contact and inter- subunit contacts -- of the Sm and Sm-like proteins which form these complexes. Using this information and the homology of these proteins to yeast Lsm1p, we predicted the residues of Lsm1p that are likely to be involved in RNA contact and inter-subunit contacts and targeted them (Figures 1a and 1b) in our first set of mutants (lsm1-6 through lsm1-9, lsm1-13 and lsm1-14).

Structural studies on several members of the Sm-like protein family have revealed that the Sm domain of these proteins folds into a conserved structure (‘Sm fold’) consisting of an N-terminal alpha helix followed by a 5-stranded, strongly bent beta sheet (KAMBACH et al. 1999; MURA et al. 2001; MURA et al. 2003; TORO et al. 2001). Here the key residues involved in contacting the RNA are located in loops 3 (between beta strands 2 and 3) and 5 (between beta strands 4 and 5) while many of the residues involved in inter- subunit interactions are located in beta strands 2, 3 and 4. The 3D structure of the Sm domain of the yeast Lsm1p generated using the homology-based modeling program 3D-

JIGSAW (BATES et al. 2001; BATES and STERNBERG 1999; CONTRERAS-MOREIRA and

BATES 2002) supports the idea that the homologous residues in Lsm1p are organized similarly (Figure 1b).

10

The Lsm1p residues implicated (based on homology) in inter-subunit contacts included both charged and hydrophobic amino acids. R59 and R62 located in loop 2 of the modeled 3D structure were mutated in the allele lsm1-6. L64 and G66 located in beta strand 2 and R62 were changed in the allele lsm1-7. Residues 101 to 104 (I, F, M and I) located in beta strand 4 were changed in lsm1-13. The charged residues R59 and R62 were replaced with alanines. On the other hand, residues 101 to 104 (I,F,M and I) and

L64 were changed to charged residues since hydrophobicity was predicted to be crucial for their function. G66 was replaced by a bulky tryptophan (Figures 1a and 1b).

Lsm1p residues implicated in RNA binding are located in loops 3 and 5 of the modeled

3D structure (Figure 1b). The loop 3 residues Y74 and N76 are mutated in lsm1-9 while the loop 5 residues R105, G106 and E107 are mutated in lsm1-14. The loop 3 residue

D72 was mutated along with R69 located in beta strand 2 in lsm1-8. N76, Y74, R105 and

E107 were predicted to form the RNA binding pocket while D72 and G106 were predicted to be important in holding N76 in the proper orientation. While G106 was changed to a tryptophan, other residues were replaced with alanines (Figures 1a and 1b).

All the clusters of charged residues in Lsm1p whose function could not be readily predicted based on were replaced with alanines to generate the mutants lsm1-1 through lsm1-5, lsm1-10 through lsm1-12, and lsm1-15 through lsm1-24

(Figure 1a). These included residues in the regions flanking or inside the Sm domain.

Changes were restricted to ≤4 neighboring residues in all the mutants.

11

Finally, in order to address the role of the regions of Lsm1p that are outside the Sm- domain, we also made 5 deletion mutants. These included lsm1-25 and lsm1-26 which had respectively 17 and 36 amino acids deleted from the N- terminus and lsm1-27 through lsm1-29 which had 55, 43 and 28 amino acids respectively deleted from the C- terminus (Figure 1a). Importantly, in these mutants, the Sm domain was left intact so that the mutant proteins are likely to fold into the proper tertiary structure that is characteristic of the Sm-like proteins.

Mutants were made as described in Methods by introducing the changes in the LSM1 gene (with its native promoter and 3’ UTR sequences) cloned in a CEN vector with

URA3 marker. The plasmids carrying the wild type and the various mutant versions of

LSM1 were transformed into the lsm1∆ strain yRP1365 so that the plasmid borne gene is the only source of Lsm1p protein in the cell. The lsm1∆ strain transformed with the empty vector, pRS416 or the recombinant vector carrying the wild type LSM1 gene were used as the negative and positive controls respectively in all experiments and referred to as lsm1∆ and wild type respectively. The mutants were studied for temperature sensitivity, mRNA decay and mRNA 3’ end protection as described below.

LSM1 mutations resulting in temperature sensitivity of growth lsm1∆ cells are temperature sensitive for growth at higher temperatures (MAYES et al.

1999). Earlier studies had suggested that the temperature sensitivity of the lsm1∆ cells is likely to be related to their mRNA 3’ end trimming phenotype (HE and PARKER 2001).

12 Therefore, as a first assay for the function of the mutant Lsm1p proteins, we studied their ability to support growth at high temperature. To this end, growth of the mutant and the wild type cells on -ura plates was examined at 25˚ and 36˚. As expected, at 25˚ both of the control strains LSM1 and lsm1∆ were able to grow while at 36˚, lsm1∆ cells were unable to grow. Analysis of the growth of the mutants revealed the following (Table 1).

First, we observed that among the three lsm1 alleles bearing mutations in the predicted

RNA binding residues (i.e., lsm1-8, lsm1-9, and lsm1-14), lsm1-8 and lsm1-14 were unable to grow at 36˚ while lsm1-9 was able to grow very slowly. These results provide the first indication that these residues are important for Lsm1p function. However, the ability of lsm1-9 cells to grow at 36˚, albeit slowly, suggests that at least some parts of the RNA binding surface can undergo mutation without affecting viability at high temperature.

Second, we observed that all the three lsm1 alleles bearing mutations in the predicted inter-subunit contact residues, lsm1-6, lsm1-7, and lsm1-13 were unable to grow at 36˚ just like lsm1∆. These results support the idea that these residues are required for Lsm1p function.

Third, we observed that large deletions in the region of Lsm1p that is C-terminal to the

Sm domain lead to temperature sensitive growth as shown by the inability of the alleles lsm1-27 and lsm1-28 to grow at 36˚. The mutant lsm1-29, which bears the shortest

13 deletion of this C-terminal region grew slowly at 36˚. These observations indicate that residues outside the Sm domain are also likely to be important for Lsm1p function.

Fourth, we observed that mutations in or deletion of the stretch of amino acids N-terminal to the Sm domain did not affect growth at any temperature suggesting that this region is not critical for Lsm1p function. Similarly, several other mutations affecting small clusters of charged residues distributed throughout the protein were all tolerated suggesting that none of these regions by itself is of major importance for the function of

Lsm1p.

Finally, although all the strains were able to grow in –ura plates at 25˚, differences in growth rates could be observed. As expected, lsm1∆ cells grew much slower than the wild type cells. Further, a subset of the mutants unable to grow at 36˚ showed slow growth at 25˚ similar to lsm1∆. They were lsm1-7, lsm1-8, lsm1-13 and lsm1-27 (not shown). These strains and lsm1-9 and lsm1-14 were also slow growing in liquid cultures at 25˚.

Effect of LSM1 mutations on mRNA decay

A key set of experiments was to examine the effect of the lsm1 mutations on mRNA decay. For this experiment, we utilized the MFA2pG reporter mRNA system. This mRNA is expressed under the control of the GAL promoter from a chromosomal location in our strains (HATFIELD et al. 1996). The MFA2pG mRNA decays by deadenylation- dependent decapping followed by 5’ to 3’ exonucleolytic digestion by Xrn1p (MUHLRAD

14 et al. 1994). The MFA2pG mRNA also contains a poly(G) insertion in its 3’ UTR that forms a strong secondary structure in vivo and blocks Xrn1p in cis. This results in the stable accumulation of the degradation intermediate called poly(G) fragment in vivo. As a result, in strains where decapping is defective, the accumulation of the poly(G) fragment is decreased. Thus, the relative levels of the poly(G) fragment and the full length mRNA at steady state in a strain can be used as a first approximation of the efficiency of decapping of this reporter mRNA in that strain (CAO and PARKER 2001;

HATFIELD et al. 1996). Therefore, we first identified the lsm1 mutants defective in mRNA decay based on their inability to the accumulate the poly(G) fragment at sufficient levels. This was followed by a direct measurement of the MFA2pG mRNA half-life in such mutants to fully confirm the mRNA decay phenotype.

