Copyright  2001 by the Genetics Society of America

The Yeast Cytoplasmic LsmI/Pat1p Complex Protects mRNA 3؅ Termini From Partial Degradation

Weihai He* and Roy Parker† *Department of Molecular and Cellular Biology and †Howard Hughes Medical Institute, University of Arizona, Tucson, Arizona 85721 Manuscript received March 15, 2001 Accepted for publication May 23, 2001

ABSTRACT A key aspect of understanding eukaryotic regulation will be the identification and analysis of that bind mRNAs and control their function. Recently, a complex of seven Lsm proteins and the Pat1p have been shown to interact with yeast mRNAs and promote mRNA decapping. In this study we present several observations to indicate that the LsmI/Pat1 complex has a second distinct function in protecting the 3Ј-UTR of mRNAs from trimming. First, in the LSM1 to LSM7, as well as PAT1, led to the accumulation of MFA2pG and PGK1pG transcripts that had been shortened by 10–20 at their 3Ј ends (referred to as trimming). Second, the trimming of these mRNAs was more severe at the high temperature, correlating with the inability of these mutant strains to grow at high temperature. In contrast, trimming did not occur in a dcp1⌬ strain, wherein the decapping enzyme is lacking. This indicates that trimming is not simply a consequence of the inhibition of mRNA decapping. Third, the temperature-sensitive growth of lsm and pat1 mutants was suppressed by mutations in the exosome or the functionally related Ski proteins, which are required for efficient 3Ј to 5Ј mRNA degradation of mRNA. Moreover, in lsm ski double mutants, higher levels of the trimmed mRNAs accumulated, indicating that exosome function is not required for mRNA trimming but that the exosome does degrade the trimmed mRNAs. These results raise the possibility that the temperature-sensitive growth of the lsm1-7 and pat1 mutants is at least partially due to mRNA trimming, which either inactivates the function of the mRNAs or makes them available for premature 3Ј to 5Ј degradation by the exosome.

HE function of eukaryotic mRNAs is controlled by (Achsel et al. 1999). There are at least two functionally Ta variety of mRNA binding proteins. Recently, a distinct Lsm complexes in yeast (for review, see He and complex of seven Lsm proteins (Lsm1p through Lsm7p) Parker 2000). A nuclear Lsm complex consisting of and the Pat1p were found to interact with yeast mRNAs Lsm2 through Lsm8 proteins is present in the nucleus, and promote mRNA degradation by enhancing the rate binds to the U6 snRNA, and functions in pre-mRNA of decapping (Bouveret et al. 2000; Tharun et al. splicing. In addition, a cytoplasmic Lsm complex con- 2000). These Lsm (Like-Sm) proteins were identified as sisting of Lsm1 through Lsm7 proteins and an addi- a family of proteins that contain the “Sm motif” found tional (Pat1p) interacts with the mRNA decay in the Sm proteins (Hermann et al. 1995; Seraphin machinery, facilitating the decapping step of mRNA 1995). The Sm proteins are a family of small proteins degradation (Hatfield et al. 1996; Bonnerot et al. 2000; that bind to the U1, U2, U4, and U5 snRNAs as a hep- Bouveret et al. 2000; Tharun et al. 2000). tameric complex (Branlant et al. 1982; Liautard et Besides their functions in pre-mRNA splicing and pro- al. 1982; Kambach et al. 1999). Sm proteins form a moting mRNA decapping, strains lacking Lsm proteins seven-member, doughnut-shaped structure through the and the Pat1p have been reported to have additional interactions between the Sm motifs (Kambach et al. phenotypes. For example, strains lacking Lsm1p or 1999). On the basis of the sequence similarities between Pat1p, which are specific to the cytoplasmic Lsm com- the Sm and the Lsm proteins, combined with coimmu- plex, are viable but fail to grow at high temperature noprecipitation experiments (Mayes et al. 1999; Sal- (Hatfield et al. 1996; Wang et al. 1996; Boeck et al. gado-Garrido et al. 1999), Lsm proteins likely assemble 1998; Tharun et al. 2000). In addition, some of these into analogous heptameric ring structures. This view is mutant strains accumulate mRNAs shortened at their supported by the finding that purified human Lsm2 3Ј end by 10–20 nucleotides. Specifically, a lsm1⌬ mutant to Lsm8 proteins form a seven-member ring structure accumulates shortened mRNA species (Boeck et al. 1998). Similar shortened MFA2 mRNA species have been noted in the lsm1, lsm5, lsm6, , and pat1 mu- Corresponding author: Roy Parker, Department of Molecular and tants (Bouveret et al. 2000). Time course experiments Cellular Biology and Howard Hughes Medical Institute, Life Sciences Ј South 404, University of Arizona, Tucson, AZ 85721. have demonstrated that these 3 shortened species arise E-mail: [email protected] by degradation of the 3Ј end of the mRNA following

