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1.
One of two general pathways of mRNA decay in the yeast Saccharomyces cerevisiae occurs by deadenylation followed by 3'-to-5' degradation of the mRNA body. Previous results have shown that this degradation requires components of the exosome and the Ski2p, Ski3p, and Ski8p proteins, which were originally identified due to their superkiller phenotype. In this work, we demonstrate that deletion of the SKI7 gene, which encodes a putative GTPase, also causes a defect in 3'-to-5' degradation of mRNA. Deletion of SKI7, like deletion of SKI2, SKI3, or SKI8, does not affect various RNA-processing reactions of the exosome. In addition, we show that a mutation in the SKI4 gene also causes a defect in 3'-to-5' mRNA degradation. We show that the SKI4 gene is identical to the CSL4 gene, which encodes a core component of the exosome. Interestingly, the ski4-1 allele contains a point mutation resulting in a mutation in the putative RNA binding domain of the Csl4p protein. This point mutation strongly affects mRNA degradation without affecting exosome function in rRNA or snRNA processing, 5' externally transcribed spacer (ETS) degradation, or viability. In contrast, the csl4-1 allele of the same gene affects rRNA processing but not 3'-to-5' mRNA degradation. We identify csl4-1 as resulting from a partial-loss-of-function mutation in the promoter of the CSL4 gene. These data indicate that the distinct functions of the exosome can be separated genetically and suggest that the RNA binding domain of Csl4p may have a specific function in mRNA degradation. 相似文献
2.
The ski7 antiviral protein is an EF1-alpha homolog that blocks expression of non-Poly(A) mRNA in Saccharomyces cerevisiae
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We mapped and cloned SKI7, a gene that negatively controls the copy number of L-A and M double-stranded RNA viruses in Saccharomyces cerevisiae. We found that it encodes a nonessential 747-residue protein with similarities to two translation factors, Hbs1p and EF1-alpha. The ski7 mutant was hypersensitive to hygromycin B, a result also suggesting a role in translation. The SKI7 product repressed the expression of nonpolyadenylated [non-poly(A)] mRNAs, whether capped or uncapped, thus explaining why Ski7p inhibits the propagation of the yeast viruses, whose mRNAs lack poly(A). The dependence of the Ski7p effect on 3' RNA structures motivated a study of the expression of capped non-poly(A) luciferase mRNAs containing 3' untranslated regions (3'UTRs) differing in length. In a wild-type strain, increasing the length of the 3'UTR increased luciferase expression due to both increased rates and duration of translation. Overexpression of Ski7p efficiently cured the satellite virus M2 due to a twofold-increased repression of non-poly(A) mRNA expression. Our experiments showed that Ski7p is part of the Ski2p-Ski3p-Ski8p antiviral system because a single ski7 mutation derepresses the expression of non-poly(A) mRNA as much as a quadruple ski2 ski3 ski7 ski8 mutation, and the effect of the overexpression of Ski7p is not obtained unless other SKI genes are functional. ski1/xrn1Delta ski2Delta and ski1/xrn1Delta ski7Delta mutants were viable but temperature sensitive for growth. 相似文献
3.