As shown in Figure 2, fragment levels relative to the full length (quantitated using the

Phosphorimager) were very low in lsm1∆ cells (~15% of total signal) compared to the wild type cells (~40% of total signal). It should be noted that because both the MFA2pG full length mRNA and the poly(G) fragment accumulate 3’ trimmed species in addition to the normal species (see below) in lsm mutants (HE and PARKER 2001), the intensities of normal and trimmed species were added up to determine the levels of the full length mRNA and the fragment.

Figure 2 also reveals the strong accumulation in the lsm1∆ strain of the deadenylated full length MFA2pG mRNA. This is characteristic of strains defective in decapping

(BEELMAN et al. 1996; DUNCKLEY and PARKER 1999; THARUN et al. 2000).

15

Examination of the various mutants revealed the following. First, mutants with lesions in the predicted RNA binding surface, lsm1-8, lsm1-9 and lsm1-14 all showed defects in mRNA decay. Each mutant showed a decreased accumulation of the mRNA decay intermediate relative to the full length mRNA. In addition, all three alleles accumulated deadenylated full length mRNAs, consistent with a defect in decapping (Figure 2).

Moreover, measurement of MFA2pG mRNA decay rate in these mutants showed a prolonged half-life indicating a clear defect in mRNA decapping (Figure 3). While lsm1-

8 showed a strong phenotype (comparable to lsm1∆), lsm1-9 and lsm1-14 showed a moderate phenotype. These results argue that RNA binding is required for Lsm1p’s role in mRNA decapping.

Second, we observed that mutations in the predicted inter-subunit contact residues, lsm1-

6, lsm1-7, and lsm1-13 all showed defects in decapping as assessed both by the levels of mRNA decay fragment and the accumulation of deadenylated full length mRNA (Figure

2). Again, measurement of the MFA2pG mRNA decay rate in these mutants revealed a prolonged half-life indicating a clear defect in mRNA decapping (Figure 3). The lsm1-7 and lsm1-13 alleles were almost as strong as a null allele, whereas the lsm1-6 allele showed a moderate phenotype. Thus, these results support the idea that maintenance of inter-subunit contacts is of critical importance for Lsm1p’s function.

Third, we observed that deletions within the C-terminal extension (lsm1-27, lsm1-28 and lsm1-29) resulted in a clear inhibition of mRNA decapping as assessed by both the

16 poly(G) fragment levels and the accumulation of deadenylated mRNA (Figure 2). In fact, even fusing a FLAG-peptide at the C-terminus of otherwise wild type Lsm1p (lsm1-31;

Table 1) led to a partial defect in mRNA decapping (Figure 2). These observations were further supported by the longer half-life of MFA2pG mRNA in these mutants (Figure 3).

These results indicate that the C-terminus has an important function in Lsm1p’s role in mRNA decapping. However, the alleles lsm1-16 through lsm1-24, which carry lesions in different clusters of charged amino acids residing in the C-terminal domain, failed to show any significant growth or mRNA decay defects indicating that smaller perturbations in this domain can be tolerated (Table 1).

Fourth, we observed that mutations (lsm1-1 through lsm1-4) or deletions (lsm1-25 and lsm1-26) in the Lsm1p primary sequence that is N-terminal to the Sm domain did not lead to any significant decapping defect as assessed from the accumulation of the poly(G) fragment or deadenylated full length mRNA (Figure 2 and Table 1). This observation was consistent with these lesions not affecting growth at high temperature (Table 1).

Similarly, mutations in the nonconserved region which is located between the Sm motifs

I and II (lsm1-11 and lsm1-12) also did not lead to any significant phenotype (Table 1).

This suggests that the residues affected in these mutants do not play a major role in

Lsm1p function.

Effect of LSM1 mutations on mRNA 3’ end protection

We also examined the effect of the lsm1 alleles on the protection of the mRNA 3’ end from a trimming reaction. Specifically, earlier studies (BOECK et al. 1998; HE and

17 PARKER 2001) showed that in lsm1 through lsm7 mutants several mRNAs accumulate species that are truncated at their 3’ ends by ~10 to 20 nucleotides (‘trimmed species’) in addition to the normal full length species in vivo. This indicates that the Lsm1p-7p complex is involved in protecting mRNA 3’ ends in vivo. In the case of MFA2pG and

PGK1pG mRNAs, poly(G) fragments generated from these mRNAs also accumulate as normal and trimmed species in the lsm mutants (HE and PARKER 2001). While the trimmed and normal species of full length mRNA run very close together in the gel, the trimmed poly(G) fragment and normal poly(G) fragment are more easily separable.

Therefore, determining the ratio of the two species of the poly(G) fragment is a convenient way to assess the degree of mRNA 3’ end protection occurring in a given strain. This was quantitated using the phosphorimager from northern blots containing steady state RNA samples. As seen in figure 2, wild type cells had very little trimmed fragment (≤10% of total fragment). However, lsm1∆ cells had high levels of trimmed fragment (~55 to 60% of the total fragment levels).

An important result was that all of the lesions that affected mRNA decapping also affected mRNA trimming. Specifically, all the mutants affecting the residues predicted to be involved in RNA and inter-subunit contacts showed significant accumulations of trimmed fragment, with lsm1-7, lsm1-8, lsm1-9, lsm1-13 and lsm1-14 showing strong defects. The mutant lsm1-6 showed a moderate accumulation of the trimmed fragment which is consistent with the moderate decapping defect seen in this mutant. Further, the

C-terminal deletion mutants lsm1-27, lsm1-28 and lsm1-29 and the C-terminal FLAG- tagged allele lsm1-31 also showed higher levels of trimmed fragment. These results

18 demonstrate a strong correlation between the impairment of mRNA decapping and the inability to protect mRNA 3’ end from trimming suggesting that these two functions are related (see discussion).

Expression of mutant Lsm1p proteins

It is possible that some of the mutants described above show a strong phenotype due to their inability to accumulate the mutant Lsm1p adequately. To test this possibility, we compared Lsm1p expression in the lsm1 mutants that are defective in mRNA decay and wild type cells by western analysis of the lysates prepared from the corresponding strains using anti-Lsm1p antiserum. Cells grown in –ura liquid medium at 25˚ and collected at log phase were used for making the lysates. As seen in Figure 4, the decay-defective mutants, lsm1-6, lsm1-8, lsm1-9, lsm1-13, and lsm1-14 expressed the mutant protein at levels comparable to wild type protein. On the other hand, the results showed that the

Lsm1p band was very weak or undetectable in the lysates of mutants lsm1-7, lsm1-27 and lsm1-28. At present it is not clear whether this is due to low accumulation of the mutant

Lsm1p in these cells or due to loss of reactivity of the mutant protein with the antibodies as a result of the mutations/deletions (especially in the case of lsm1-27 and lsm1-28 which have large deletions).

Effect of mutations affecting the predicted RNA contacting surfaces of Lsm1p on the localization of the Lsm proteins to P-bodies

Recent studies have suggested that decapping and the 5’ to 3’ degradation of mRNA occur in discrete cytoplasmic structures called P-bodies where the Lsm1p through Lsm7p

19 proteins and many other decay factors including the decapping enzyme are concentrated in both yeast and mammalian cells (COUGOT et al. 2004; INGELFINGER et al. 2002; SHETH and PARKER 2003; VAN DIJK et al. 2002). It is not known how the Lsm1p through

Lsm7p proteins are recruited to the P-bodies although studies in mammalian cells have suggested that these proteins need to be a part of the Lsm1p-7p complex to get efficiently recruited to the P-bodies (INGELFINGER et al. 2002). One possibility is that the interaction of the Lsm1p-7p complex with RNA is important for the recruitment of the

Lsm1p-7p complex to the P-bodies. In order to test this possibility, we performed experiments to determine if lesions in the predicted RNA binding surfaces of Lsm1p affect the recruitment of either Lsm1p or the other Lsm proteins to the P-bodies.