Genetics 158: 1445–1455 (August 2001) 1446 W. He and R. Parker deadenylation (Boeck et al. 1998; Schwartz and Par- (Schena and Yamamoto 1988). The MFA2pG region was am- plified using oPR1043 (5Ј GTT AGT CTT TTT TTT AGT TTT ker 2000). We will refer to the specific removal of 10–20 Ј Ј AAA ACA CCA AGC CAG CGA GCT ATC ATC TTC 3 ) and nucleotides from the 3 end of the mRNA as trimming. oRP1044 (5Ј GTT TTC CCA GTC ACG AC 3Ј) from vector In this study we address the roles of the cytoplasmic pRP1044 (pCD61). The two PCR products share a homolo- Lsm complex in preventing mRNA trimming and the gous region, while each of them has a region homologous to relationship of this function to its role in promoting the vector pRP11. The two pieces of PCR products and linear- mRNA decapping. We demonstrate that defects in any ized vector pRP11 were cotransformed into yeast to allow homologous recombination. The recombined plasmids were component of the cytoplasmic LsmI/Pat1p complex, isolated from yeast cells, amplified in , and con- but not defects in decapping per se, lead to trimming of firmed by restriction enzyme digestion. multiple mRNAs in a process that is accelerated at higher RNA analysis: Temperature-shift experiments were per- temperatures. Moreover, inhibition of cytoplasmic 3Ј to 5Ј formed by growing cells at 24Њ in media containing 2% galac- ϭ degradation by the exosome does not prevent trimming, tose until early log phase (OD600 0.3). Then the culture was split into two, one kept at 24Њ, and the other shifted to 37Њ. but partially suppresses the temperature-sensitive growth After an hour, cells were harvested and immediately frozen of the lsm and pat1 strains and leads to the accumulation in dry ice. of higher levels of trimmed mRNAs. These results indi- Total RNA isolation and Northern analysis were performed cate that the LsmI/Pat1p complex has a distinct role according to standard protocols (Caponigro et al. 1993). The in preventing mRNA trimming. They also suggest that used as probes in the Northern analysis are: oPR140 (5Ј ATA TTG ATT AGA TCA GGA ATT CC 3Ј) for the temperature-sensitive growth of lsm1-7 and pat1 mu- MFA2pG; oPR141 (5Ј AAT TGA TCT ATC GAG GAA TTC C tants is at least partially due to mRNA trimming, which 3Ј) for PGK1pG; oRP100 (5Ј GTCTAGCCGCGAGGAAGG 3Ј) either inactivates the function of the mRNAs or makes for signal recognition particle (SRP); oRP98 (5Ј CTT GGA them available for premature 3Ј to 5Ј degradation by CCC GTA AGT TTC AC 3Ј) for GAL10; oRP1041 for the 5Ј → Ј the exosome. end poly(G) fragment of MFA2pG (5 CCA AAT TCC TAG ATC TCT TGG 3Ј); oPR154 to detect the 5Ј end → poly(G) fragment of PGK1pG (Muhlrad and Parker 1994); oRP1048 for 25S rRNA (5Ј CTA AGT CGT ATA CAA ATG 3Ј); oRP1050 MATERIALS AND METHODS for 18S rRNA (5Ј GGA CGT AAT CAA CGC AAG 3Ј); and Media and yeast strains: Yeast media were prepared ac- oRP924 for 5.8S rRNA (van Hoof et al. 2000). cording to standard methods. Cells were grown in YEP rich RNase H reactions were done as previously described medium or complete minimal (CM) drop-out medium to (Muhlrad and Parker 1992). The oligos used in RNase H reactions to reduce the size of mRNAs are as follows: oligo maintain plasmids. Most yeast strains, unless indicated, con- Ј Ј tained GAL1 upstream activating sequence (UAS) regulated oRP70 (5 CGG ATA AGA AAG CAA CAC CTG G 3 ) for PGK1pG; oRP97 (5Ј GTA TCT ACA AGG CTC GAT TG 3Ј) MFA2pG and PGK1pG genes and were grown in medium con- Ј taining 2% galactose to induce the of reporter for GAL10; oRP1049 for 25S rRNA (5 CAA TTC GCC AGC AAG CAC 3Ј); and oRP1051 for rRNA 18S (5Ј CGC TTA CTA genes MFA2pG and PGK1pG. Conditional or lsm4 mutants Ј (GAL UAS-controlled LSM3 and LSM4) were transformed GGA ATT CCT C 3 ). with a glyceraldehyde-3- dehydrogenase (GPD) pro- moter-regulated MFA2pG plasmid, grown in CM drop-out me- dium containing 2% galactose, and then shifted to CM drop- RESULTS out medium containing 2% dextrose for 14 hr to shut off the transcription of LSM3 or LSM4 (Mayes et al. 1999). Strains defective in the LsmI/Pat1p complex accumu- Yeast strains used in this study are listed in Table 1. Most late 3؅ trimmed mRNAs: To examine how general the strains used in this study are in the genetic background of accumulation of 3Ј shortened mRNAs is in strains defec- yRP841, with mutations introduced either by transformation tive in components of the LsmI/Pat1p complex, we or by repeated backcrosses. The lsm3, lsm4, and -1 strains Ј used for examining mRNA trimming are in a different genetic examined the 3 end of several mRNAs in a variety of background and were compared to their isogenic wild types. yeast strains. These included strains defective in the All double mutants were constructed by crossing the respective LSM1 through the LSM7 genes, as well as a strain lacking single-mutant strains. PCR analysis was used to identify the Pat1p, which associates with the Lsm1–7p complex. In lsm7⌬::HIS3 . Primers oRP1046 (5Ј CCT TGT TGT Ј Ј these experiments we examined the structure of the CGT ACT GTC 3 ) and oRP1047 (5 GGA CAC TGA GTT Њ Њ TCG AAA 3Ј) were used to amplify the LSM7 region. The mRNAs at both 24 and after a 1-hr incubation at 37 . HIS3 replacement of the LSM7 open reading frame changes Examination of the PGK1pG mRNA showed that full- the length of the PCR products. The ski4-1 mutation abolishes length