The Ski complex (composed of Ski3p, Ski8p, and the DEVH ATPase Ski2p) is a central component of the 3'-5' cytoplasmic mRNA degradation pathway in yeast. Although the proteins of the complex interact with each other as well as with Ski7p to mediate degradation by exosome, a 3'-exonuclease complex, the nature of these interactions is not well understood. Here we explore interactions within the Ski complex and between the Ski complex and Ski7p using a directed two-hybrid approach combined with coimmunoprecipitation experiments. We also test the functional significance of these interactions in vivo. Our results suggest that within the Ski complex, Ski3p serves as a scaffold protein with its C terminus interacting with Ski8p, and the sub-C terminus interacting with Ski2p, while no direct interaction between Ski2p and Ski8p was found. Ski7p interacts with the Ski complex via its interaction with Ski8p and Ski3p. In addition, inactivating the Ski complex by mutating conserved residues in the DEVH helicase motif of Ski2 did not abrogate its interaction with Ski7p, indicating that Ski2p function is not necessary for this interaction. 相似文献
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A mutant killer plasmid whose replication depends on a chromosomal "superkiller" mutation 总被引:3,自引:1,他引:3
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Yeast strains carrying a 1.5 x 10(6) molecular weight linear double-stranded RNA in virus-like particles (M dsRNA, the killer plasmid or virus) secrete a toxin that is lethal to strains not carrying this plasmid. Recessive mutations in any of four chromosomal genes (called ski1-ski4) result in increased production of toxin activity. We report here a mutation of the killer plasmid (called [KIL-sd] for ski-dependent) that makes the killer plasmid dependent for its replication on the presence of a chromosomal mutation in any ski gene. Thus, the [KIL-sd] plasmid is lost from SKI(+) strains. When the wild-type killer plasmid, [KIL-k], is introduced into a ski2-2 [KIL-o] strain, the killer plasmid changes to a [KIL-sd] plasmid. This may represent a specific form of mutagenesis or selective replication in the ski2-2 strain of [KIL-sd] variants (mutants) in the normal [KIL-k] population. The ski2-1 and ski2-3 mutations do not convert [KIL-k] to [KIL-sd], but ski2-3 does allow maintenance of the [KIL-sd] plasmid. The [KIL-sd] plasmid thus lacks a plasmid site or product needed for replication in wild-type cells. 相似文献
6.
Zhang X Nakashima T Kakuta Y Yao M Tanaka I Kimura M 《Protein science : a publication of the Protein Society》2008,17(1):136-145
The Ski complex composed of Ski2p, Ski3p, and Ski8p plays an essential role in the 3' to 5' cytoplasmic mRNA degradation pathway in yeast. Ski2p is a putative RNA helicase, belonging in the DExD/H-box protein families and conserved in eukarya as well as in archaea. The gene product (Ph1280p) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 shows sequence homology with Ski2p, sharing 22.6% identical amino acids with a central region of Ski2p. In order to gain structural information about the Ski2p-like RNA helicase, we overproduced Ph1280p in Escherichia coli cells, and purified it to apparent homogeneity. Ph1280p exhibits DNA/RNA-dependent ATPase activity with an optimal temperature at approximately 90 degrees C. The crystal structure of Ph1280p has been solved at a resolution of 3.5 A using single-wavelength anomalous dispersion (SAD) and selenomethionyl (Se-Met)-substituted protein. Ph1280p comprises four subdomains; the two N-terminal subdomains (N1 and N2) fold into an RecA-like architecture with the conserved helicase motifs, while the two C-terminal subdomains (C1 and C2) fold into alpha-helical structures containing a winged helix (WH)-fold and helix-hairpin-helix (HhH)-fold, respectively. Although the structure of each of the Ph1280p subdomains can be individually superimposed on the corresponding domains in other helicases, such as the Escherichia coli DNA helicase RecQ, the relative orientation of the helicase and C-terminal subdomains in Ph1280p is significantly different from that of other helicases. This structural feature is implicated in substrate specificity for the Ski2-like helicase and would play a critical role in the 3' to 5' cytoplasmic mRNA degradation in the Ski complex. 相似文献
7.
