- Email: [email protected]
Yeast mRNA Decapping Enzyme
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PROCESSING AND DEGRADAT1VE ENDORIBONUCLEASES
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[I 8] Yeast mRNA Decapping Enzyme B y TRAVIS DUNCKLEY a n d ROY PARKER
Introduction An important step in gene regulation occurs at the level of mRNA stability (for reviews, see Refs.l-3). In the yeast Saccharomyces cerevisiae mRNAs are degraded through two general pathways. The predominant decay pathway occurs via shortening of the poly(A) tail, followed by removal of the 5' cap structure by the Dcplp decapping enzyme.4 Decapping of the mRNA exposes the body of the message to Xrn 1p-mediated 5'----~3'-exonucleolytic degradation.5- 8 The second general decay pathway occurs by poly(A) shortening followed by 3'-+5'-exonucleolytic degradation of the mRNA. 8'9 Decapping is a key step in the major pathway of 5'---~3' mRNA decay in yeast because removal of the cap structure is a prerequisite to 5'---~3'-exoribonucleolytic degradation of the mRNA. 4-6 Different mRNAs also have message-specific rates of decapping, suggesting that the process of removing the 5' cap structure is controlled.7,8 Moreover, the process of mRNA surveillance, wherein aberrant mRNAs are recognized, occurs by rapid deadenylation-independent decapping.~° A yeast decapping enzyme, encoded by the DCP1 gene, has been identified and shown to be required for all decapping in yeast.4,11 In this article we briefly review the process of mRNA decapping including the properties of Dcplp and the proteins that modulate decapping rates in vivo (for a more comprehensive review of decapping see Tucker and Parker12). In addition, we describe techniques for analyzing mRNA decapping in vitro. Dcplp: The Enzyme Purification of Dcplp and subsequent in vitro characterization of the enzyme have yielded several important observations. 11,13 First, the Dcpl protein I j. Ross, Microbiol. Rev. 59, 423 (1995). 2 G. Caponigro and R. Parker, Microbiol. Rev. 60, 233 (1996). 3 A. Jacobson and S. W. Peltz, Annu. Rev. Biochem. 65, 693 (1996). 4 C. Beelman, A. Stevens, G. Caponigro, T. LaGrandeur, L. Hatfield, D. Fortner, and R. Parker, Nature (London) 283, 642 (1996). 5 C. J. Decker and R. Parker, Genes Dev. 7, 1632 (1993). 6 C. L. Hsu and A. Stevens, Mol. Cell BioL 13, 4826 (1993). 7 D. Muhlrad, C. Decker, and R. Parker, Genes Dev. 8, 855 (1994). 8 D. Muhlrad, C. Decker, and R. Parker, Mol. Cell. BioL 15, 2145 (1995). 9 j. S. Anderson and R. Parker, EMBO J. 17, 1497 (1998). 10 D. Muhlrad and R. Parker, Nature (London) 370, 578 (1994).
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23
71
I DCPI (NC 001147.1) Candida albieans (54/66) Arabidopsis (AAC17938.1) (22/51) Drosophila (AAF47089.1) (28/56) Homo Sapiens (CAB77023,1) (26/51)
I
ELS~t3~IllI~N~t~
r , ~ E l l r l g EIl~a'Ual~
FIG. 1. Proteins related to Dcplp. Shown is an alignment with Dcplp and several related proteins in the database. Only the most highly conserved region of the protein is shown. Percentage identity and similarity are indicated in parentheses. Boxed residues denote completely conserved residues that are known to be critical for Dcplp functionJ 4 Lighter shaded boxes indicate similarity and darker boxes indicate identity. Only amino acids conserved between Dcplp and at least two other homologs are shaded. The Candida albicans sequence was obtained from the Candida database (http://alces.med. umn.edu/Candida, html ).
is sufficient for the decapping of mRNAs in vitro. This indicates that Dcplp is the yeast mRNA decapping enzyme. Second, Dcplp cleaves cap structures to the products mVGDP and the full-length mRNA containing a 5'-phosphate. Third, Dcplp preferentially cleaves substrates containing 7-methylguanine over those containing an unmethylated cap. This demonstrates that the 7-methyl modification on the cap structure contributes to the specificity of Dcplp. Fourth, Dcplp shows enhanced decapping activity on longer mRNA substrates. In vitro, Dcplp does not efficiently decap messenger RNAs shorter than 25 nucleotides in length, indicating that Dcp 1p likely recognizes a portion of the mRNA in addition to the 7-methyl modification. Consistent with Dcplp requiring recognition of the mRNA body, the in vitro activity of Dcplp is inhibited by the addition of uncapped mRNA but not by the addition of cap analog, m7GpppG.11 Fifth, the DCP 1 protein requires divalent cation in the form of either Mg 2+ or Mn 2+, with a preference for magnesium, l 1 Sixth, purified Dcplp for model substrates shows a Km of approximately 15 riM.11'13Last, Dcp lp is known to be a phosphoprotein when purified from yeast, although the precise modification and its significance for enzymatic activity are currently unknown. Nevertheless, a requirement for posttranslational modification may explain why Dcplp expressed in Escherichia coli is enzymatically inactive. Because decapping of mRNAs is likely a conserved process it is anticipated that there will be homologs of Dcplp in other eukaryotes. Examination of the available databases identifies a family of proteins that are similar to Dcplp (Fig. 1). Importantly for Dcplp, mutational analyses have demonstrated that altering the residues conserved among this family of proteins has yielded the strongest loss of function alleles in Dcplp (Tharun and Parkerl4; boxed residues in Fig. 1). This 11 T. E. LaGrandeur and R. Parker, EMBO J. 17, 1487 (1998). 12 M. Tucker and R. Parker, Annu. Rev, Biochem. 69, 571 (2000). 13 A. Stevens, Biochem. Biophys. Res. Commun. 96, 1150 (1980). 14 S. Tharun and R. Parker, Genetics 151, 1273 (1999).