transcripts shortened from the 3Ј end were seen the recognition site for restriction endonuclease StyI. The in the lsm1⌬ strains at 24Њ (Figure 1A). Since these ف ski4-1 allele was identified by PCR amplification using oRP943 and oRP944, followed by StyI as described previously (van trimmed PGK1 mRNAs are 10 nucleotides smaller Hoof et al. 2000). than the full-length mRNA (FL), we refer to this species Plasmid construction: The GPD promoter controlled as FL(3Ј-10). At 24Њ, the PGK1 FL(3Ј-10) species was not MFA2pG plasmid (pRP1043) was constructed by homologous observed in the wild type, dcp1⌬,orinthelsm2ts, lsm5ts, recombination. The GPD promoter region was PCR amplified lsm6⌬, and lsm7⌬ mutants. In contrast, after a 1-hr incu- with primer pairs oPR1045 (5Ј GGA AAC AGC TAT GAC CAT Ј bation at 37Њ, the amount of the PGK1 FL(3Ј-10) in- GAT TAC GAA TTC GCA TTA TCA ATA CTC GCC 3 ) and ⌬ Ј oPR1042 (5Ј GTT ATT GTT GTA TGA AGA TGA TAG CTC creased in the lsm1 strain, and the PGK1 FL(3 -10) GCT GGC TTG GTG TTT TAA AAC 3Ј) from vector PG-1 mRNA was detected in the lsm2ts, lsm5ts, lsm6⌬, lsm7⌬, Lsm/Pat1 and mRNA 3Ј Degradation 1447 (1998) (1998) (1998) (2000) (1996) (1996) Parker Parker Parker (1998) (1998) (1996) (2000) (2000) (2000) (2000) (2000) (2000) (1995) (1995) (1999) (1999) Source and and and et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. This study This study This study This study This study This study This study This study This study This study van Hoof Beelman This study Anderson Cooper Pannone Anderson Cooper Mayes Mayes Pannone Tharun Anderson Hatfield Hatfield Tharun Tharun Tharun Tharun Tharun ) ::TRP1 ::LEU2 ⌬ ⌬ ::TRP1 ::URA3 ⌬ ⌬ ::LEU2 ::RP1 ski2 GAL-HA-LSM3 ::URA3 lsm1 ⌬ ⌬ ⌬ ::TRP1 ski4-1 pRP949 ::TRP1 ski3 ::TRP1 pRP949 ::LEU2 ski4-1 ::URA3 ::HIS3 ski4-1 ::TRP1 ::TRP1 ski4-1 ::TRP1 ski8 ::HIS3 ::LEU2 ::TRP1 ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ pAEM64 ( ::HIS3 ski4-1 pAEM55 ::HIS3 pAEM55 ::HIS3 ⌬ ⌬ ⌬ ::TRP1 ⌬ TABLE 1 ::LEU2/PGKpG/MFA2pG ski2 ::LEU2 ::TRP1 ::LEU2/PGKpG/MFA2pG lsm1 ::URA3 ⌬ ::LEU2/PGKpG/MFA2pG dcp1 ⌬ ⌬ ⌬ ⌬ ⌬ Strains used in this study ::LEU2/PGKpG/MFA2pG pat1 ::LEU2/PGKpG/MFA2pG lsm5 ::LEU2/PGKpG/MFA2pG lsm1 ::LEU2/PGKpG/MFA2pG lsm5 ::LEU2/PGKpG/MFA2pG lsm7 ::LEU2/PGKpG/MFA2pG lsm1 ::LEU2/PGKpG/MFA2pG lsm1 ::LEU2/PGKpG/MFA2pG ski4-1 ::LEU2/PGKpG/MFA2pG dcp1 ::LEU2/PGKpG/MFA2pG pat1 ::LEU2/PGKpG/MFA2pG ::LEU2/PGKpG/MFA2pG lsm1 ::LEU2/PGKpG/MFA2pG ::LEU2/PGKpG/MFA2pG lsm6 ::LEU2/PGKpG/MFA2pG lsm1 ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ⌬ ::LEU2/PGKpG/MFA2pG ::LEU2/PGKpG/MFA2pG lsm2 ::LEU2/PGKpG/MFA2pG lsm7 ⌬ ⌬ ⌬ 1 trp1-289 ura33-52 1 trp1-289 ura33-52 (LEU2-GAL-LSM4) ⌬ ⌬ 1 his3-11,-15 ura3-1 leu2-3,-112 ade2-1 can-100 lsm3 1 his3-11-15 ura3-1 leu2-3,-112 ade2-1 can-100 ⌬ ⌬ trp1 leu2-3,112 his4-539 ura3-52trp1 cup1 leu2-3,112 his4-539 lys2-201trp1 ura3-52 leu2-3,112 pat1 his4-539 ura3-52trp1 cup1 leu2-3,112 ura3-52 cup1 trp1 leu2-3,112 his4-539 ura3-52 cup1 trp1 leu2-3,112 lys2-201 ura3-52 cup1 ade1-101 his3- trp1 leu2-3,112 ura3-52 his4-539 lys2-201 ski3 trp1 trp1 his4-539 trp1 leu2-3,112 lys2-201 ura3-52 cup1 trp1 leu2-3,112 lys2-201 ura3-52trp1 cup1 leu2-3,112 lys2-201 ura3-5trp1 cup1 leu2-3,112 ura3-52 cup1 trp1 leu2-3,112 his4-539 ura3-52trp1 cup1 leu2-3,112 lys2-201 ura3-52trp1 cup1 leu2-3,112 ura3-52 cup1 trp1 leu2-3,112 ura3-52 his4-539 lys2-201 cup1 trp1 leu2-3,112 ura3-52 his4-539 cup1 trp1 leu2-3,112 ura3-52 his4-539 cup1 trp1 leu2-3,112 his4-539 ura3-52 cup1 trp1 leu2-3,112 his4-539 ura3-52 cup1 trp1 leu2-3,112 his4-539 ura3-52 lys2-201cup1 trp1 leu2-3,112 ura3-52 his4-539 cup1 trp1 leu2-3,112 his4-539 lys2-201 ura3-52 ski8 trp1 leu2 his3 ura3 lys2 ade2 lsm8-1 trp1 leu2 his3 ura3 lys2 ade2 ade1-101 his3- trp1 leu2-3,112 his4-539 ura3-52 cup1 trp1 leu2-3,112 his4-539 ura3-52 cup1 ␣ ␣ ␣ ␣ a a ␣ a ␣ a a a ␣ a a ␣ a ␣ ␣ a ␣ ␣ ␣ ␣ ␣ ␣ ␣ ␣ a a MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT MAT yRP1631 yRP1371 yRP1633 yRP1368 yRP1626 yRP1410 yRP1635 yRP1555 yRP1625 yRP1540 yRP1424 yRP1070 yRP1647 yRP1194 BP4 W303 CY3 yRP1193 AEMY31 MCY4 BMA64 yRP1192 yRP841 yRP1365 yRP1366 yRP1367 yRP1369 yRP1370 yRP1372 StrainyRP840 Genotype 1448 W. He and R. Parker

Figure 1.—Mutations in the LsmI/Pat1p complex cause trimming of mRNAs. Northern blots show (A) the trimming of the PGK1pG mRNA, (B) the trimming of the MFA2pG mRNA in a variety of strains, and (C) the trimming of the MFA2pG mRNA in strains depleted for Lsm3p or Lsm4p. The diagrams on the right illustrate the general structures of the RNA species. FL, full- .and 20 nucleotides, respectively 10ف length mRNA; FR, poly(G) fragment; (3Ј-10) and (3Ј-20), mRNA species shortened by The mRNAs were probed with oligos that specifically hybridize to the poly(G) junction with the 3Ј-flanking region (Hatfield et al. 1996). The position of hybridization is indicated by the black bar underneath the diagrammed . The M lane denotes the molecular markers, marker sizes are labeled on the left, and the fragment panels are overexposed to show the identities of fragments. and pat1⌬ mutant strains. The increase in trimmed by an 18- poly(G) tract inserted in the 3Ј- mRNA species at the high temperature suggested that untranslated region (UTR) of the PGK1pG mRNA. The this process is accelerated at the high temperature (see poly(G) tract forms a strong tertiary structure and blocks below). exonucleolytic degradation of the reporter mRNAs The shortening of the 3Ј end of the mRNA can also (Vreken et al. 1992; Decker and Parker 1993). This be observed on an intermediate of mRNA decay trapped allows the accumulation of a mRNA fragment that has Lsm/Pat1 and mRNA 3Ј Degradation 1449