Ski6p Is a Homolog of RNA-Processing Enzymes That Affects Translation of Non-Poly(A) mRNAs and 60S Ribosomal Subunit Biogenesis 总被引:11,自引:6,他引:5
Lionel Benard Kathleen Carroll Rosaura C. P. Valle Reed B. Wickner 《Molecular and cellular biology》1998,18(5):2688-2696
We mapped and cloned SKI6 of Saccharomyces cerevisiae, a gene that represses the copy number of the L-A double-stranded RNA virus, and found that it encodes an essential 246-residue protein with homology to a tRNA-processing enzyme, RNase PH. The ski6-2 mutant expressed electroporated non-poly(A) luciferase mRNAs 8- to 10-fold better than did the isogenic wild type. No effect of ski6-2 on expression of uncapped or normal mRNAs was found. Kinetics of luciferase synthesis and direct measurement of radiolabeled electroporated mRNA indicate that the primary effect of Ski6p was on efficiency of translation rather than on mRNA stability. Both ski6 and ski2 mutants show hypersensitivity to hygromycin, suggesting functional alteration of the translation apparatus. The ski6-2 mutant has normal amounts of 40S and 60S ribosomal subunits but accumulates a 38S particle containing 5′-truncated 25S rRNA but no 5.8S rRNA, apparently an incomplete or degraded 60S subunit. This suggests an abnormality in 60S subunit assembly. The ski6-2 mutation suppresses the poor expression of the poly(A)− viral mRNA in a strain deficient in the 60S ribosomal protein L4. Thus, a ski6 mutation bypasses the requirement of the poly(A) tail for translation, allowing better translation of non-poly(A) mRNA, including the L-A virus mRNA which lacks poly(A). We speculate that the derepressed translation of non-poly(A) mRNAs is due to abnormal (but full-size) 60S subunits. 相似文献
8.
Linking the 3′ Poly(A) Tail to the Subunit Joining Step of Translation Initiation: Relations of Pab1p, Eukaryotic Translation Initiation Factor 5B (Fun12p), and Ski2p-Slh1p
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Anjanette Searfoss Thomas E. Dever Reed Wickner 《Molecular and cellular biology》2001,21(15):4900-4908
The 3' poly(A) structure improves translation of a eukaryotic mRNA by 50-fold in vivo. This enhancement has been suggested to be due to an interaction of the poly(A) binding protein, Pab1p, with eukaryotic translation initiation factor 4G (eIF4G). However, we find that mutation of eIF4G eliminating its interaction with Pab1p does not diminish the preference for poly(A)(+) mRNA in vivo, indicating another role for poly(A). We show that either the absence of Fun12p (eIF5B), or a defect in eIF5, proteins involved in 60S ribosomal subunit joining, specifically reduces the translation of poly(A)(+) mRNA, suggesting that poly(A) may have a role in promoting the joining step. Deletion of two nonessential putative RNA helicases (genes SKI2 and SLH1) makes poly(A) dispensable for translation. However, in the absence of Fun12p, eliminating Ski2p and Slh1p shows little enhancement of expression of non-poly(A) mRNA. This suggests that Ski2p and Slh1p block translation of non-poly(A) mRNA by an effect on Fun12p, possibly by affecting 60S subunit joining. 相似文献
9.
Decoying the cap- mRNA degradation system by a double-stranded RNA virus and poly(A)- mRNA surveillance by a yeast antiviral system. 总被引:21,自引:6,他引:15
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D C Masison A Blanc J C Ribas K Carroll N Sonenberg R B Wickner 《Molecular and cellular biology》1995,15(5):2763-2771
The major coat protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae covalently binds m7 GMP from 5' capped mRNAs in vitro. We show that this cap binding also occurs in vivo and that, while this activity is required for expression of viral information (killer toxin mRNA level and toxin production) in a wild-type strain, this requirement is suppressed by deletion of SKI1/XRN1/SEP1. We propose that the virus creates decapped cellular mRNAs to decoy the 5'-->3' exoribonuclease specific for cap- RNA encoded by XRN1. The SKI2 antiviral gene represses the copy numbers of the L-A and L-BC viruses and the 20S RNA replicon, apparently by specifically blocking translation of viral RNA. We show that SKI2, SKI3, and SKI8 inhibit translation of electroporated luciferase and beta-glucuronidase mRNAs in vivo, but only if they lack the 3' poly(A) structure. Thus, L-A decoys the SKI1/XRN1/SEP1 exonuclease directed at 5' uncapped ends, but translation of the L-A poly(A)- mRNA is repressed by Ski2,3,8p. The SKI2-SKI3-SKI8 system is more effective against cap+ poly(A)- mRNA, suggesting a (nonessential) role in blocking translation of fragmented cellular mRNAs. 相似文献
10.