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suggests that, although the overall similarity is not high, these proteins may be functional homologs of Dcp lp. Additional Decapping Factors In addition to Dcp Ip, several additional proteins have been shown to affect the process of mRNA decapping (Table I15-26). Among these is the DCP2-encoded protein. The DCP2-encoded protein was identified as a high-copy suppressor of a conditional dcpl mutantJ 5 Subsequent analysis of the dcp2A mutant demonstrated that Dcp2p is absolutely required for decapping of both normal and aberrant mRNAs. Dcp2p physically associates with the Dcplp decapping enzyme and it appears that the interaction between Dcplp and Dcp2p is required for the activation of Dcp I p (Dunckley and Parker 15; and our unpublished observations, 2000). The specific mechanism whereby Dcp2p activates Dcplp is unclear. However, because Dcp2p possesses a functional MutT motif, the Dcp2p-mediated activation of Dcp Ip likely requires cleavage of an as yet unknown pyrophosphate bond (for a review of the MutT motif see Bessman et al., 27 and Koonin2S). The Lsm (like Sin) proteins represent another set of proteins that influence the mRNA decapping rate. There are seven Lsm proteins (Lsml to Lsm7) that affect mRNA decappingJ 6-1s The Lsm proteins are related to the Sm proteins, which are components of small nuclear RNAs (snRNAs) and appear to form a sevenmembered protein complex that binds to mRNA to promote mRNA decappingJ 7,J8 In addition, the Lsm protein complex involved in mRNA decay stably interacts with the PATI/MRT] gene product, which is an additional decapping factor. 17,18,29However, neither the Lsm complex nor Patl/Mrtlp is required for decapping through the mRNA surveillance pathway. Thus, the patl/Mrtl-Lsm complex functions in 15 T. Dunckley and R. Parker, EMBO J. 18, 5411 (1999). t6 R. Boeck, B. Lapeyre, C. E. Brown, and A. B. Sachs, Mol. Cell Biol. 18, 5062 (1998). 17 S. Tharun, W. He, A. E. Mayes, P. Lennertz, J. D. Beggs, and R. Parker, Nature (London) 404, 515 (2000). 18 E. Bouveret, G. Rigaut, A. Shevchenko, M. Wilm, and B. Serapin, EMBOJ. 19, 1661 (2000). J9 S. W. Peltz, A. H. Brown, and A. Jacohson, Genes Dev. 7, 1737 (1993). 20 y. Cui, K. W. Hagan, S. Zhang, and S. W. Peltz, Genes Dev. 9, 423 (1995). 21 E He and A. Jacobson, Genes Dev. 9, 437 (1995). 22 G. Caponigro and R. Parker, Genes Dev. 9, 2421 (1995). 23 D. C. Schwartz and R. Parker, Mol. Cell Biol. 19, 5247 (1999). 24 D. C. Schwartz and R. Parker, submitted (2001). 25 S. Zbang, C. J. Williams, K. Hagan, and S. W. Peltz, biol. Cell, Biol. 19, 7568 (1999). 26 D. Zuk, J. E Belk, and A. Jacobson, Genetics 153, 35 (1999). 27 M. J. Bessman, D. N. Frick, and S. E O'Handley, J. Biol. Chem. 27I, 25059 (1996). 28 E. g. Koonin, NucleieAcids Res. 21, 4847 (1993). 29 L. Hatfield, C. A. Beelman, A. Stevens, and R. Parker, Mol. Cell. Biol. 16, 5830 (1996).
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TABLE I PROTEINS THAT AFFECT m R N A DECAPPING
Gene DCP2 LSM1 - L S M 7 PAT1/MRT1 UPF1, UPF2, UPF3 PAB1 elF-4E VPS16 GRC5, SLA2, MRT4, THS1
Protein function
Refs.
Activates Dcp l p Protein complex that binds mRNA to stimulate decapping Interacts with Lsm complex to stimulate decapping Activates mRNA surveillance Inhibits decapping Binding to cap inhibits mRNA decapping rate Mutation inhibits decapping activity Affects decapping rates
" b c d e
f g h
a Dunckley and Parker (1999). 15 b Boeck e t al. (1998), 16 Tharun et al. (2000), 17 and Bouveret e t al. (2000)J 8 ¢ Tharun et al. (2000) 17 and Bouveret e t al. (2000)) 8 d Peltz et al. (1993), 19 Cui et al. (1995), 2° and He and Jacobson (1995). 2j e Caponigro and Parker (1995). 22 f S c h w a r t z and Parker (1999) 23 and Schwartz and Parker (2000). 24 g Zang e t al. (1999). 25 h Zuk et al. (1999). 26
the deadenylation-dependent decapping pathway to bind to mRNAs to promote decapping by a yet to be determined mechanism. A different set of three proteins functions exclusively to activate decapping through the mRNA surveillance pathway (reviewed in Jacobson and Peltz3). These proteins, referred to as Upfl, Upf2, and Upf3, interact with each other as well as with translation termination factors. 3° This has led to the idea that the Upf protein complex binds to the translation termination machinery and signals a rapid, deadenylation-independent decapping event if translation termination is premature. Translation and Decapping In addition to the activators of decapping discussed above, translation initiation factors are in some cases inhibitors of decapping. For example, the poly(A) tail and the associated poly(A) binding protein inhibit decapping. 22 Similarly, several lines of evidence suggest that there is a competition between the cap binding protein eIF-4E and Dcpl for the cap structure. For example, eIF-4E inhibits decapping in a purified system, z4 Moreover, in vivo mutations in eIP-4E increase the rates of 30 K. Czaplinski, M. J. Ruiz-Echevarria, S. V. Paushkin, X. Han, Y. Weng, H. A. Perlick, H. C. Dietz, M. D. Ter-Avanesyan, and S. W. Peltz, Genes Dev. 12, 1665 (1998).