First we examined the intracellular localization of the GFP-tagged forms of the mutant proteins Lsm1-9p and Lsm1-14p (which bear lesions in the predicted RNA binding surface of Lsm1p) or wild type Lsm1p. We did the studies with cells subjected to stress

(incubation in glucose-free medium for 10 minutes) since P-bodies are larger and more easily visualized in stressed cells than in cells that are not subjected to stress (TEIXEIRA et al. 2004). We observed that both Lsm1-9p and Lsm1-14p localized to P-bodies just like the wild type Lsm1p protein (Figure 5). This indicates that the RNA binding surface of

Lsm1p is not absolutely required for the recruitment of Lsm1p to the P-bodies.

Next, we examined how defects in Lsm1p affect the localization of other components of the Lsm1p-7p complex. We examined the localization of Lsm2p-GFP or Lsm7p-GFP in

LSM1, lsm1-9, lsm1-14 and lsm1∆ cells that were subjected to stress. We observed that

20 neither Lsm7p-GFP nor Lsm2p-GFP was localized to P-bodies in lsm1∆ cells (Figure 6).

However, both Lsm2p-GFP and Lsm7p-GFP localized to P-bodies in the lsm1-9 and lsm1-14 mutants (Figure 6). Therefore, these results suggest that even when the direct contacts between Lsm1p and RNA are impaired, Lsm1p could be recruited to the P- bodies by virtue of its interactions with the other subunits of the Lsm1p-7p complex.

21 DISCUSSION

The mechanism by which the Lsm1p-7p complex activates mRNA decapping is not known. Since Lsm1p is the distinguishing subunit of this complex, we have analyzed the critical functional features of this protein. Our results support the idea that the functioning of the Lsm1p-7p complex is similar to the other Sm-like protein complexes at a mechanistic level. When the Lsm1p residues that are likely to function in inter-subunit contacts and RNA binding were predicted based on the homology of Lsm1p to other Sm- like proteins (for whom such residues were identified experimentally) and mutagenized, it resulted in mRNA decay defects. This indicates that the organization of such crucial residues is similar between Lsm1p and other Sm-like proteins. Further this is also consistent with the idea that just like the other Sm-like protein complexes, the Lsm1p-7p complex also needs to directly bind to the RNA substrate to act on it (i.e., activate decapping).

Our studies indicate that most of the residues that are crucial for Lsm1p function reside in the conserved regions, Sm motifs I and II, of the Lsm1p protein. Mutations affecting any small set of neighboring residues located outside these regions did not lead to any significant mRNA decay defects although large deletions in the region C-terminal to the

Sm domain led to significant mRNA decay defects (Table 1). Thus, the Lsm1p protein appears to have two critical regions for function.

22 Several observations indicate that the stretch of amino acids N-terminal to the Sm domain of Lsm1p (residues 1 to 26) is not required for Lsm1p function. First, deletions or point mutations in this region do not affect growth at high temperature (Table 1). Second, they also do not affect mRNA decapping as assessed by the ability to accumulate poly(G) fragment or deadenylated mRNA (Table 1 and Figure 2). Third, this region is almost completely absent in the human homolog of Lsm1p although the region C-terminal to the

Sm domain is reasonably well conserved in the human protein (Figure 1c). In fact, even in fungal species that are very closely related to S. cerevisiae, the N-terminal extension is not highly conserved.

Our results argue that the C-terminal extension of Lsm1p is required for its function.

Lsm1p has a 61 amino acids-long region C-terminal to the Sm domain that is absent in other members of the Sm-like protein family. The following observations indicate that this region is important. First, deletions in this region (lsm1-27, lsm1-28 and lsm1-29) inhibited mRNA decay (Figures 2 and 3). Although the western analyses indicate that the mutant (truncated) Lsm1p accumulation may be severely reduced in lsm1-27 and lsm1-28, they reveal normal accumulation of Lsm1-29p, indicating that al least the phenotype of lsm1-29 is attributable to the deletion (Figure 4). Second, the allele lsm1-31, which is a fusion of the FLAG peptide at the C-terminus of an otherwise wild type

Lsm1p was also defective in mRNA decay and makes normal levels of the fusion protein

(Figures 2, 3 and 4). Importantly, fusion of the FLAG epitope at the N-terminus of

Lsm1p has no effect on mRNA decay or mRNA 3’ end protection (not shown). Finally, all the C-terminal deletion mutants were also defective in mRNA 3’ end protection

23 (Figure 2). The functional importance of Lsm1p’s C-terminal extension is also supported by the observation that it is reasonably well conserved in human Lsm1p (Figure 1c).

Further, genetic interaction studies of Boeck et al (1998)., have shown that an lsm1 allele

(spb8-1) with intact Sm domain but affected in the C-terminal domain due to a frame shift mutation is able to suppress the lethality of pab1∆ like several other decapping mutants including lsm1∆ (BOECK et al. 1998; CAPONIGRO and PARKER 1995; WYERS et al. 2000). Surprisingly, our studies also indicate that smaller perturbations in the C- terminal extension of Lsm1p can be tolerated since the alleles lsm1-15 through lsm1-24

(each of which carry changes in a small set of neighboring charged residues in the region

C-terminal to the Sm domain) do not show any defect in mRNA decay. This could be because multiple redundant sites in this region mediate functionally important interactions perhaps as part of a larger protein-protein interaction surface.

Our results indicate that the residues of Lsm1p that are predicted (based on homology) to be involved in inter-subunit contacts are indeed crucial for Lsm1p function. This is shown by the significant mRNA decay defect exhibited by the mutants in which these residues are changed (lsm1-6, lsm1-7 and lsm1-13, see Figures 2 and 3). Although western analyses revealed that the accumulation of the mutant Lsm1p may be severely impaired in lsm1-7, they showed normal levels of accumulation in the case of Lsm1-6p and Lsm1-13p. Since the residues mutated in these alleles are homologous to the experimentally determined inter-subunit contact residues in other Sm-like proteins, these observations are consistent with the idea that homologous regions serve similar functions in the different Sm-like proteins. Importantly, these results suggest that Lsm1p needs to

24 be a part of the Lsm1p-7p complex to execute its function efficiently. Recent studies have identified cytoplasmic structures, referred to as P-bodies, that are the sites of decapping (COUGOT et al. 2004; SHETH and PARKER 2003). Mutations in human Lsm4p that are predicted to disrupt its interactions with other Lsm proteins inhibit its entry into

P-bodies (INGELFINGER et al. 2002). Therefore, it is possible that in lsm1-6 and lsm1-13, the defect in decapping is due to the inefficient targeting of the Lsm1p to P-bodies.

Despite the clear importance of the Sm domain for Lsm1p function, mutations in the region that separates the Sm motifs I and II failed to have any effect on mRNA decay.

This region corresponds to the loop 4 in the modeled tertiary structure of Lsm1p (Figure

1b). Both lsm1-11 and lsm1-12, which bear lesions in this region, were able to grow at

36˚ and accumulate wild type levels of poly(G) fragment (Table 1). This observation is consistent with the fact that neither the primary sequence nor the length of this region is conserved among the members of the Sm-like protein family. Given this, the primary function of this loop may be to provide a structural turn between the third and fourth beta sheets in the core structure.

The Sm and Sm-like protein complexes are known to bind directly to their substrate RNA molecules as shown by several studies (ACHSEL et al. 1999; ACHSEL et al. 2001; MOLLER et al. 2002; RAKER et al. 1999; SCHUMACHER et al. 2002; TORO et al. 2001). Our studies suggest that in an analogous manner, the Lsm1p-7p complex also could bind directly to mRNA. This is supported by the observation that all the three mutants affected in the residues predicted to play a key role in RNA binding (lsm1-8, lsm1-9 and lsm1-14) are

25 impaired in mRNA decay. This indicates that the ability to directly interact with mRNA is important for the mRNA decapping function of the Lsm1p-7p complex. Further these results also suggest that the Lsm1p-7p complex is similar to the Sm complex and the other Sm-like protein complexes in the organization of its RNA contacting surfaces and the manner in which it contacts RNA.