The structure of Ski8p, a protein regulating mRNA degradation: Implications for WD protein structure 总被引:1,自引:0,他引:1
Ski8p is a 44-kD protein that primarily functions in the regulation of exosome-mediated, 3'--> 5' degradation of damaged mRNA. It does so by forming a complex with two partner proteins, Ski2p and Ski3p, which complete a complex that is capable of recruiting and activating the exosome/Ski7p complex that functions in RNA degradation. Ski8p also functions in meiotic recombination in complex with Spo11 in yeast. It is one of the many hundreds of primarily eukaryotic proteins containing tandem copies of WD repeats (also known as WD40 or beta-transducin repeats), which are short ~40 amino acid motifs, often terminating in a Trp-Asp dipeptide. Genomic analyses have demonstrated that WD repeats are found in 1%-2% of proteins in a typical eukaryote, but are extremely rare in prokaryotes. Almost all structurally characterized WD-repeat proteins are composed of seven such repeats and fold into seven-bladed beta propellers. Ski8p was thought to contain five WD repeats on the basis of primary sequence analysis implying a five-bladed propeller. The 1.9 A crystal structure unexpectedly exhibits a seven-bladed propeller fold with seven structurally authentic WD repeats. Structure-based sequence alignments show additional sequence diversity in the two undetected repeats. This demonstrates that many WD repeats have not yet been identified in sequences and also raises the possibility that the seven-bladed propeller may be the predominant fold for this family of proteins. 相似文献
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13.
Yeast strains carrying a 1.5 X 10(6)-dalton double-stranded RNA in virus-like particles secrete a protein toxin which is lethal to strains not carrying this species of double-stranded RNA. We find that recessive mutations in any of four chromosomal genes result in the superkiller phenotype, i.e., increased secretion of killer toxin activity by strains carrying the killer genome. These genes are designated ski1 through ski4 (for superkiller), ski3 and ski4 are located on chromosome XIV, and ski1 is on chromosome VII. A ski1 mutation results in a decreased rate of cell growth. The kex1 and kex2 mutations are epistatic to each ski mutation. 相似文献
14.
Cheng Z Liu Y Wang C Parker R Song H 《Protein science : a publication of the Protein Society》2004,13(10):2673-2684
Ski8p is a WD-repeat protein with an essential role for the Ski complex assembly in an exosome-dependent 3'-to-5' mRNA decay. In addition, Ski8p is involved in meiotic recombination by interacting with Spo11p protein. We have determined the crystal structure of Ski8p from Saccharomyces cerevisiae at 2.2 A resolution. The structure reveals that Ski8p folds into a seven-bladed beta propeller. Mapping sequence conservation and hydrophobicities of amino acids on the molecular surface of Ski8p reveals a prominent site on the top surface of the beta propeller, which is most likely involved in mediating interactions of Ski8p with Ski3p and Spo11p. Mutagenesis combined with yeast two-hybrid and GST pull-down assays identified the top surface of the beta propeller as being required for Ski8p binding to Ski3p and Spo11p. The functional implications for Ski8p function in both mRNA decay and meiotic recombination are discussed. 相似文献
15.
Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome 总被引:9,自引:1,他引:8
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RNA interference (RNAi) is a conserved RNA silencing pathway that leads to sequence-specific mRNA decay in response to the presence of double-stranded RNA (dsRNA). Long dsRNA molecules are first processed by Dicer into 21-22-nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multimeric RNA-induced silencing complex (RISC) that cleaves mRNAs at a site determined by complementarity with the siRNAs. Following this initial endonucleolytic cleavage, the mRNA is degraded by a mechanism that is not completely understood. We investigated the decay pathway of mRNAs targeted by RISC in Drosophila cells. We show that 5' mRNA fragments generated by RISC cleavage are rapidly degraded from their 3' ends by the exosome, whereas the 3' fragments are degraded from their 5' ends by XRN1. Exosome-mediated decay of the 5' fragments requires the Drosophila homologs of yeast Ski2p, Ski3p, and Ski8p, suggesting that their role as regulators of exosome activity is conserved. Our findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation. 相似文献
16.