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decapping and can suppress partial loss of function alleles in Dcplp. 23'24 These observations imply that more highly translated mRNAs are stable, at least in part, because they are bound to initiation factors more frequently than are poorly translated mRNAs, thereby slowing the decapping rate for highly translated mRNAs. An important goal of future work is to understand the relationship between the decapping activator proteins and the inhibition of decapping imposed by the translation initiation machinery. Methods A useful tool for the analysis of Dcp 1 has been the ability to assay the decapping reaction in vitro. This has allowed an analysis of the effects of various proteins on the activity of Dcplp and has enabled the analysis of the effects of various dcpl mutants on enzymatic activity. A simple in vitro decapping assay utilizing synthetic, cap-labeled substrates and examining the reaction products by thin-layer chromatography has been developed by modifications of the methods of Stevens31 and Zhang et al.32 This assay can be performed with purified Dcp lp, and also proteins that affect decapping such as Dcp2p 15 or elF-4E, 24 thereby assessing their effects on Dcplp activity. In addition, such an assay can be applied to crude extracts, thereby allowing the analysis of decapping in other organisms, 32 or biochemical mutant screens. 25 Below we describe the in vitro decapping assay from the initial purification of Dcplp to the analysis of the reaction products. Methods for each of these procedures have been published previously.4,11,14,31,32 However, we present them here, with modifications to the Dcplp purification protocol, as a convenient reference. P u r i f i c a t i o n of D c p 1 p Dcp lp has been purified from yeast by using a number of different epitope tags. The best yields have been obtained from overexpressing, from the glyceraldehyde phosphate dehydrogenase (GPD) promoter, the Dcplp protein containing a hexahistidine (His6) epitope at its N terminus. The following purification procedure should yield between 1 and 5 mg of highly purified Dcplp. 1. Grow 2 liters of a yeast strain expressing the Dcplp fusion protein from the GPD promoter to an OD600 of 1.0 to 1.5. Pellet the cells and wash once with 30 ml of cold (4 °) doubly distilled H20. Resuspend the washed cell pellet in 6 ml of lysis buffer [50 mM NaPO4 (pH 8.0), 300 mM NaC1, 2 mM 2-mercaptoethanol, 1 tablet
31A. Stevens,Mol. Cell. Biol. 8, 2005 (1988). 32S. Zhang,C. J. Williams,M. Wormington,A. Stevens,and S. W. Peltz,Methods 17, 46 (1999).
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of EDTA-free protease inhibitor cocktail from Roche/Boehringer Mannheim (Indianapolis, IN)]. 2. Add 4.5 ml of acid-treated glass beads. Lyse the cells by vortexing for 30 sec at 4 ° and placing the tube on ice for 30 sec. Repeat this for 10 cycles of vortexing and cooling. 3. Centrifuge the lysate at 4 ° for 20 min at 15000 rpm. Add the supernatant fraction to 1 ml of Ni2+-nitrilotriacetic acid (NTA) agarose that has been equilibrated in lysis buffer. Incubate the slurry at 4 ° for 1 hr with gentle agitation. Load the slurry onto a 15-ml column. Reapply the flow-through to the column and then wash sequentially with 15 ml of buffer 1 (lysis buffer without protease inhibitors), 30 ml of buffer 2 [50 mM NaPO4 (pH 6.0), 1 M NaC1, 5 mM 2-mercaptoethanol], and 10 ml of buffer 3 [50 mM NaPO4 (pH 7.0), 25 mM NaC1, 5 mM 2-mercaptoethanol, 10% (v/v) glycerol]. For the final two washes use 10 ml of buffer 3 containing 50 mM imidazole and then 3 ml of buffer 3 containing 75 mM imidazole. The low concentrations of imidazole in the last two washes help to increase the purity of the preparation by eluting proteins that may bind nonspecifically to Ni 2+. 4. Elute the His6-Dcplp from the column by washing with 4 ml of buffer 3 containing 250 mM imidazole. Finally, because the imidazole that is required for the elution of hexahistidine fusion proteins can interfere with the in vitro decapping assay described below, the purified Dcplp should be dialyzed against 4 liters of 5 mM Tris-HC1 (pH 7.6), 10 mM NaC1, 200 mM dithiothreitol (DTT). This sample may then be concentrated to 500/zl. For storage in aliquots at - 8 0 ° nonidet P-40 (NP-40) and glycerol should then be added to a final concentration of 0.1 and 20% (v/v), respectively. P r e p a r a t i o n o f S u b s t r a t e RNA Any mRNA may be in vitro transcribed and used for a decapping assay because the decapping product is the same for any mRNA. u 1. Transcribe the mRNA by standard in vitro transcription methods. To ensure that all of the mRNA is of uniform length, the RNA should be gel purified in a 6% (w/v) denaturing polyacrylamide gel. By UV shadowing, the full-length mRNA can be observed. The full-length mRNA is excised from the gel and eluted by any standard technique. The eluted mRNA should be phenol-chloroform extracted and ethanol precipitated. 2. Cap the mRNA in vitro, using [a-32p] GTP, a methyl donor for formation of the 7-methyl modification on the cap, and vaccinia virus guanylyltransferase (GibcoBRL, Gaithersburg, MD). Specifically, the capping reaction may be performed as follows: combine 25 pmol of mRNA, 45 pmol of [a-32P]GTE 4.5 #1 of 10x capping buffer [500 mM Tris-HCl (pH 7.7), 20 mM MgCI2, 10 mM DTT, 60 mM KC1], 3 #1 of 10 mM S-adenosylmethionine, 1 /zl (40 units) of rRNasin, 14 tzl