Studies in mammalian cells showed that human Lsm4p cannot be recruited to P-bodies if its interactions with the other subunits of the Lsm1p-7p complex are disrupted by mutations (INGELFINGER et al. 2002). Our results in figure 6 show that Lsm2p-GFP and

Lsm7p-GFP are not concentrated in P-bodies in lsm1∆ cells indicating that the recruitment of these proteins to P-bodies is dependent on Lsm1p. Thus, together these results support the idea that the recruitment of any of the subunits of the Lsm1p-7p is dependent on the ability of that subunit to be incorporated into the Lsm1p-7p complex.

Therefore, it is likely that the Lsm1p-7p complex is recruited as a unit to the P-bodies.

On the other hand, our results also indicate that mutations disrupting the predicted RNA binding surfaces of Lsm1p do not affect the recruitment of the mutant Lsm1p or the other subunits of the Lsm1p-7p complex to the P-bodies (Figures 5 and 6). This supports the idea that the mutations in lsm1-9 and lsm1-14 affect the inter-subunit interactions and hence the assembly of the Lsm1p-7p complex only minimally if at all. Since both lsm1-9 and lsm1-14 mutants are defective in mRNA decay and 3’ end protection, these results further suggest that the Lsm1p can be recruited to the P-bodies by virtue of its interactions with the other subunits of the Lsm1p-7p complex even when the direct interactions between Lsm1p and RNA are disrupted.

26

Our results document a strong correlation between the ability of Lsm1p to promote decapping, and its ability to protect the 3’ end from trimming. The key observation is that all the mutants that are defective in mRNA 3’ protection are also defective in mRNA decay (Table 1 and Figures 2 and 3). This is consistent with the model that an initial step in the activation of decapping is the interaction of the Lsm1p-7p complex with the 3’ end of the mRNA substrate and the 3’ end protection is a consequence of such an interaction.

An unresolved issue is the binding specificity of the Lsm1-7p complex and where on the mRNA this complex may interact. Several observations support the hypothesis that the

Lsm1p-7p complex directly interacts with 3’ ends of deadenylated mRNAs in vivo. (i)

Co-immunoprecipitation studies indicate that Lsm1p and Lsm5p specifically associate with deadenylated mRNAs in vivo (THARUN and PARKER 2001a). (ii) The poly(G) decay intermediate of MFA2pG mRNA, which lacks more than 50% of the 5’ portion of the mRNA (including the whole of the ORF and a part of the 3’ UTR), also strongly co- immunoprecipitates with Lsm1p. This suggests that the 3’ UTR of this mRNA contains a possible Lsm1p-7p complex binding site. (iii) The Lsm2p-8p and the Lsm2p-7p complexes which are closely related to the Lsm1p-7p complex bind to the 3’ ends of their substrates, the U6 snRNA and snR5 respectively (ACHSEL et al. 1999; FERNANDEZ et al.

2004). (iv) The fact that several deadenylated mRNAs accumulate as 3’ trimmed species

(truncated at their 3’ ends by ~10 to 20 nucleotides) in cells lacking any of the Lsm1p through Lsm7p proteins is consistent with a lack of protection to the 3’ ends of deadenylated mRNAs in these cells. (v) The Sm-like protein complexes in general

27 (including the Lsm2p-8p complex) are known to bind preferentially to U-rich regions of

RNA (ACHSEL et al. 1999; ACHSEL et al. 2001; RAKER et al. 1999; SCHUMACHER et al.

2002; TORO et al. 2001) and a significant percentage of yeast mRNAs contain a short stretch of U residues at their 3’ end (GRABER et al. 1999). Given the fact that the Lsm1p-

7p complex interacts with and affects the decay of multiple mRNAs (THARUN et al.

2000; THARUN and PARKER 2001a), this supports the idea that the Lsm1p-7p complex binds to the 3’ ends of deadenylated mRNAs in vivo.

Based on these observations we propose that following deadenylation, the Lsm1p-7p complex directly interacts with the 3’ end of the deadenylated mRNAs which results in the protection of the mRNA 3’ end. Further, this interaction also triggers mRNP rearrangement events that ultimately result in the activation of decapping. Importantly, mRNA 3’ end protection by itself could be a crucial cellular function of the Lsm1p-7p complex since it could potentially regulate the rate of 3’ to 5’ decay of the mRNA. Such a control could be very important in the case of mRNAs whose major mode of decay is the 3’ to 5’ decay pathway. Consistent with this, the studies of He and Parker (2001) suggest that the temperature sensitivity of lsm1∆ cells is due to the increased susceptibility to (exosome and ski proteins-mediated) 3’ to 5’ degradation of some mRNAs that are essential at high temperature. Thus, for such mRNAs, the Lsm1p-7p complex could have a protective/stabilizing (rather than decay-promoting) function.

Although recent studies have suggested that mRNA decay occurs in P-bodies (COUGOT et al. 2004; INGELFINGER et al. 2002; SHETH and PARKER 2003; VAN DIJK et al. 2002), it is not known what fraction of cellular mRNAs degrade outside the P-bodies. Interestingly

28 the ski and exosomal proteins (which mediate the 3’ to 5’ mRNA decay) are not concentrated in P-bodies but are uniformly distributed (ALLMANG et al. 1999; SHETH and

PARKER 2003; VAN HOOF et al. 2000; ZANCHIN and GOLDFARB 1999) in the cytoplasm suggesting that 3’ to 5’ decay is likely to occur outside the P-bodies. Thus, the major outcome of Lsm1p-7p binding could be different for mRNAs that degrade outside

(stabilization) and inside (activation of decapping and decay) the P-bodies.

29 Acknowledgements

This work was supported by funds from the National Institutes of Health (GM45443) and the Uniformed Services University of the Health Sciences (C071GY).

30 Materials and Methods

Yeast strains, plasmids and mutagenesis

All the mutant versions of LSM1 were made following the Quikchange mutagenesis protocol (Stratagene) using plasmid pST11 (carrying the wild type LSM1 gene) as the template. This generated the plasmids, pST21 through pST49 and pST18 each of which carried a different lsm1 allele (Table 1). pST11 was made by amplifying the LSM1 gene

(containing the ORF along with 619 and 543 nucleotides respectively of the 5’ and 3’ flanking regions) by polymerase chain reaction from yeast genomic DNA and cloning it into pRS416 which is a CEN vector with URA3 marker (SIKORSKI and HIETER 1989).

All the plasmids were sequenced to make sure that unintended mutations are not present in the LSM1 gene. Plasmids carrying wild type (pST11) and mutant versions of LSM1 were transformed into the lsm1∆ strain yRP1365 (MATα trp1 leu2 ura3 lys2 cup1∆::LEU2(PM) lsm1∆::TRP1) (THARUN et al. 2000) and the resulting transformants were used for the experiments. yRP1365 carrying pRS416 served as the negative control.

For the experiments in figure 5, an lsm1∆ strain expressing GFP fusions of LSM1 or lsm1-9 or lsm1-14 under LSM1’s native promoter from a CEN vector was used. Plasmid pRP1176 (COLLER and PARKER 2004a) is a CEN vector expressing LSM1-GFP from

LSM1’s native promoter. Mutations of lsm1-9 and lsm1-14 were introduced into the

LSM1-GFP cassette of this plasmid by Quikchange mutagenesis protocol (Stratagene) to generate plasmids pST91 and pST92 respectively. pRP1176, pST91 and pST92 were separately transformed into the Euroscarf lsm1∆ strain, ES11301 (MATα, his3, leu2, ura3,

31 lys2, lsm1∆::NEOr) to get the strains needed for the experiments shown in Figure 5.