Figure 1.—Continued.

been decapped and degraded from 5Ј to 3Ј to the 5Ј media. Under this condition, MFA2 transcripts trimmed side of the poly(G) tract. Similar to previous results from the 3Ј end appeared [Figure 1C, FL(3Ј-10)]. This (Boeck et al. 1998; Bouveret et al. 2000; Schwartz indicates that the lsm3 and lsm4 mutations lead to trim- and Parker 2000), we observed that the PGK1 poly(G) ming of the MFA2 transcript. nucleotides at the The above experiments indicate that lesions in the 10ف fragment was also shortened by 3Ј end in the lsm2ts, lsm5ts, lsm6⌬, and lsm7⌬ mutant cytoplasmic LsmI/Pat1p complex lead to accumulation strains at 37Њ, and in the lsm1⌬ and pat1⌬ strains at of mRNAs shortened at their 3Ј ends. To test whether both temperatures, but not in the wild type at either the nuclear Lsm complex was involved in this process, temperature (Figure 1A). The levels of poly(G) frag- we examined the trimming phenotype in a lsm8-1 mu- ment (FR) and trimmed poly(G) fragment [FR(3Ј-10)] tant, which is a partial loss of function allele in this gene were low in the lsm2ts and lsm5ts mutants and absent (Pannone et al. 1998). The Lsm8 protein is a unique from the dcp1⌬ strain due to the inhibition of decapping member of the nuclear Lsm complex. There was no in these mutants (Figure 1A). trimming of the MFA2 mRNA in the lsm8-1 mutant (data Similar, but slightly different, results were obtained not shown). This result indicates that protecting the when the 3Ј end of the MFA2 mRNA was examined in 3Ј-UTR of mRNAs is a distinct function of the cyto- the same strains (Figure 1B). The trimmed full-length plasmic LsmI/Pat1p complex. MFA2pG mRNA was not detected in the lsm2ts, lsm5ts, 3؅ mRNA trimming is not simply due to an inhibition lsm6⌬, and lsm7⌬ mutants at 24Њ, with only a small of decapping: The above results document that defects amount present at 37Њ. In contrast, a 3Ј shortened full- in the LsmI/Pat1p complex lead to the accumulation length MFA2pG mRNA [FL(3Ј-10)] was detected in the of mRNAs shortened from the 3Ј end. This effect is seen lsm1⌬ and the pat1⌬ mutants, most notably at 37Њ (Fig- with the MFA2 and PGK1 mRNAs, and we have also seen ure 1B). No trimming of the full-length MFA2pG mRNA similar results with the GAL10 mRNA (data not shown). was observed either in the wild-type or the dcp1⌬ mutant In principle, the accumulation of the 3Ј trimmed species (Figure 1B). This latter observation suggests that simply could be caused in two different ways. First, because blocking decapping does not account for the mRNA defects in the LsmI/Pat1p complex cause an inhibition trimming. Two forms of the trimmed MFA2 poly(G) of decapping, this defect in decapping could simply and 20 nucleotides, re- allow sufficient time for mRNAs to be trimmed at the 10ف fragments, shortened by spectively, were clearly seen in the lsm1⌬, lsm6⌬, lsm7⌬, 3Ј end. However, this possibility is inconsistent with the and pat1⌬ mutants [Figure 1B, FR(3Ј-10) and FR(3Ј-20)]. observation that a dcp1⌬ strain, which has a strong block The wild-type cells showed a low level of trimmed MFA2 to decapping, does not accumulate 3Ј trimmed species poly(G) fragment FR(3Ј-10) at 37Њ (Figure 1B), indicat- (Beelman et al. 1996; Figure 1, A and B). An alternative ing that high temperature makes the 3Ј-UTR of MFA2 possibility is that the protection of the 3Ј end of mRNAs poly(G) fragment accessible to trimming even in the from trimming is a second distinct function of the LsmI/ wild-type cells. Pat1p complex, independent of this complex’s role in We also used the MFA2pG transcript to determine promoting decapping. To more rigorously distinguish whether strains defective in Lsm3p and Lsm4p would between these possibilities, we examined the difference show trimming of mRNAs. To do this, we used cells in in mRNA trimming between LSM1 and lsm1⌬ strains which Lsm3p and Lsm4p were expressed from a GAL in a dcp1⌬ background where decapping is completely promoter and were depleted following a shift to glucose inhibited. Since in a dcp1⌬ background all decapping 1450 W. He and R. Parker

Figure 2.—3Ј trimming is not simply due to an inhibition of decapping. North- ern blots show (top) the MFA2 and (bot- tom) the PGK1 mRNAs in the wild type (as a control), dcp1⌬, lsm1⌬ dcp1⌬, and lsm1⌬ strains. The mRNAs were probed with oligos (oRP154 for PGK1 and oRP1041 for MFA2) that specifically hy- bridize to the poly(G) junction and the 5Ј- flanking region (Muhlrad and Parker 1994; Anderson and Parker 1998).