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of doubly distilled H20, and 6/zl (20 units) of guanylyltransferase and incubate the reaction for 2 hr at 37 °. Add 150 #1 of RNase-free TE and remove the unincorporated GTP by purification over a Sephadex G-50 column. To ensure that the capped RNA is not degraded during purification, we often spin the RNA directly into phenol-chloroform preequilibrated in TE and quickly extract the samples. To increase the yield of capped mRNA, wash the column once with 100/zl of TE and collect the elution as described previously in phenol-chloroform. The purity of the substrate can by analyzed by polyethyleneimine (PEI)-cellulose thin-layer chromatography (TLC) (described below) or polyacrylamide gel electrophoresis. I n Vitro D e c a p p i n g A s s a y
The in vitro decapping assay requires three components: a decapping protein, a cap-labeled mRNA substrate, and decapping buffer. This assay monitors the release of the decapping product, r:a7GDP, from the cap-labeled mRNA substrate. The reaction is performed as follows: combine 1.5/zl of 10x decapping buffer [500 mM HEPES (pH 7.0), 10 mM MgC12, 10 mM DTT, 0.5% (v/v) NP-40], 1 #1 of purified Dcplp (100 ng), 1 /zl of capped mRNA (30,000 cpm), and 11.5 #1 of doubly distilled H20. Incubate at 30 ° for 20 min. Stop the reaction by adding 1 #1 of 500 mM EDTA. To ensure a strong signal, spot at least 10,000 cpm onto a PEI--cellulose TLC plate that has been prerun for several minutes (see below). To analyze decapping over a time course (Fig. 2B), remove aliquots of the reaction at A
B
7mGDP PG
'qF'- origin neg HISDep 1p
neg 0' 2'
4' 6'
8' 10'
FIG.2. In vitro decappingassays.(A) A representativedecappingassayusingpurifiedHis6-Dcplp and the decappingprotocoldescribed herein is shown. The His6-Dcplp sampleshowsthe amount of mTGDPreleasedaftera 20-minreaction.Indicatedto the side of the TLC plate for comparisonare the relativemobilitiesof other guaninenucleotidederivatives.(B) A representativedecappingtimecourse is shown. Protein purificationand decappingassays wereperformedas described in Methods.
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the desired times and rapidly stop the reaction by adding EDTA and placing the aliquots on ice. Thin-Layer Chromatography Thin-layer chromatography (TLC) is a relatively rapid and straightforward technique for separating small molecules on the basis of their relative hydrophobicity. To use this technique to analyze the products of a decapping reaction, the PEI-cellulose TLC plates must be prerun in 450 mM ammonium sulfate for 5 min. After the prerunning, the individual decapping reactions are spotted near the bottom of the TLC plate slightly above the running buffer (450 mM ammonium sulfate). The plates are then incubated in the buffer for 2 hr. With PEI-cellulose TLC, more positively charged molecules migrate more rapidly up the TLC plate. This allows the separation of the decapping product from the full-length mRNA, which remains at the origin (Fig. 2A). The relative positions where various other nucleotides run in this type of TLC plate is shown in Fig. 2A. This protocol can be adapted in several ways, depending on the objective of the experiment. For example, to rapidly screen yeast strains for decapping defects, the assay can be performed with 20 to 50 /xg of a crude yeast cell extract. 32 This assay also is applicable to studying the decapping activity of Xenopus laevis oocyte extracts. 32 That cell types other than yeast possess mRNA decapping activity highlights the point that mRNA decapping is a conserved process.
[19] RNA Lariat Debranching Enzyme By SIEW LOON OoI, CHARLESDANN III, KIEBANGNAM, DANIELJ. LEAHY, MASAD J. DAMHA, and JEF D. BOEKE
Introduction Nucleic acids are usually linked by 3'-5' phosphodiester bonds. Interestingly, in both prokaryotes and eukaryotes, there exist low levels of nucleic acids containing 2'-5' phosphodiester bonds. This linkage results in unusual branched nucleic acids that can form either a fork or lariat structure (Fig. 1A and B). Examples of branched RNAs include intron lariats in eukaryotes, 1 Y-like transplicing