Strains used in experiments for Figure 6 were made by introducing each of the plasmids pST11 or pST 29 or pST34 (coding for LSM1, lsm1-9 and lsm1-14 respectively; see table

1) separately into the strain yRP 2008 (genotype same as ES11301 except that it is MATa and LSM7-GFP-HIS3) or yRP 2010 (genotype same as ES11301 except that it is MATa and LSM2-GFP-HIS3). yRP 2008 and yRP 2010 were made by crossing the LSM2-GFP and LSM7-GFP strains of Euroscarf with the Euroscarf lsm1∆ strain ES11301. .

RNA and protein analyses and microscopy

Transcription shut-off experiments, RNA preparation, northern analysis, determination of

MFA2pG mRNA half-life, quantitation of poly(G) fragment accumulation, preparation of yeast cell lysates and western analysis for Lsm1p were all performed as described earlier

(THARUN et al. 2000; THARUN and PARKER 1999; THARUN and PARKER 2001a). For all experiments, cells were grown at 25˚ in –ura medium and collected at log phase. For examination of P-bodies (experiments in figures 5 and 6), cells grown to log phase were collected, incubated in glucose-free medium for 10 minutes and then observed using confocal microscope. Confocal microscopy was done as described (SHETH and PARKER

2003; TEIXEIRA et al. 2004).

32 REFERENCES

ABLER, M. L., and P. J. GREEN, 1996 Control of mRNA stability in higher plants. Plant Mol Biol 32: 63-78. ACHSEL, T., H. BRAHMS, B. KASTNER, A. BACHI, M. WILM et al., 1999 A doughnut- shaped heteromer of human Sm-like proteins binds to the 3'-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro. Embo J 18: 5789-5802. ACHSEL, T., H. STARK and R. LUHRMANN, 2001 The Sm domain is an ancient RNA- binding motif with oligo(U) specificity. Proc Natl Acad Sci U S A 98: 3685-3689. ALLMANG, C., E. PETFALSKI, A. PODTELEJNIKOV, M. MANN, D. TOLLERVEY et al., 1999 The yeast exosome and human PM-Scl are related complexes of 3' --> 5' exonucleases. Genes Dev 13: 2148-2158. ANDERSON, J. S. J., and R. P. PARKER, 1998 The 3' to 5' degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3' to 5' exonucleases of the exosome complex. Embo J 17: 1497-1506. BATES, P. A., L. A. KELLEY, R. M. MACCALLUM and M. J. STERNBERG, 2001 Enhancement of protein modeling by human intervention in applying the automatic programs 3D-JIGSAW and 3D-PSSM. Proteins Suppl 5: 39-46. BATES, P. A., and M. J. STERNBERG, 1999 Model building by comparison at CASP3: using expert knowledge and computer automation. Proteins Suppl 3: 47-54. BEELMAN, C. A., A. STEVENS, G. CAPONIGRO, T. E. LAGRANDEUR, L. HATFIELD et al., 1996 An essential component of the decapping enzyme required for normal rates of mRNA turnover. Nature 382: 642-646. BOECK, R., B. LAPEYRE, C. E. BROWN and A. B. SACHS, 1998 Capped mRNA degradation intermediates accumulate in the yeast spb8-2 mutant. Mol Cell Biol 18: 5062-5072. BONNEROT, C., R. BOECK and B. LAPEYRE, 2000 The two proteins Pat1p (Mrt1p) and Spb8p interact in vivo, are required for mRNA decay, and are functionally linked to Pab1p. Mol Cell Biol 20: 5939-5946. BOUVERET, E., G. RIGAUT, A. SHEVCHENKO, M. WILM and B. SERAPHIN, 2000 A Sm- like protein complex that participates in mRNA degradation. Embo J 19: 1661- 1671. CAO, D., and R. PARKER, 2001 Computational modeling of eukaryotic mRNA turnover. Rna 7: 1192-1212. CAPONIGRO, G., and R. PARKER, 1995 Multiple functions for the poly(A)-binding protein in mRNA decapping and deadenylation in yeast. Genes Dev 9: 2421-2432. COLLER, J., and R. PARKER, 2004a Activators of mRNA decapping are translational repressors. Submitted. COLLER, J., and R. PARKER, 2004b EUKARYOTIC mRNA DECAPPING. Annu Rev Biochem 73: 861-890. COLLER, J. M., N. K. GRAY and M. P. WICKENS, 1998 mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation. Genes Dev 12: 3226-3235. COLLER, J. M., M. TUCKER, U. SHETH, M. A. VALENCIA-SANCHEZ and R. PARKER, 2001 The DEAD box helicase, Dhh1p, functions in mRNA decapping and interacts with both the decapping and deadenylase complexes. RNA 7: 1717-1727.

33 COLLINS, B. M., S. J. HARROP, G. D. KORNFELD, I. W. DAWES, P. M. CURMI et al., 2001 Crystal structure of a heptameric Sm-like protein complex from archaea: implications for the structure and evolution of snRNPs. J Mol Biol 309: 915-923. CONTRERAS-MOREIRA, B., and P. A. BATES, 2002 Domain fishing: a first step in protein comparative modelling. Bioinformatics 18: 1141-1142. COOPER, M., L. H. JOHNSTON and J. D. BEGGS, 1995 Identification and characterization of Uss1p (Sdb23p): a novel U6 snRNA-associated protein with significant similarity to core proteins of small nuclear ribonucleoproteins. Embo J 14: 2066- 2075. COUGOT, N., S. BABAJKO and B. SERAPHIN, 2004 Cytoplasmic foci are sites of mRNA decay in human cells. J Cell Biol 165: 31-40. COUTTET, P., M. FROMONT-RACINE, D. STEEL, R. PICTET and T. GRANGE, 1997 Messenger RNA deadenylylation precedes decapping in mammalian cells. Proc Natl Acad Sci U S A 94: 5628-5633. DECKER, C. J., and R. PARKER, 1993 A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. Genes Dev 7: 1632-1643. DECKER, C. J., and R. PARKER, 2002 mRNA decay enzymes: decappers conserved between yeast and mammals. Proc Natl Acad Sci U S A 99: 12512-12514. DUNCKLEY, T., and R. PARKER, 1999 The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. Embo J 18: 5411-5422. DUNCKLEY, T., M. TUCKER and R. PARKER, 2001 Two related proteins, Edc1p and Edc2p, stimulate mRNA decapping in Saccharomyces cerevisiae. Genetics 157: 27-37. FERNANDEZ, C. F., B. K. PANNONE, X. CHEN, G. FUCHS and S. L. WOLIN, 2004 An Lsm2-Lsm7 Complex in Saccharomyces cerevisiae Associates with the Small Nucleolar RNA snR5. Mol Biol Cell 15: 2842-2852. FISCHER, N., and K. WEIS, 2002 The DEAD box protein Dhh1 stimulates the decapping enzyme Dcp1. Embo J 21: 2788-2797. GAO, M., C. J. WILUSZ, S. W. PELTZ and J. WILUSZ, 2001 A novel mRNA-decapping activity in HeLa cytoplasmic extracts is regulated by AU-rich elements. Embo J 20: 1134-1143. GRABER, J. H., C. R. CANTOR, S. C. MOHR and T. F. SMITH, 1999 In silico detection of control signals: mRNA 3'-end-processing sequences in diverse species. Proc Natl Acad Sci U S A 96: 14055-14060. GUHANIYOGI, J., and G. BREWER, 2001 Regulation of mRNA stability in mammalian cells. Gene 265: 11-23. GUMBS, A. A., C. BASSI, P. S. MOORE, M. FALCONI, I. FRIGERIO et al., 2002 Overexpression of the Sm-like proto-oncogene in primary and metastatic pancreatic endocrine tumors. Jop 3: 109-115. HATFIELD, L., C. A. BEELMAN, A. STEVENS and R. PARKER, 1996 Mutations in trans- acting factors affecting mRNA decapping in Saccharomyces cerevisiae. Mol Cell Biol 16: 5830-5838. HE, W., and R. PARKER, 2001 The yeast cytoplasmic LsmI/Pat1p complex protects mRNA 3' termini from partial degradation. Genetics 158: 1445-1455.