is inhibited, any difference between LSM1 and lsm1⌬ two important observations. First, in contrast to other strains must be due to an effect distinct from one affect- lesions that affect decapping, such as the dcp1⌬ (Ander- ing decapping. As shown in Figure 2, the MFA2 mRNA son and Parker 1998) or the dcp2⌬ (Dunckley et al. and the PGK1 mRNA were not trimmed in the dcp1⌬ 2001), the lsm1⌬ was not synthetically lethal with any mutant. However, mRNAs were still trimmed in the of the ski mutations. Because the lsm1⌬ is not a complete lsm1⌬ dcp1⌬ mutant. On the basis of this result, we block to decapping, this observation is consistent with suggest that the LsmI/Pat1p complex has a distinct func- only a low level of mRNA degradation being required tion in protecting the 3Ј ends of mRNAs from some for viability (Dunckley and Parker 1999; Dunckley type of exo- or endonucleolytic degradation. et al. 2001). The second important observation was that -The ski mutations that cause defects in the 3؅ to 5؅ the ski2⌬, ski4-1, ski3⌬, ski7⌬, and ski8⌬ mutations par mRNA degradation suppress the temperature-sensitive tially suppressed the temperature sensitivity of the lsm1⌬ growth of lsm and pat1 mutants: The observation that mutant at 37Њ (Figure 3A; A. van Hoof and R. Parker, the 3Ј trimming of mRNAs is increased at high tempera- unpublished results). This suggests that the tempera- ture in the mutant strains suggests a possible explana- ture-sensitive growth of the lsm1⌬ strain is at least par- tion for the growth phenotypes of strains defective in tially due to the 3Ј to 5Ј degradation of mRNAs. the LsmI/Pat1p complex. Lsm1p and Pat1p, the two To test the generality of the suppression of the lsm1⌬ proteins specific for the cytoplasmic LsmI/Pat1p com- thermosensitivity by lesions in the SKI genes, we deter- plex, are both required only for growth at 37Њ. [Deletion mined whether the growth defect at 37Њ caused by the of other Lsm genes is either lethal (LSM2, LSM3, LSM4, pat1⌬, lsm2ts, lsm5ts, and lsm7⌬ lesions could also be sup- LSM5, and LSM8) or also causes temperature-sensitive pressed by defects in the SKI genes. For this analysis we growth (LSM6 and LSM7), although the interpretation used the ski4-1 allele, which is a point mutation in a of these phenotypes is more complicated since these core component of the exosome and has the strongest proteins are also components of a nuclear Lsm complex effect on mRNA turnover of the ski mutations (van (Mayes et al. 1999).] The observation that the trimming Hoof et al. 2000). In this case, we observed that the of PGK1pG and MFA2pG mRNAs is increased at 37Њ lsm2ts ski4-1, lsm5ts ski4-1, and lsm7⌬ ski4-1 double mutants suggests that the inability of at least the lsm1⌬ and pat1⌬ could all grow at 37Њ, although not as well as a wild-type strains to grow at the higher temperature might be par- strain (Figure 3B). We also observed that the ski4-1 tially due to the enhanced mRNA trimming. A predic- lesion could very weakly suppress the temperature-sensi- tion of this hypothesis is that blocking the 3Ј to 5Ј degra- tive growth of the pat1⌬ strain (Figure 3B). Taking these dation of mRNA might rescue the temperature-sensitive observations together, the ski mutations (ski2⌬, ski3⌬, growth. ski4-1, ski7⌬, and ski8⌬) suppress the temperature-sensi- To test this hypothesis, we created double-mutant tive growth of the lsm and pat1 mutants. strains carrying the lsm1⌬ and a second lesion affecting The ski mutations cause increased accumulation of the 3Ј to 5Ј degradation of mRNA. For this analysis, we trimmed mRNAs in a lsm1⌬ mutant: To determine the used lesions in the SKI2, SKI3, SKI4, SKI7, and SKI8 mechanism by which the ski mutations were suppressing genes, all of which are required for the 3Ј to 5Ј degrada- the lsm1⌬ and pat1⌬ temperature-sensitive growth we tion of mRNA following deadenylation by the exosome examined the 3Ј ends of the MFA2pG and PGK1pG complex (Anderson and Parker 1998; van Hoof et al. mRNAs in the lsm1⌬ ski2⌬, lsm1⌬ ski3⌬, lsm1⌬ ski4-1, 2000). The creation of these double mutants revealed and lsm1⌬ ski8⌬ double mutants. This analysis led to Lsm/Pat1 and mRNA 3Ј Degradation 1451

mRNAs are, at least in part, being degraded 3Ј to 5Ј by the exosome (see discussion). Interestingly, the ski2⌬ (data not shown) and the ski4-1 (Figure 4) mutants also generated the trimmed poly(G) fragments like the lsm1⌬ mutant. Two observa- tions argue that the trimmed poly(G) species from the ski2⌬ and the ski4-1 mutants have the same structures as those from the lsm1⌬ mutant. First, both the PGK1 and the MFA2 trimmed poly(G) fragments migrated in the same way in the ski2⌬, ski4-1, and lsm1⌬ mutants. Second, the lsm1⌬ ski2⌬ and lsm1⌬ ski4-1 double mu- tants produce the same trimmed species as the single mutants. This suggests that when the competing 3Ј to 5Ј decay pathway mediated by the exosome is blocked, a low level of trimming can occur even in the presence of the LsmI/Pat1 complex. The ski mutations inhibit the 3؅ to 5؅ degradation of trimmed mRNAs: The increased amount of trimmed species in the lsm1⌬ ski double mutants argues that at least some of the trimmed full-length mRNAs are being degraded by the exosome. Since this model predicts that mRNAs are being de- graded 3Ј to 5Ј in the lsm1⌬ strain, we should be able Figure 3.