1 H. D o m d e y , B. Apostol, R. J. Lin, A. N e w m a n , E. Brody, and J. Abelson, Cell 39, 611 (1984).
METHODSIN ENZYMOLOGY,VOL. 342
Copyright© 2001 by AcademicPress All rights of reproductionin any form reserved. 0076-6879/01$35.00
PROCESSING AND DEGRADAT1VE ENDORIBONUCLEASES
[ 1 8]
[I 8] Yeast mRNA Decapping Enzyme B y TRAVIS DUNCKLEY a n d ROY PARKER
Introduction An important step in gene regulation occurs at the level of mRNA stability (for reviews, see Refs.l-3). In the yeast Saccharomyces cerevisiae mRNAs are degraded through two general pathways. The predominant decay pathway occurs via shortening of the poly(A) tail, followed by removal of the 5' cap structure by the Dcplp decapping enzyme.4 Decapping of the mRNA exposes the body of the message to Xrn 1p-mediated 5'----~3'-exonucleolytic degradation.5- 8 The second general decay pathway occurs by poly(A) shortening followed by 3'-+5'-exonucleolytic degradation of the mRNA. 8'9 Decapping is a key step in the major pathway of 5'---~3' mRNA decay in yeast because removal of the cap structure is a prerequisite to 5'---~3'-exoribonucleolytic degradation of the mRNA. 4-6 Different mRNAs also have message-specific rates of decapping, suggesting that the process of removing the 5' cap structure is controlled.7,8 Moreover, the process of mRNA surveillance, wherein aberrant mRNAs are recognized, occurs by rapid deadenylation-independent decapping.~° A yeast decapping enzyme, encoded by the DCP1 gene, has been identified and shown to be required for all decapping in yeast.4,11 In this article we briefly review the process of mRNA decapping including the properties of Dcplp and the proteins that modulate decapping rates in vivo (for a more comprehensive review of decapping see Tucker and Parker12). In addition, we describe techniques for analyzing mRNA decapping in vitro. Dcplp: The Enzyme Purification of Dcplp and subsequent in vitro characterization of the enzyme have yielded several important observations. 11,13 First, the Dcpl protein I j. Ross, Microbiol. Rev. 59, 423 (1995). 2 G. Caponigro and R. Parker, Microbiol. Rev. 60, 233 (1996). 3 A. Jacobson and S. W. Peltz, Annu. Rev. Biochem. 65, 693 (1996). 4 C. Beelman, A. Stevens, G. Caponigro, T. LaGrandeur, L. Hatfield, D. Fortner, and R. Parker, Nature (London) 283, 642 (1996). 5 C. J. Decker and R. Parker, Genes Dev. 7, 1632 (1993). 6 C. L. Hsu and A. Stevens, Mol. Cell BioL 13, 4826 (1993). 7 D. Muhlrad, C. Decker, and R. Parker, Genes Dev. 8, 855 (1994). 8 D. Muhlrad, C. Decker, and R. Parker, Mol. Cell. BioL 15, 2145 (1995). 9 j. S. Anderson and R. Parker, EMBO J. 17, 1497 (1998). 10 D. Muhlrad and R. Parker, Nature (London) 370, 578 (1994).
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I DCPI (NC 001147.1) Candida albieans (54/66) Arabidopsis (AAC17938.1) (22/51) Drosophila (AAF47089.1) (28/56) Homo Sapiens (CAB77023,1) (26/51)
I
ELS~t3~IllI~N~t~
r , ~ E l l r l g EIl~a'Ual~
FIG. 1. Proteins related to Dcplp. Shown is an alignment with Dcplp and several related proteins in the database. Only the most highly conserved region of the protein is shown. Percentage identity and similarity are indicated in parentheses. Boxed residues denote completely conserved residues that are known to be critical for Dcplp functionJ 4 Lighter shaded boxes indicate similarity and darker boxes indicate identity. Only amino acids conserved between Dcplp and at least two other homologs are shaded. The Candida albicans sequence was obtained from the Candida database (http://alces.med. umn.edu/Candida, html ).
is sufficient for the decapping of mRNAs in vitro. This indicates that Dcplp is the yeast mRNA decapping enzyme. Second, Dcplp cleaves cap structures to the products mVGDP and the full-length mRNA containing a 5'-phosphate. Third, Dcplp preferentially cleaves substrates containing 7-methylguanine over those containing an unmethylated cap. This demonstrates that the 7-methyl modification on the cap structure contributes to the specificity of Dcplp. Fourth, Dcplp shows enhanced decapping activity on longer mRNA substrates. In vitro, Dcplp does not efficiently decap messenger RNAs shorter than 25 nucleotides in length, indicating that Dcp 1p likely recognizes a portion of the mRNA in addition to the 7-methyl modification. Consistent with Dcplp requiring recognition of the mRNA body, the in vitro activity of Dcplp is inhibited by the addition of uncapped mRNA but not by the addition of cap analog, m7GpppG.11 Fifth, the DCP 1 protein requires divalent cation in the form of either Mg 2+ or Mn 2+, with a preference for magnesium, l 1 Sixth, purified Dcplp for model substrates shows a Km of approximately 15 riM.11'13Last, Dcp lp is known to be a phosphoprotein when purified from yeast, although the precise modification and its significance for enzymatic activity are currently unknown. Nevertheless, a requirement for posttranslational modification may explain why Dcplp expressed in Escherichia coli is enzymatically inactive. Because decapping of mRNAs is likely a conserved process it is anticipated that there will be homologs of Dcplp in other eukaryotes. Examination of the available databases identifies a family of proteins that are similar to Dcplp (Fig. 1). Importantly for Dcplp, mutational analyses have demonstrated that altering the residues conserved among this family of proteins has yielded the strongest loss of function alleles in Dcplp (Tharun and Parkerl4; boxed residues in Fig. 1). This 11 T. E. LaGrandeur and R. Parker, EMBO J. 17, 1487 (1998). 12 M. Tucker and R. Parker, Annu. Rev, Biochem. 69, 571 (2000). 13 A. Stevens, Biochem. Biophys. Res. Commun. 96, 1150 (1980). 14 S. Tharun and R. Parker, Genetics 151, 1273 (1999).