The yeast Ski complex assists the exosome in the degradation of mRNA. The Ski complex consists of three components; Ski2, Ski3, and Ski8, believed to be present in a 1:1:1 stoichiometry. Measuring the mass of intact isolated endogenously expressed Ski complexes by native mass spectrometry we unambiguously demonstrate that the Ski complex has a hetero-tetrameric stoichiometry consisting of one copy of Ski2 and Ski3 and two copies of Ski8. To validate the stoichiometry of the Ski complex, we performed tandem mass spectrometry. In these experiments one Ski8 subunit was ejected concomitant with the formation of a Ski2/Ski3/Ski8 fragment, confirming the proposed stoichiometry. To probe the topology of the Ski complex we disrupted the complex and mass analyzed the thus formed subcomplexes, detecting Ski8-Ski8, Ski2-Ski3, Ski8-Ski2, and Ski8-Ski8-Ski2. Combining all data we construct an improved structural model of the Ski complex. 相似文献
17.
Superkiller mutations in Saccharomyces cerevisiae suppress exclusion of M2 double-stranded RNA by L-A-HN and confer cold sensitivity in the presence of M and L-A-HN. 总被引:30,自引:15,他引:15
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In an mktl host, L-A-HN double-stranded RNA excludes M2 double-stranded RNA at 30 degrees C but not at 20 degrees C. Recessive mutations suppressing the exclusion of M2 by L-A-HN in an mktl host include six ski (superkiller) genes, three of which (ski6, ski7 and ski8) are new genes. The dominant mutations in one gene (MKS50) and recessive mutations in at least two genes (mks1 and mks2) suppress M2 exclusion by L-A-HN but do not show other characteristics of ski mutations and thus define a new class of killer-related chromosomal genes. Mutations in ski2, ski3, ski4, ski6, ski7, and ski8 result in increased M copy number at 30 degrees C and prevent the cells from growing at 8 degrees C. Elimination of M double-stranded RNA from a cold-sensitive ski- strain results in the loss of cold sensitivity. ski- [KIL-sd1] strains lack L-A-HN, carry L-A-E, and have a lower M1 copy number than do ski- [KIL-k1] strains and are only slightly cold sensitive. The LTS5 (=MAK6) product is required both for low temperature growth and for M1 maintenance or replication. We propose that the elevated levels of M in ski- strains divert the host LTS5 product away from the host and to the M replication process. We also suggest that the essential role of L-A in M replication is protection of M double-stranded RNA from the negative influence of SKI+ products. 相似文献
18.
Meiotic recombination initiates with double-strand breaks (DSBs) catalyzed by Spo11 in conjunction with accessory proteins whose roles are not understood. Two-hybrid analysis reveals a network of interactions connecting the yeast DSB proteins to one another. Of these proteins, Ski8 was known to function in cytoplasmic RNA metabolism, suggesting that its role in recombination might be indirect. However, obligate partners of Ski8 in RNA metabolism are dispensable for recombination and Ski8 relocalizes to the nucleus and associates with chromosomes specifically during meiosis. Interaction of Ski8 with Spo11 is essential for DSB formation and Ski8 relocalization. Thus, Ski8 plays distinct roles in RNA metabolism and, as a direct partner of Spo11, in DSB formation. Ski8 works with Spo11 to recruit other DSB proteins to meiotic chromosomes, implicating Ski8 as a scaffold protein mediating assembly of a multiprotein complex essential for DSB formation. 相似文献
19.
c-Jun associates with the oncoprotein Ski and suppresses Smad2 transcriptional activity 总被引:3,自引:0,他引:3
Pessah M Marais J Prunier C Ferrand N Lallemand F Mauviel A Atfi A 《The Journal of biological chemistry》2002,277(32):29094-29100