—The temperature-sensitive growth of lsm and Ј pat1 mutants can be suppressed by the ski mutations defective to detect a RNA fragment degraded to the 3 side of in the 3Ј to 5Ј mRNA degradation. (A) The temperature- the poly(G) tract (Anderson and Parker 1998). In sensitive growth of the lsm1⌬ mutant was suppressed by the addition, the production of this 3Ј degraded mRNA ⌬ ⌬ ⌬ ski2 , ski3 , ski4-1, and ski8 mutations. (B) The temperature- should be dependent on the Ski proteins. To test this sensitive growth of the lsm2ts, lsm5ts, lsm7⌬, and pat1⌬ mutants Ј → was suppressed by the ski4-1 mutation. prediction, we looked at the 5 end poly(G) fragment of the PGK1pG mRNA generated by the 3Ј to 5Ј mRNA degradation using a probe hybridizing to the poly(G) tract and the 5Ј-flanking region (Muhlrad et al. 1994, two important observations. First, we observed that trim- 1995). The 5Ј end → poly(G) fragment phenotypes were ming of the PGK1pG and the MFA2pG mRNAs still oc- similar at 24Њ (Figure 5) and 37Њ (data not shown). -60ف curred in the double mutants, because both trimmed Upon treatment with RNase H and oPR70, the full-length and trimmed poly(G) fragments of MFA2pG nucleotides long 5Ј end → poly(G) fragment was not and PGK1pG mRNAs were detected at 24Њ (Figure 4) Њ ⌬ ⌬ detected in the wild-type cells (Figure 5). This is so and 37 (data not shown). Therefore the ski2 , ski3 , because the majority of the mRNA is processed through ⌬ ski4-1, and ski8 mutations do not prevent trimming in the 5Ј to 3Ј mRNA degradation pathway and any 5Ј ⌬ the lsm1 mutant. This implies that the ski mutations end → poly(G) fragment produced is rapidly degraded do not suppress the temperature-sensitive growth of through the 5Ј to 3Ј degradation pathway. There was ⌬ lsm1 by preventing trimming. Another implication of no 5Ј end → poly(G) fragment in the ski2⌬ mutant this observation is that trimming does not require the (Figure 5), which is known to be defective in the 3Ј to SKI-dependent exosome activities and that there must 5Ј degradation. The 5Ј end → poly(G) fragment accumu- be other nucleases that perform the trimming reaction. lated in the lsm1⌬ and dcp1⌬ mutants (Figure 5), because A second important observation was that there were it could not be efficiently degraded through the 5Ј to increased levels of trimmed MFA2pG and PGK1pG 3Ј degradation pathway. There was no 5Ј end → poly(G) mRNAs and mRNA fragments in the double mutants fragment in the lsm1⌬ ski2⌬ double mutants (Figure 5), as compared to the lsm1⌬ alone (Figure 4, A and B). indicating that the trimmed species cannot be degraded In addition, a second trimmed species shortened by by the 3Ј to 5Ј degradation pathway. This implies that -nucleotides was more prevalent in the double mu- the ski2⌬ mutation might suppress the temperature 20ف tant, both for the full-length mRNAs and for the mRNA sensitive growth of lsm1⌬ by preventing the 3Ј to 5Ј fragment [FL(3Ј-20) and FR(3Ј-20), Figure 4, A and B]. degradation of the trimmed species (see discussion). This increased level of the trimmed mRNA fragments is consistent with the previous work demonstrating that DISCUSSION these types of mRNA fragments are degraded 3Ј to 5Ј by the exosome (Anderson and Parker 1998). The The cytoplasmic LsmI/Pat1p complex has a distinct -increased levels of trimmed full-length mRNAs in the function in stabilizing the 3؅ terminus of mRNAs: Sev double mutants imply that the trimmed full-length eral lines of evidence suggest that the LsmI/Pat1p com- 1452 W. He and R. Parker

Figure 4.—ski mutations cause in- creased accumulation of trimmed mRNAs in a lsm1⌬ mutant. Northern blots show the (A) MFA2pG mRNA and the (B) PGK1pG mRNA at 24Њ. The asterisks in- dicate the trimmed full-length mRNA species. The ladder of PGK1pG decay in- termediates migrating between the full- length and the poly(G) fragment in the double mutants results from a block of both the 5Ј to 3Ј and the 3Ј to 5Ј mRNA degradation pathways as seen in the dcp1-2 ski8⌬ (Anderson and Parker 1998) and lsm1⌬ ski2⌬ mutants (Figure 5). Species migrating faster than the trimmed poly(G) fragments are typical of mRNA decay intermediates found in the 3Ј to 5Ј decay mutants (Anderson and Parker 1998). Lsm/Pat1 and mRNA 3Ј Degradation 1453

Figure 5.—The ski mutations inhibit the 3Ј to 5Ј degradation of trimmed species. Shown is Northern blot analysis of PGK1pG mRNA at 24Њ. The mRNA was internally cleaved with RNase H/oRP70, probed with oligo oRP154 specifically hybridized to the poly(G) junction and the 5Ј-flanking region to detect the decay intermediate of 3Ј to 5Ј degradation, the 5Ј end → poly(G) frag- ment. The lower panel is overexposed to show the fragments. The bands underneath the 5Ј end → poly(G) fragments are due to nonspecific hybridization.