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suggests that, although the overall similarity is not high, these proteins may be functional homologs of Dcp lp. Additional Decapping Factors In addition to Dcp Ip, several additional proteins have been shown to affect the process of mRNA decapping (Table I15-26). Among these is the DCP2-encoded protein. The DCP2-encoded protein was identified as a high-copy suppressor of a conditional dcpl mutantJ 5 Subsequent analysis of the dcp2A mutant demonstrated that Dcp2p is absolutely required for decapping of both normal and aberrant mRNAs. Dcp2p physically associates with the Dcplp decapping enzyme and it appears that the interaction between Dcplp and Dcp2p is required for the activation of Dcp I p (Dunckley and Parker 15; and our unpublished observations, 2000). The specific mechanism whereby Dcp2p activates Dcplp is unclear. However, because Dcp2p possesses a functional MutT motif, the Dcp2p-mediated activation of Dcp Ip likely requires cleavage of an as yet unknown pyrophosphate bond (for a review of the MutT motif see Bessman et al., 27 and Koonin2S). The Lsm (like Sin) proteins represent another set of proteins that influence the mRNA decapping rate. There are seven Lsm proteins (Lsml to Lsm7) that affect mRNA decappingJ 6-1s The Lsm proteins are related to the Sm proteins, which are components of small nuclear RNAs (snRNAs) and appear to form a sevenmembered protein complex that binds to mRNA to promote mRNA decappingJ 7,J8 In addition, the Lsm protein complex involved in mRNA decay stably interacts with the PATI/MRT] gene product, which is an additional decapping factor. 17,18,29However, neither the Lsm complex nor Patl/Mrtlp is required for decapping through the mRNA surveillance pathway. Thus, the patl/Mrtl-Lsm complex functions in 15 T. Dunckley and R. Parker, EMBO J. 18, 5411 (1999). t6 R. Boeck, B. Lapeyre, C. E. Brown, and A. B. Sachs, Mol. Cell Biol. 18, 5062 (1998). 17 S. Tharun, W. He, A. E. Mayes, P. Lennertz, J. D. Beggs, and R. Parker, Nature (London) 404, 515 (2000). 18 E. Bouveret, G. Rigaut, A. Shevchenko, M. Wilm, and B. Serapin, EMBOJ. 19, 1661 (2000). J9 S. W. Peltz, A. H. Brown, and A. Jacohson, Genes Dev. 7, 1737 (1993). 20 y. Cui, K. W. Hagan, S. Zhang, and S. W. Peltz, Genes Dev. 9, 423 (1995). 21 E He and A. Jacobson, Genes Dev. 9, 437 (1995). 22 G. Caponigro and R. Parker, Genes Dev. 9, 2421 (1995). 23 D. C. Schwartz and R. Parker, Mol. Cell Biol. 19, 5247 (1999). 24 D. C. Schwartz and R. Parker, submitted (2001). 25 S. Zbang, C. J. Williams, K. Hagan, and S. W. Peltz, biol. Cell, Biol. 19, 7568 (1999). 26 D. Zuk, J. E Belk, and A. Jacobson, Genetics 153, 35 (1999). 27 M. J. Bessman, D. N. Frick, and S. E O'Handley, J. Biol. Chem. 27I, 25059 (1996). 28 E. g. Koonin, NucleieAcids Res. 21, 4847 (1993). 29 L. Hatfield, C. A. Beelman, A. Stevens, and R. Parker, Mol. Cell. Biol. 16, 5830 (1996).
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TABLE I PROTEINS THAT AFFECT m R N A DECAPPING
Gene DCP2 LSM1 - L S M 7 PAT1/MRT1 UPF1, UPF2, UPF3 PAB1 elF-4E VPS16 GRC5, SLA2, MRT4, THS1
Protein function
Refs.
Activates Dcp l p Protein complex that binds mRNA to stimulate decapping Interacts with Lsm complex to stimulate decapping Activates mRNA surveillance Inhibits decapping Binding to cap inhibits mRNA decapping rate Mutation inhibits decapping activity Affects decapping rates
" b c d e
f g h
a Dunckley and Parker (1999). 15 b Boeck e t al. (1998), 16 Tharun et al. (2000), 17 and Bouveret e t al. (2000)J 8 ¢ Tharun et al. (2000) 17 and Bouveret e t al. (2000)) 8 d Peltz et al. (1993), 19 Cui et al. (1995), 2° and He and Jacobson (1995). 2j e Caponigro and Parker (1995). 22 f S c h w a r t z and Parker (1999) 23 and Schwartz and Parker (2000). 24 g Zang e t al. (1999). 25 h Zuk et al. (1999). 26
the deadenylation-dependent decapping pathway to bind to mRNAs to promote decapping by a yet to be determined mechanism. A different set of three proteins functions exclusively to activate decapping through the mRNA surveillance pathway (reviewed in Jacobson and Peltz3). These proteins, referred to as Upfl, Upf2, and Upf3, interact with each other as well as with translation termination factors. 3° This has led to the idea that the Upf protein complex binds to the translation termination machinery and signals a rapid, deadenylation-independent decapping event if translation termination is premature. Translation and Decapping In addition to the activators of decapping discussed above, translation initiation factors are in some cases inhibitors of decapping. For example, the poly(A) tail and the associated poly(A) binding protein inhibit decapping. 22 Similarly, several lines of evidence suggest that there is a competition between the cap binding protein eIF-4E and Dcpl for the cap structure. For example, eIF-4E inhibits decapping in a purified system, z4 Moreover, in vivo mutations in eIP-4E increase the rates of 30 K. Czaplinski, M. J. Ruiz-Echevarria, S. V. Paushkin, X. Han, Y. Weng, H. A. Perlick, H. C. Dietz, M. D. Ter-Avanesyan, and S. W. Peltz, Genes Dev. 12, 1665 (1998).