34 HENTZE, M. W., 1991 Determinants and regulation of cytoplasmic mRNA stability in eukaryotic cells. Biochim Biophys Acta 1090: 281-292. HERMANN, H., P. FABRIZIO, V. A. RAKER, K. FOULAKI, H. HORNIG et al., 1995 snRNP Sm proteins share two evolutionarily conserved sequence motifs which are involved in Sm protein-protein interactions. Embo J 14: 2076-2088. HSU, C. L., and A. STEVENS, 1993 Yeast cells lacking 5'-->3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure. Mol Cell Biol 13: 4826-4835. INGELFINGER, D., D. J. ARNDT-JOVIN, R. LUHRMANN and T. ACHSEL, 2002 The human LSm1-7 proteins colocalize with the mRNA-degrading enzymes Dcp1/2 and Xrnl in distinct cytoplasmic foci. Rna 8: 1489-1501. KAMBACH, C., S. WALKE, R. YOUNG, J. M. AVIS, E. DE LA FORTELLE et al., 1999 Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. Cell 96: 375-387. KHANNA, R., and M. KILEDJIAN, 2004 Poly(A)-binding-protein-mediated regulation of hDcp2 decapping in vitro. Embo J 23: 1968-1976. KSHIRSAGAR, M., and R. PARKER, 2004 Identification of Edc3p as an enhancer of mRNA decapping in Saccharomyces cerevisiae. Genetics 166: 729-739. LERNER, M. R., and J. A. STEITZ, 1981 Snurps and scyrps. Cell 25: 298-300. LIU, H., N. D. RODGERS, X. JIAO and M. KILEDJIAN, 2002 The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases. Embo J 21: 4699-4708. LUHRMANN, R., B. KASTNER and M. BACH, 1990 Structure of spliceosomal snRNPs and their role in pre-mRNA splicing. Biochim Biophys Acta 1087: 265-292. LYKKE-ANDERSEN, J., 2002 Identification of a human decapping complex associated with hUpf proteins in nonsense-mediated decay. Mol Cell Biol 22: 8114-8121. MAYES, A. E., L. VERDONE, P. LEGRAIN and J. D. BEGGS, 1999 Characterization of Sm- like proteins in yeast and their association with U6 snRNA. Embo J 18: 4321- 4331. MOLLER, T., T. FRANCH, P. HOJRUP, D. R. KEENE, H. P. BACHINGER et al., 2002 Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction. Mol Cell 9: 23-30. MUHLRAD, D., C. J. DECKER and R. PARKER, 1994 Deadenylation of the unstable mRNA encoded by the yeast MFA2 gene leads to decapping followed by 5'-->3' digestion of the transcript. Genes Dev 8: 855-866. MUHLRAD, D., C. J. DECKER and R. PARKER, 1995 Turnover mechanisms of the stable yeast PGK1 mRNA. Mol Cell Biol 15: 2145-2156. MUHLRAD, D., and R. PARKER, 1992 Mutations affecting stability and deadenylation of the yeast MFA2 transcript. Genes Dev 6: 2100-2111. MURA, C., D. CASCIO, M. R. SAWAYA and D. S. EISENBERG, 2001 The crystal structure of a heptameric archaeal Sm protein: Implications for the eukaryotic snRNP core. Proc Natl Acad Sci U S A 98: 5532-5537. MURA, C., A. KOZHUKHOVSKY, M. GINGERY, M. PHILLIPS and D. EISENBERG, 2003 The oligomerization and ligand-binding properties of Sm-like archaeal proteins (SmAPs). Protein Sci 12: 832-847.

35 PANNONE, B. K., S. D. KIM, D. A. NOE and S. L. WOLIN, 2001 Multiple functional interactions between components of the Lsm2-Lsm8 complex, U6 snRNA, and the yeast La protein. Genetics 158: 187-196. PANNONE, B. K., D. XUE and S. L. WOLIN, 1998 A role for the yeast La protein in U6 snRNP assembly: evidence that the La protein is a molecular chaperone for RNA polymerase III transcripts. Embo J 17: 7442-7453. PELTZ, S. W., G. BREWER, P. BERNSTEIN, P. A. HART and J. ROSS, 1991 Regulation of mRNA turnover in eukaryotic cells. Crit Rev Eukaryot Gene Expr 1: 99-126. PICCIRILLO, C., R. KHANNA and M. KILEDJIAN, 2003 Functional characterization of the mammalian mRNA decapping enzyme hDcp2. Rna 9: 1138-1147. RAKER, V. A., K. HARTMUTH, B. KASTNER and R. LUHRMANN, 1999 Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and thermodynamically stable manner. Mol Cell Biol 19: 6554-6565. RAMIREZ, C. V., C. VILELA, K. BERTHELOT and J. E. MCCARTHY, 2002 Modulation of eukaryotic mRNA stability via the cap-binding translation complex eIF4F. J Mol Biol 318: 951-962. SALGADO-GARRIDO, J., E. BRAGADO-NILSSON, S. KANDELS-LEWIS and B. SERAPHIN, 1999 Sm and Sm-like proteins assemble in two related complexes of deep evolutionary origin. Embo J 18: 3451-3462. SCHUMACHER, M. A., R. F. PEARSON, T. MOLLER, P. VALENTIN-HANSEN and R. G. BRENNAN, 2002 Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein. Embo J 21: 3546-3556. SCHWARTZ, D., C. J. DECKER and R. PARKER, 2003 The enhancer of decapping proteins, Edc1p and Edc2p, bind RNA and stimulate the activity of the decapping enzyme. Rna 9: 239-251. SCHWARTZ, D. C., and R. PARKER, 1999 Mutations in translation initiation factors lead to increased rates of deadenylation and decapping of mRNAs in Saccharomyces cerevisiae. Mol Cell Biol 19: 5247-5256. SCHWARTZ, D. C., and R. PARKER, 2000 mRNA decapping in yeast requires dissociation of the cap binding protein, eukaryotic translation initiation factor 4E. Mol Cell Biol 20: 7933-7942. SCHWEINFEST, C. W., M. W. GRABER, J. M. CHAPMAN, T. S. PAPAS, P. L. BARON et al., 1997 CaSm: an Sm-like protein that contributes to the transformed state in cancer cells. Cancer Res 57: 2961-2965. SERAPHIN, B., 1992 The HIT protein family: a new family of proteins present in prokaryotes, yeast and mammals. DNA Seq 3: 177-179. SERAPHIN, B., 1995 Sm and Sm-like proteins belong to a large family: identification of proteins of the U6 as well as the U1, U2, U4 and U5 snRNPs. Embo J 14: 2089- 2098. SHETH, U., and R. PARKER, 2003 Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300: 805-808. SIKORSKI, R. S., and P. HIETER, 1989 A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19-27.