plex has a specific role in stabilizing the 3Ј termini of et al. 1999; Pannone et al. 2001). This raises the possibil- mRNAs. Initially, this was suggested by the observation ity that both the nuclear and the cytoplasmic Lsm com- that defects in, or depletion of, Lsm1p to Lsm7p, or plexes function to protect the 3Ј ends of RNAs from Pat1p lead to the accumulation of mRNA degradative reactions. -nucleotides (Figure Previous work has observed that inhibition of decap 20–10ف trimmed into the 3Ј-UTR 1 and Boeck et al. 1998; Bouveret et al. 2000; Schwartz ping by the addition of cycloheximide led to the accu- and Parker 2000). This effect is observed with several mulation of 3Ј trimmed MFA2 and PGK1 mRNAs (Boeck mRNAs, including the MFA2pG, PGK1pG, and GAL10 et al. 1998). This led the authors to the reasonable inter- transcripts (Figure 1 and data not shown; Boeck et al. pretation that the trimmed species arose because of an 1998; Bouveret 2000; Schwartz and Parker 2000). inhibition of decapping. However, this is inconsistent Two observations indicate that the shortening of the with our observation that a dcp1⌬ strain does not accu- mRNAs’ 3Ј end is due to the absence of the LsmI/Pat1p mulate trimmed species (Figures 1 and 2). There are complex and is not a general consequence of the partial several possible explanations for this apparent contra- inhibition of decapping that also occurs in these mu- diction. For example, since the mechanism by which tants. First, dcp1⌬ strains, which are completely blocked cycloheximide inhibits mRNA decapping is unclear, it for decapping, do not accumulate the 3Ј trimmed may be that this drug indirectly affects the interaction mRNAs for the MFA2, PGK1, and GAL10 mRNAs (Figure of the LsmI/Pat1p complex with mRNAs. This would 1 and data not shown). Second, the 3Ј trimmed mRNAs both inhibit decapping and lead to mRNA trimming. accumulate in a dcp1⌬ lsm1⌬ double mutant (Figure 2). Alternatively, it may be that in different strain back- On the basis of these observations, we argue that the grounds, or for different mRNAs, the susceptibility of LsmI/Pat1p complex has a distinct function to protect the mRNAs to trimming may be different. This is based the 3Ј termini of the mRNAs from a trimming reaction. on the observation that in our strain background we do The mechanism by which the LsmI/Pat1p complex not observe trimming of the MFA2 transcript following protects the 3Ј termini of mRNAs from degradation is the addition of cycloheximide (Beelman and Parker currently unclear. The simplest hypothesis is that this 1994). complex binds to the mRNAs in this region and steri- Shortening of the mRNA 3؅ end occurs by an unknown cally inhibits an exo- or endonuclease. This possibility mechanism: Our results suggest that the shortening of is supported, but not proven, by two observations. First, the 3Ј end observed in the lsm and pat1 mutant strains it is known that the LsmI/Pat1p complex binds to is different from the previously observed mRNA degra- mRNAs (Tharun et al. 2000). Second, the analogous dation processes. The 3Ј to 5Ј degradation of the mRNA nuclear Lsm complex is known to bind near the 3Ј end body following deadenylation requires the exosome as of the U6 snRNA (Achsel et al. 1999; Mayes et al. 1999). well as the Ski2p, Ski3p, Ski7p, and Ski8p (Anderson Moreover, the binding of the nuclear Lsm complex to and Parker 1998; van Hoof et al. 2000). We have dem- the U6 snRNA appears to be required for the stability onstrated that the accumulation of the 3Ј trimmed spe- of the U6 snRNA (Cooper et al. 1995; Mayes et al. 1999; cies is independent of these proteins (Figures 4 and 5). Pannone et al. 2001). Specifically, overexpression of U6 However, because the levels of the trimmed species are snRNA can rescue the temperature-sensitive growth of increased in the ski2⌬, ski4-1, lsm1⌬ ski2⌬, lsm1⌬ ski3⌬, mutants defective in the nuclear Lsm complex (Mayes lsm1⌬ ski4-1, and lsm1⌬ ski8⌬ strains, the degradation 1454 W. He and R. Parker of the trimmed species occurs, at least in part, by the trimmed into the coding region or subjected to deleteri- normal exosome-mediated 3Ј to 5Ј decay pathway of ous degradation as a consequence of trimming, this mRNA. These results indicate that a different, as-yet- would be sufficient to explain the temperature-depen- unidentified exo- or endonuclease is able to remove dent growth phenotypes. An alternative possibility is the 3Ј terminal portion of the mRNA, but is generally that trimming of mRNAs compromises the function of inhibited from further degradation of the mRNA. This at least one essential mRNA in some other manner. For raises the interesting implication that there is a specific example, trimming might affect or localiza- organization of the 3Ј end of the mRNP that can allow tion of some mRNAs. In this view, the suppression would mRNA trimming to only a limited extent. Moreover, occur because stabilization of the trimmed species because multiple mRNAs show the same behavior this would allow for prolonged time for increased function would have to be a shared feature of mRNP organiza- of the transcript, thereby compensating indirectly