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[ 18]
decapping and can suppress partial loss of function alleles in Dcplp. 23'24 These observations imply that more highly translated mRNAs are stable, at least in part, because they are bound to initiation factors more frequently than are poorly translated mRNAs, thereby slowing the decapping rate for highly translated mRNAs. An important goal of future work is to understand the relationship between the decapping activator proteins and the inhibition of decapping imposed by the translation initiation machinery. Methods A useful tool for the analysis of Dcp 1 has been the ability to assay the decapping reaction in vitro. This has allowed an analysis of the effects of various proteins on the activity of Dcplp and has enabled the analysis of the effects of various dcpl mutants on enzymatic activity. A simple in vitro decapping assay utilizing synthetic, cap-labeled substrates and examining the reaction products by thin-layer chromatography has been developed by modifications of the methods of Stevens31 and Zhang et al.32 This assay can be performed with purified Dcp lp, and also proteins that affect decapping such as Dcp2p 15 or elF-4E, 24 thereby assessing their effects on Dcplp activity. In addition, such an assay can be applied to crude extracts, thereby allowing the analysis of decapping in other organisms, 32 or biochemical mutant screens. 25 Below we describe the in vitro decapping assay from the initial purification of Dcplp to the analysis of the reaction products. Methods for each of these procedures have been published previously.4,11,14,31,32 However, we present them here, with modifications to the Dcplp purification protocol, as a convenient reference. P u r i f i c a t i o n of D c p 1 p Dcp lp has been purified from yeast by using a number of different epitope tags. The best yields have been obtained from overexpressing, from the glyceraldehyde phosphate dehydrogenase (GPD) promoter, the Dcplp protein containing a hexahistidine (His6) epitope at its N terminus. The following purification procedure should yield between 1 and 5 mg of highly purified Dcplp. 1. Grow 2 liters of a yeast strain expressing the Dcplp fusion protein from the GPD promoter to an OD600 of 1.0 to 1.5. Pellet the cells and wash once with 30 ml of cold (4 °) doubly distilled H20. Resuspend the washed cell pellet in 6 ml of lysis buffer [50 mM NaPO4 (pH 8.0), 300 mM NaC1, 2 mM 2-mercaptoethanol, 1 tablet
31A. Stevens,Mol. Cell. Biol. 8, 2005 (1988). 32S. Zhang,C. J. Williams,M. Wormington,A. Stevens,and S. W. Peltz,Methods 17, 46 (1999).
[ 18]
YEAST mRNA DECAPPINGENZYME
231
of EDTA-free protease inhibitor cocktail from Roche/Boehringer Mannheim (Indianapolis, IN)]. 2. Add 4.5 ml of acid-treated glass beads. Lyse the cells by vortexing for 30 sec at 4 ° and placing the tube on ice for 30 sec. Repeat this for 10 cycles of vortexing and cooling. 3. Centrifuge the lysate at 4 ° for 20 min at 15000 rpm. Add the supernatant fraction to 1 ml of Ni2+-nitrilotriacetic acid (NTA) agarose that has been equilibrated in lysis buffer. Incubate the slurry at 4 ° for 1 hr with gentle agitation. Load the slurry onto a 15-ml column. Reapply the flow-through to the column and then wash sequentially with 15 ml of buffer 1 (lysis buffer without protease inhibitors), 30 ml of buffer 2 [50 mM NaPO4 (pH 6.0), 1 M NaC1, 5 mM 2-mercaptoethanol], and 10 ml of buffer 3 [50 mM NaPO4 (pH 7.0), 25 mM NaC1, 5 mM 2-mercaptoethanol, 10% (v/v) glycerol]. For the final two washes use 10 ml of buffer 3 containing 50 mM imidazole and then 3 ml of buffer 3 containing 75 mM imidazole. The low concentrations of imidazole in the last two washes help to increase the purity of the preparation by eluting proteins that may bind nonspecifically to Ni 2+. 4. Elute the His6-Dcplp from the column by washing with 4 ml of buffer 3 containing 250 mM imidazole. Finally, because the imidazole that is required for the elution of hexahistidine fusion proteins can interfere with the in vitro decapping assay described below, the purified Dcplp should be dialyzed against 4 liters of 5 mM Tris-HC1 (pH 7.6), 10 mM NaC1, 200 mM dithiothreitol (DTT). This sample may then be concentrated to 500/zl. For storage in aliquots at - 8 0 ° nonidet P-40 (NP-40) and glycerol should then be added to a final concentration of 0.1 and 20% (v/v), respectively. P r e p a r a t i o n o f S u b s t r a t e RNA Any mRNA may be in vitro transcribed and used for a decapping assay because the decapping product is the same for any mRNA. u 1. Transcribe the mRNA by standard in vitro transcription methods. To ensure that all of the mRNA is of uniform length, the RNA should be gel purified in a 6% (w/v) denaturing polyacrylamide gel. By UV shadowing, the full-length mRNA can be observed. The full-length mRNA is excised from the gel and eluted by any standard technique. The eluted mRNA should be phenol-chloroform extracted and ethanol precipitated. 2. Cap the mRNA in vitro, using [a-32p] GTP, a methyl donor for formation of the 7-methyl modification on the cap, and vaccinia virus guanylyltransferase (GibcoBRL, Gaithersburg, MD). Specifically, the capping reaction may be performed as follows: combine 25 pmol of mRNA, 45 pmol of [a-32P]GTE 4.5 #1 of 10x capping buffer [500 mM Tris-HCl (pH 7.7), 20 mM MgCI2, 10 mM DTT, 60 mM KC1], 3 #1 of 10 mM S-adenosylmethionine, 1 /zl (40 units) of rRNasin, 14 tzl