36 TEIXEIRA, D., U. SHETH, M. A. VALENCIA-SANCHEZ, M. BRENGUES and R. PARKER, 2004 Processing bodies require RNA for assembly and contain non-translating mRNAs. RNA In press. THARUN, S., W. HE, A. E. MAYES, P. LENNERTZ, J. D. BEGGS et al., 2000 Yeast Sm-like proteins function in mRNA decapping and decay. Nature 404: 515-518. THARUN, S., and R. PARKER, 1999 Analysis of mutations in the yeast mRNA decapping enzyme. Genetics 151: 1273-1285. THARUN, S., and R. PARKER, 2001a Targeting an mRNA for decapping: displacement of translation factors and association of the Lsm1p-7p complex on deadenylated yeast mRNAs. Mol Cell 8: 1075-1083. THARUN, S., and R. PARKER, 2001b Turnover of mRNA in eukaryotic cells, pp. 245-257 in RNA, edited by D. SOLL, S. NISHIMURA and P. MOORE. Pergamon. TORO, I., S. THORE, C. MAYER, J. BASQUIN, B. SERAPHIN et al., 2001 RNA binding in an Sm core domain: X-ray structure and functional analysis of an archaeal Sm protein complex. Embo J 20: 2293-2303. TSENG-ROGENSKI, S. S., J. L. CHONG, C. B. THOMAS, S. ENOMOTO, J. BERMAN et al., 2003 Functional conservation of Dhh1p, a cytoplasmic DExD/H-box protein present in large complexes. Nucleic Acids Res 31: 4995-5002. TUCKER, M., M. A. VALENCIA-SANCHEZ, R. R. STAPLES, J. CHEN, C. L. DENIS et al., 2001 The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Cell 104: 377-386. URLAUB, H., V. A. RAKER, S. KOSTKA and R. LUHRMANN, 2001 Sm protein-Sm site RNA interactions within the inner ring of the spliceosomal snRNP core structure. Embo J 20: 187-196. VAN DIJK, E., N. COUGOT, S. MEYER, S. BABAJKO, E. WAHLE et al., 2002 Human Dcp2: a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures. Embo J 21: 6915-6924. VAN DIJK, E., H. LE HIR and B. SERAPHIN, 2003 DcpS can act in the 5'-3' mRNA decay pathway in addition to the 3'-5' pathway. Proc Natl Acad Sci U S A 100: 12081- 12086. VAN HOOF, A., R. R. STAPLES, R. E. BAKER and R. PARKER, 2000 Function of the ski4p (Csl4p) and Ski7p proteins in 3'-to-5' degradation of mRNA. Mol Cell Biol 20: 8230-8243. VILELA, C., C. VELASCO, M. PTUSHKINA and J. E. MCCARTHY, 2000 The eukaryotic mRNA decapping protein Dcp1 interacts physically and functionally with the eIF4F translation initiation complex. Embo J 19: 4372-4382. WANG, Z., X. JIAO, A. CARR-SCHMID and M. KILEDJIAN, 2002 The hDcp2 protein is a mammalian mRNA decapping enzyme. Proc Natl Acad Sci U S A 99: 12663- 12668. WANG, Z., and M. KILEDJIAN, 2001 Functional link between the mammalian exosome and mRNA decapping. Cell 107: 751-762. WILUSZ, C. J., M. GAO, C. L. JONES, J. WILUSZ and S. W. PELTZ, 2001 Poly(A)-binding proteins regulate both mRNA deadenylation and decapping in yeast cytoplasmic extracts. Rna 7: 1416-1424.

37 WYERS, F., M. MINET, M. E. DUFOUR, L. T. VO and F. LACROUTE, 2000 Deletion of the PAT1 gene affects translation initiation and suppresses a PAB1 gene deletion in yeast. Mol Cell Biol 20: 3538-3549. ZANCHIN, N. I., and D. S. GOLDFARB, 1999 Nip7p interacts with Nop8p, an essential nucleolar protein required for 60S ribosome biogenesis, and the exosome subunit Rrp43p. Mol Cell Biol 19: 1518-1525.

38 FIGURE LEGENDS

Figure 1. Regions of LSM1 targeted in our study. a, Position of the mutated residues in the primary sequence. Regions of primary sequence targeted in the mutant alleles, lsm1-

1 through lsm1-24 are underlined. The allele number is given directly below the part of primary sequence targeted in each of these mutant alleles. Residues replacing the wild type sequence are indicated in italics above the appropriate wild type residues. Triangles following (alleles #25 and #26) or preceding (#27 through #29) allele numbers point to the last of the residues deleted from the N and C-termini respectively in those deletion alleles. Amino acid residues shown in bold and italicized bold letters are predicted to be involved in inter-subunit or RNA contacts respectively. Regions of Lsm1p predicted to form the secondary structural elements (N-terminal α helix, α1, and the five β strands, β1 through β5) characteristic of the Sm and Lsm proteins are also indicated. Primary sequence stretches covered by long and short gray boxes are Sm motifs I and II respectively of the Sm domain. Amino acid residue numbers are indicated on the right side of each line. b, Location of the residues implicated in inter-subunit and RNA contacts in the predicted three dimensional structure of the Sm domain of Lsm1p.

Tertiary structure of the Sm domain of Lsm1p was generated using the homology-based modeling program 3D-JIGSAW (BATES et al. 2001; BATES and STERNBERG 1999;

CONTRERAS-MOREIRA and BATES 2002) using the known tertiary structure of the archaebacterial Sm-like protein, Smap1 from Pyrobaculum aerophilum as the template.

Regions in green and pink contain residues implicated in inter-subunit contacts and RNA contacts respectively. Residues targeted in our study are indicated in bold letters.

39 Various secondary structural elements like the N-terminal alpha helix (α1), the five beta strands (β1 through β5) and the loop regions are also indicated. c, Alignment of yeast

Lsm1p with human Lsm1p using the ClustalW program. Long and short gray boxes cover Sm motifs I and II of yeast Lsm1p. Residues in bold or italicized bold letters indicate those predicted to be involved in inter-subunit and RNA contacts respectively.

Portions of the primary sequence forming the Sm domain, and the N- and C-terminal extensions are indicated above the primary sequence. The bottom line in each block indicates the yeast Lsm1p residues that are either identical (*) or replaced with a similar residue (o) in the human Lsm1p. Amino acid residue numbers are indicated on the left and right (for the lower block) of the primary sequence. For both proteins, the entire primary sequence is shown.

Figure 2. Relative levels of poly(G) fragment and full length MFA2pG mRNA in lsm1 mutants. Cells were grown in –ura medium with galactose as the sole carbon source to log phase when they were collected and RNA extracted. RNA was separated on polyacrylamide gel and subjected to northern analysis using oligo probe for MFA2pG mRNA. Bands were visualized and quantitated using the phosphorimager. lsm1 alleles carried by the strains from which RNA was made are indicated above the lanes.

Predicted function of the residues targeted in the important mutants and the nature of the deletions in the deletion mutants are also indicated above the allele names. All strains are deleted for the chromosomal copy of LSM1 and carry the indicated alleles on a CEN vector as mentioned in Methods. The lsm1∆ strain carries empty vector. LSM1+dT indicates RNA from wild type cells subjected to oligo dT mediated RNaseH reaction

40 before running on the gel. Positions of normal and trimmed full length MFA2pG mRNA and poly(G) fragment are indicated on the left. %poly(G) fragment and %trimmed poly(G) fragment are given below the lanes. %poly(G) fragment is calculated by taking the total signal (i.e., total full length plus total poly(G) fragment) as 100%. %trimmed poly(G) fragment is calculated by taking the total poly(G) fragment (i.e., trimmed fragment plus normal fragment) as 100%.

Figure 3. MFA2pG mRNA half-life in lsm1 mutants. Cells were grown as mentioned for Figure 2. At log phase, they were collected and resuspended in fresh -ura medium containing glucose but no galactose. Aliquots of culture were removed at specified time intervals (indicated above the lanes) following shift to glucose medium and cells were collected from them. RNA was extracted from these cells and subjected to northern analysis after separation on polyacrylamide gels as mentioned for Figure 2. Bands were visualized and the intensity of full length MFA2pG mRNA bands quantitated using the phosphorimager. Blots were stripped and reprobed for 7S RNA. 7S RNA bands were also quantitated in each lane with phosphorimager and those values were used for normalizing the MFA2pG full length mRNA levels. Calculated half-life values are given on the right side of each panel. lsm1 alleles carried by the strains used for the experiments are indicated on the left.

Figure 4. Comparison of the Lsm1p protein levels in wild type and lsm1 mutant cells.

Cells were grown in –ura liquid medium at 25˚ and collected at log phase. Lysates were made from the various strains (indicated on top of the lanes) and subjected to western

41 analysis as described in Methods to visualize the Lsm1p protein. Species of higher mobility seen in several lanes (marked by arrow heads) are presumably degraded fragments of Lsm1p. The mutant protein produced in lsm1-13 shows an aberrant lower mobility than wild type Lsm1p in spite of being equal to the wild type protein in length.

Figure 5. Determination of the effect of mutations affecting the predicted RNA binding surface of Lsm1p on the localization of Lsm1p to P-bodies. Yeast cells expressing GFP fusions of LSM1 or lsm1-9 or lsm1-14 (indicated on the left) were observed by confocal microscopy as described in methods.