for tion. the defect in mRNA function. Future experiments ex- Phenotypic consequences of lsm and pat1 mutations: amining the function and metabolism of the trimmed Several observations suggest that at least part of the mRNAs should help to distinguish these possibilities. reason that the lsm1⌬ and pat1⌬ strains die at high Ј We thank Ambro van Hoof and Sundaresan Tharun for their helpful temperature is inappropriate RNA degradation in a 3 to comments and discussions. We thank C. J. Decker for the generous 5Ј direction. We observed that the temperature-sensitive gift of the pCD61 plasmid. This work was supported by a grant from growth of the lsm1⌬ and pat1⌬ strains could be at least the National Institutes of Health (GM-45443) and funds from the partially suppressed by lesions in the known 3Ј to 5Ј Howard Hughes Medical Institute to R.P. cytoplasmic mRNA degradation machinery. There are two general possibilities for how the lesions in 3Ј to 5Ј mRNA decay machinery could suppress the tempera- LITERATURE CITED ture sensitivity of the lsm and pat1⌬ strains. First, it could Achsel, T., H. Brahms, B. Kastner, A. Bachi, M. Wilm et al., 1999 be that the cytoplasmic exosome degrades an as-yet- A doughnut-shaped heteromer of human Sm-like proteins binds to-be-identified essential cytoplasmic noncoding RNA to the 3Ј-end of U6 snRNA, thereby facilitating U4/U6 duplex whose stability requires the Lsm proteins. To date, we formation in vitro. EMBO J. 18: 5789–5802. Ј Anderson, J. S. J., and R. P. Parker, 1998 The 3Ј to 5Ј degradation have not seen any differences in the 3 ends of stable of yeast mRNAs is a general mechanism for mRNA turnover that cytoplasmic 5.8S, 18S, and 25S rRNAs and SCR1 RNA requires the SKI2 DEVH box protein and 3Ј to 5Ј exonucleases (a small cytoplasmic RNA that is a component of the of the exosome complex. EMBO J. 17: 1497–1506. Beelman, C. A., and R. Parker, 1994 Differential effects of transla- SRP; Hann and Walter 1991) in mutants defective tional inhibition in cis and in trans on the decay of the unstable in the LsmI/Pat1p complex (data not shown). These yeast MFA2 mRNA. J. Biol. Chem. 269: 9687–9692. observations are consistent with the possibility that the Beelman, C. A., A. Stevens, G. Caponigro, T. E. LaGrandeur, L. Hatfield et al., 1996 An essential component of the decapping LsmI/Pat1p complex specifically protects the 10–20 nu- enzyme required for normal rates of mRNA turnover. [see com- cleotides at the 3Ј end of mRNAs. ments] Nature 382: 642–646. An alternative possibility for the requirement for Lsm Boeck, R., B. Lapeyre, C. E. Brown and A. B. Sachs, 1998 Capped mRNA degradation intermediates accumulate in the yeast spb82 proteins for growth at high temperature is that the mutant. Mol. Cell. Biol. 18: 5062–5072. LsmI/Pat1p complex inhibits the trimming of certain Bonnerot, C., R. Boeck and B. Lapeyre, 2000 The two proteins mRNAs, whose function would be inactivated by 3Ј trim- Pat1p (Mrt1p) and Spb8p interact in vivo, are required for mRNA ming. In this view, the ski mutations would suppress the decay, and are functionally linked to Pab1p. Mol. Cell. Biol. 20: 5939–5946. temperature sensitivity of the lsm and pat1 lesions by Bouveret, E., G. Rigaut, A. Shevchenko, M. Wilm and B. Seraphin, stabilizing the trimmed mRNAs. A prediction of the 2000 A Sm-like protein complex that participates in mRNA above hypothesis is that trimmed mRNAs might show degradation. EMBO J. 19: 1661–1671. Ј Ј Branlant, C., A. Krol, J. P. Ebel, E. Lazar, B. Haendler et al., increased rates of 3 to 5 degradation by the exosome. 1982 U2 RNA shares a structural with U1, U4, and U5 However, so far we have been unable to directly demon- RNAs. EMBO J. 1: 1259–1265. strate such a change in the rate of 3Ј to 5Ј mRNA decay Caponigro, G., D. Muhlrad and R. Parker, 1993 A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharo- for the MFA2pG transcript in a strain where trimming myces cerevisiae: a stimulatory role for rare codons. Mol. Cell. is occurring (A. van Hoof and R. Parker, unpublished Biol. 13: 5141–5148. observation). This suggests that if trimming does lead Cooper, M., L. H. Johnston and J. D. Beggs, 1995 Identification Ј Ј and characterization of Uss1p (Sdb23p): a novel U6 snRNA- to increased rates of 3 to 5 mRNA degradation it is a associated protein with significant similarity to core proteins of relatively small effect (Ͻ150% of the rate of full-length small nuclear ribonucleoproteins. EMBO J. 14: 2066–2075. mRNA based on kinetic modeling; C. Cao and R. Par- Decker, C. J., and R. Parker, 1993 A turnover pathway for both ker, unpublished observations), or is more pronounced stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. Genes Dev. 7: 1632–1643. on specific mRNAs. In support of mRNA-specific effects Dunckley, T., and R. Parker, 1999 The DCP2 protein is required of trimming, we have observed that some mRNAs are for mRNA decapping in and contains trimmed by Ͼ30 nucleotides into the 3Ј-UTR in lsm1⌬ a functional MutT motif. EMBO J. 18: 5411–5422. Dunckley, T., M. Tucker and R. Parker, 2001 Two related pro- strains (W. Olivas and R. 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