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[18]
of doubly distilled H20, and 6/zl (20 units) of guanylyltransferase and incubate the reaction for 2 hr at 37 °. Add 150 #1 of RNase-free TE and remove the unincorporated GTP by purification over a Sephadex G-50 column. To ensure that the capped RNA is not degraded during purification, we often spin the RNA directly into phenol-chloroform preequilibrated in TE and quickly extract the samples. To increase the yield of capped mRNA, wash the column once with 100/zl of TE and collect the elution as described previously in phenol-chloroform. The purity of the substrate can by analyzed by polyethyleneimine (PEI)-cellulose thin-layer chromatography (TLC) (described below) or polyacrylamide gel electrophoresis. I n Vitro D e c a p p i n g A s s a y
The in vitro decapping assay requires three components: a decapping protein, a cap-labeled mRNA substrate, and decapping buffer. This assay monitors the release of the decapping product, r:a7GDP, from the cap-labeled mRNA substrate. The reaction is performed as follows: combine 1.5/zl of 10x decapping buffer [500 mM HEPES (pH 7.0), 10 mM MgC12, 10 mM DTT, 0.5% (v/v) NP-40], 1 #1 of purified Dcplp (100 ng), 1 /zl of capped mRNA (30,000 cpm), and 11.5 #1 of doubly distilled H20. Incubate at 30 ° for 20 min. Stop the reaction by adding 1 #1 of 500 mM EDTA. To ensure a strong signal, spot at least 10,000 cpm onto a PEI--cellulose TLC plate that has been prerun for several minutes (see below). To analyze decapping over a time course (Fig. 2B), remove aliquots of the reaction at A
B
7mGDP PG
'qF'- origin neg HISDep 1p
neg 0' 2'
4' 6'
8' 10'
FIG.2. In vitro decappingassays.(A) A representativedecappingassayusingpurifiedHis6-Dcplp and the decappingprotocoldescribed herein is shown. The His6-Dcplp sampleshowsthe amount of mTGDPreleasedaftera 20-minreaction.Indicatedto the side of the TLC plate for comparisonare the relativemobilitiesof other guaninenucleotidederivatives.(B) A representativedecappingtimecourse is shown. Protein purificationand decappingassays wereperformedas described in Methods.
[19]
RNA LARIATDEBRANCHINGENZYME
233
the desired times and rapidly stop the reaction by adding EDTA and placing the aliquots on ice. Thin-Layer Chromatography Thin-layer chromatography (TLC) is a relatively rapid and straightforward technique for separating small molecules on the basis of their relative hydrophobicity. To use this technique to analyze the products of a decapping reaction, the PEI-cellulose TLC plates must be prerun in 450 mM ammonium sulfate for 5 min. After the prerunning, the individual decapping reactions are spotted near the bottom of the TLC plate slightly above the running buffer (450 mM ammonium sulfate). The plates are then incubated in the buffer for 2 hr. With PEI-cellulose TLC, more positively charged molecules migrate more rapidly up the TLC plate. This allows the separation of the decapping product from the full-length mRNA, which remains at the origin (Fig. 2A). The relative positions where various other nucleotides run in this type of TLC plate is shown in Fig. 2A. This protocol can be adapted in several ways, depending on the objective of the experiment. For example, to rapidly screen yeast strains for decapping defects, the assay can be performed with 20 to 50 /xg of a crude yeast cell extract. 32 This assay also is applicable to studying the decapping activity of Xenopus laevis oocyte extracts. 32 That cell types other than yeast possess mRNA decapping activity highlights the point that mRNA decapping is a conserved process.
[19] RNA Lariat Debranching Enzyme By SIEW LOON OoI, CHARLESDANN III, KIEBANGNAM, DANIELJ. LEAHY, MASAD J. DAMHA, and JEF D. BOEKE
Introduction Nucleic acids are usually linked by 3'-5' phosphodiester bonds. Interestingly, in both prokaryotes and eukaryotes, there exist low levels of nucleic acids containing 2'-5' phosphodiester bonds. This linkage results in unusual branched nucleic acids that can form either a fork or lariat structure (Fig. 1A and B). Examples of branched RNAs include intron lariats in eukaryotes, 1 Y-like transplicing
1 H. D o m d e y , B. Apostol, R. J. Lin, A. N e w m a n , E. Brody, and J. Abelson, Cell 39, 611 (1984).
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