Figure 6. Determination of the effect of mutations affecting the predicted RNA binding surface of Lsm1p on the localization of Lsm2p and Lsm7p to P-bodies. Wild type

(LSM1) or mutant (lsm1∆ or lsm1-9 or lsm1-14) cells (indicated on top) expressing GFP tagged versions of either Lsm2p (top panel) or Lsm7p (bottom panel) were observed by confocal microscopy as described in methods.

42

AAA A AA AA A A A MSANSKDRNQSNQDAKRQQQNFPKKISEGEADLYLDQYNFTTTA 44 1 2 3 4 25∆ 26∆ =α1=

A D W AAA AA A A A A A AA AA AIVSSVDRKIFVLLRDGRMLFGVLRTFDQYANLILQDCVERIYF 88 5 6 7 8 9 10 =α1== ==β1== ===β2======β3====

AWA AA A AAAA REKD A A AAAA A AA SEENKYAEEDRGIFMIRGENVVMLGEVDIDKEDQPLEAMERIPF 132 11 12 13 14 15 16 17 ∆27 ∆28 ====β4======β5==

AA A A AAAA AAA AA AA AA A AA A KEAWLTKQKNDEKRFKEETHKGKKMARHGIVYDFHKSDMY 172 18 19 20 21 22 23 24 ∆29

Figure 1a Loop5 R105 Loop3 E107 R59 Loop2 G106 D72 Y74

A75 β5 R62 N76 β1 I104 β2 Loop4 M103 I78 L64 β3 α1 F102 L79 β4 Q80

Loop1 G66 I101

Figure 1b

├▬▬▬▬▬▬▬▬▬N-terminal extension▬▬▬▬▬▬▬▬▬┼▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬Sm Domain▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬ Yeast Lsm1p 1 MSANSKDRNQSNQDAKRQQQNFPKKISEGEADLYLDQYNFTTTAAIVSSVDRKIFVLLRDGRMLFGVLRTFDQYANLILQDCVERIYFSEENKYAEEDR Human Lsm1p 1 ------MNYMPGTASLIEDIDKKHLVLLRDGRTLIGFLRSIDQFANLVLHQTVERIHVG--KKYGDIPR ** oo o*o* ******* * * **o **o***o* **** **oo *

▬▬▬▬▬Sm domain▬┼▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬C-terminal extension▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬▬┤ Yeast Lsm1p 100 GIFMIRGENVVMLGEVDIDKE-DQPLEAMERIPFKEAWLTKQKNDEKRFKEETHKGKKMARHGIVYDFHKSDMY--- 172 Human Lsm1p 62 GIFVVRGENVVLLGEIDLEKESDTPLQQVS----IEEILEEQR-VEQQTKLEAEKLKVQALKDRGLSIPRADTLDEY 133 ***oo******o***o*oo** * ** o * * *o * * * * * * o o *

Figure 1c G) A inter-subunit N-terminal C-terminal contact RNA binding minal FL deletions deletions er

lsm1-6 lsm1-7 lsm1-13lsm1-8 lsm1-9 lsm1-14lsm1∆ LSM1 LSM1+dTlsm1-1lsm1-2 lsm1-25lsm1-26lsm1-27lsm1-28lsm1-29lsm1-31LSM1 (C-t lsm1∆

MFA2pG normal full length [ mRNA { trimmed

poly(G) normal [ fragment { trimmed % poly(G) fragment 21 21 16 14 13 14 13 37 35 43 36 37 24 15 20 20 41 15

%poly(G) fragment 27 55 48 50 43 40 53 10 12 10 6 5 34 41 29 34 7 59 in trimmed form

Figure 2 Time after glucose addition (minutes) Half-life 0 2 4 6 8 10 15 20 30 50 (minutes)

LSM1 6.0

lsm1∆ 15.5

lsm1-6 11.0

lsm1-7 16.1

lsm1-8 14.5

lsm1-9 12.1

lsm1-13 15.0 lsm1-14 12.5

lsm1-31 11.0

9.2 lsm1-27

15.0 lsm1-28 lsm1-29 12.1

Figure 3 lsm1∆LSM1lsm1-31lsm1-27lsm1-28lsm1-29 lsm1∆LSM1lsm1-6lsm1-7lsm1-8lsm1-9lsm1-13lsm1-14

<

< <

< < < < < <

Figure 4 LSM1 -GFP

lsm1-9 -GFP

lsm1-14 -GFP

Figure 5 lsm1∆ LSM1 lsm1-9 lsm1-14

LSM2-GFP

LSM7-GFP

Figure 6 Table 1. Description of the changes introduced in the various alleles and their phenotypes.

Mutations in the Sm domain

(a) lsm1 alleles affected in predicted inter-subunit contact residues

LSM1 Plasmid Amino acid Growth mRNA mRNA 3’

allele borne residue changes at 36ûCa decayb end

protectionb lsm1-6 pST26 R59, D60 and R62 to A’s - ++ ++ lsm1-7* pST27 R62, L64 and G66 to A, D and W - + +

respectively lsm1-13 pST33 I101, F102, M103, and I104 to R, E, - + +

K and D respectively

(b) lsm1 alleles affected in predicted RNA-contact residues lsm1-8 pST28 R69 and D72 to A’s - + + lsm1-9 pST29 Y74 and N76 to A’s -/+ ++ + lsm1-14 pST34 R105, G106, and E107 to A, W and - ++ +

A respectively

(c) lsm1 alleles affected in other Sm domain residues lsm1-5 pST25 D51, R52 and K53 to A’s + +++ +++ lsm1-11 pST31 E90, E91 and K93 to A’s + +++ +++ lsm1-12 pST32 E96, E97, D98, and R99 to A’s + +++ +++ lsm1-15 pST35 E114 and D116 to A’s + +++ +++

Mutations outside the Sm domain

(a) lsm1 alleles with mutations in the N-terminal extension lsm1-1 pST21 K6, D7 and R8 to A’s + +++ +++ lsm1-2 pST22 D14, K16 and R17 to A’s + +++ +++ lsm1-3 pST23 K24 and K25 to A’s + +++ +++ lsm1-4 pST24 E28, E30 and D32 to A’s + +++ +++

(b) lsm1 allels with mutations in the C-terminal extension lsm1-16 pST36 D118, K119, E120, and D121 to A’s + +++ +++ lsm1-17 pST37 E125, E128 and R129 to A’s + +++ +++ lsm1-18 pST38 K133 and E134 to A’s + +++ +++ lsm1-19 pST39 K139 and K141 to A’s + +++ +++ lsm1-20 pST40 D143, E144, K145, and R146 to A’s + +++ +++

respectively lsm1-21 pST41 K148, E149, and E150 to A’s + +++ +++ lsm1-22 pST42 H152, K153, K155 and K156 to A’s + +++ +++ lsm1-23 pST43 R159 and H160 to A’s + +++ +++ lsm1-24 pST44 D165, H167, K168 and D170 to A’s + +++ +++

(c) lsm1 alleles with N-terminal truncations lsm1-25 pST45 N-terminus to R17 deleted + +++ +++ lsm1-26 pST46 N-terminus to D36 deleted + +++ +++

(d) lsm1 alleles with C-terminal truncations/insertion lsm1-27* pST47 D118 to C-terminus (55 residues) - ++ ++

deleted lsm1-28* pST48 I130 to C-terminus (43 residues) - + +

deleted lsm1-29 pST49 K145 to C-terminus (28 residues) -/+ ++ ++

deleted lsm1-31 pST18 2x FLAG epitope fusion at the C- - ++ ++

terminus

*mutant Lsm1p protein band was very weak or undetectable (by western analysis) in the lysates of these alleles. a growth, no growth and growth that is significantly slower than wild type cells are respectively indicated by +, - and -/+ symbols. b Strong (comparable to lsm1∆) and moderate block to these functions are indicated by ‘+’ and

‘++’ respectively. ‘+++’ indicates normal (wild type) function.