Drosophila 5′ → 3′-Exoribonuclease Pacman

Drosophila 5' --~ 3'-EXORIBONUCLEASEPacman

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B y IGOR V. CHERNUKHIN, JULIAN E. SEAGO, a n d SARAH F. NEWBURY

Introduction Early development in multicellular organisms is programmed by maternal gene products that are transported into the egg before fertilization. The initial coordinates of the body plan in the fertilized embryo are entirely dependent on maternal products, as no zygotic transcription occurs. Therefore up to a particular stage of development (cellularization in Drosophila, 2.5 hr after fertilization), the developmental program is entirely dependent on posttranscriptional control, in particular RNA localization, translation, and RNA stability. Of these three controlling events, the least is known about the control of RNA stability. Analysis of early development in Drosophila has shown that RNA localization, control of translation, and mRNA stability are intimately linked.l-3 Generally, translational repression leads to degradation of an RNA, and failure of an RNA to localize correctly also leads to its degradation. For example, the binding of Smaug protein to unlocalized nanos RNA causes it to be translationally repressed and then degraded. 4 After the initial coordinates of the developing embryo have been determined, the RNAs responsible for early development are specifically degraded and embryonic transcription commences. Work on the yeast Saccharomyces cerevisiae has identified many ribonucleases and associated factors that control mRNA decay, RNA splicing, and rRNA processing. In yeast, it has been shown that 3' --+ 5' degradation/processing of RNA requires the exosome, which is a complex of at least 10 proteins. 5"6 Degradation of RNA in a 5' ~ 3' direction occurs by initial decapping of the mRNA, followed by 5' --+ 3' degradation of the RNA by Xrnlp. The two decapping proteins (Dcplp and Dcp2p) and Xrnlp have been shown to be complexed to seven Lsm proteins, which are likely to form a ring encircling the RNA 7-1° Because many of these I D. Curtis, R. Lehmann, and E D. Zamore, Cell 81, 171 (1995). 2 A. Jacobsen and S. Pelz, Annu. Rev. Biochem. 65, 693 (1996). 3 E M. Macdonald and C. A. Smibert, Curr. Opin. Genet. Dev. 6, 403 (1996). 4 C. A. Smibert, J. E. Wilson, K. Kerr, and E M. Macdonald, Genes Dev. 10, 2600 (1996). 5 C. Allmang, E. Petfalski, A. Podtelejnikov, M. Mann, D. Tollervey, and E Mitchell, Genes Dev. 13, 2148 (1999). 6 p. Mitchell, E. Petfalski, A. Shevchenko, M. Mann, and D. Tollervey, Cell 91, 457 (1997). 7 Z. Dunckley and R. Parker, EMBO J. 18, 5411 (1999). 8 C. A. Beelman, A. Stevens, G. Camponigro, T. E. LaGrandeur, L. Hatfield, D. M. Fortner, and R. Parker, Nature (London) 382, 642 (1996). 9 S. Tharun, W. He, A. E. Mayes, P. Lennertz, J. D. Beggs, and R. Parker, Nature (London) 404, 515 (2000).

METHODSIN ENZYMOLOGY,VOL.342

Copyright© 2001 by AcademicPress All rightsof reproductionin anyformreserved. 0076-6879/01$35.00

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proteins are conserved in higher eukaryotes, it is likely that the mechanism of RNA processing/degradation is similar, although components of the complexes may vary between species. To understand the role of RNA stability in development, a number of approaches can be used. Once the genes encoding particular ribonucleases or associated factors have been identified, then expression of the RNA during development can be determined. In addition, a portion of the protein can be expressed and antibodies raised so that the spatial and temporal distribution of the encoded protein can be determined. We have used these techniques to show that the 5' ~ 3'exoribonuclease Pacman is differentially expressed during development. II In a genetically tractable organism such as Drosophila, it is then often possible to identify a mutation in the gene of interest (FlyBase, http://flybase.bio.indiana.edu, or http://fly.ebi.ac.uk:7081/). Alternatively, the gene of interest can be ectopically expressed at particular developmental stages or in different tissues and the consequences of misexpression analyzed.12 Biochemical methods provide an alternative and complementary approach to the understanding of the mechanisms of action of these enzymes. The mechanisms whereby ribonucleases target and degrade RNA are not well understood and the way in which these enzymes may interact with each other or with other components of the cell is not at all clear. Biochemical characterization of these enzymes is essential in order to determine their active sites, the regions of the proteins that recognize RNA, and the domains of the proteins that interact with each other. The understanding of the ways in which these ribonucleases interact with each other and their target RNAs will not only shed light on the mechanism of gene regulation but is also likely to be crucial in understanding the link between translation, localization, and RNA stability. This chapter concentrates on the methods we have used to express a Drosophila recombinat 5' ~ 3'-exoribonuclease, purify the protein, and analyze its activity in vitro. We offer these protocols as a starting point for investigators wishing to perform similar experiments on similar proteins from higher organisms. Identification of Ribonucleases

and Associated Factors

A number of approaches can be used to identify ribonucleases and associated factors in multicellular organisms. For example, if a particular region of RNA is known to be necessary and sufficient to promote its own degradation, 13 then this

JOE. Bouveret, G. Rigaut, A. Shevchenko,M. Wilm, and B. Seraphin, EMBOJ. 19, 1661 (2000). Il D. D. Till, B. Linz, J. E. Seago, S. J. Elgar, P. E. Marujo, M. Elias, J. A. McClellan, C. M. Arraiano, J. E. G. McCarthy,and S. E Newbury,Mech. Dev. 79, 51 (1998). 12p. p. D'Avinoand C. S. Thummel, Methods Enzymol. 194, 565 (1999). 13A. Bashirullah, S. R. Halsell, R. L. Cooperstock, M. Kloc, A. Karaiskakis, W. W. Fisher, W. Fu, J. K. Hamilton, L. D. Etkin, and H. D. Lipshitz, EMBOZ 18, 2610 (1999).

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RNA sequence can be used as a "bait" in cross-linking analysis to identify interaction proteins. However, we have used the "candidate gene" approach, which has proved fruitful with the increasing information available through genomic databases and also because of the evolutionary conservation between ribonucleases. This approach is particularly useful where genetic screening would be difficult because the genes involved have pleiotropic effects and/or are functionally redundant. Many ribonucleases or ribonuclease motifs show evolutionary conservation from the bacteria Escherichia coli through to humans. In a remarkable database analysis, where the amino acid sequences of prokaryotic ribonucleases such as RNase II were used to identify similar genes in other organisms, many 3' ~ 5'ribonucleases were identified or predicated in a range of eukaryotes. 14 However, for some ribonucleases, such as RRP4, homologs are not found in prokaryotes but are nevertheless well conserved from yeast to humans. 6 This conservation means that identifying ribonucleases in the organism of choice (where sequence data are available) is comparatively straightforward. In our experience it is advisable to search using conserved motifs and avoid regions with repeated motifs [such as tetratricopeptide repeat (TPR) motifs]. Searching the Drosophila database not only reveals the genomic location of the gene of interest, but also the expressed sequence tags (ESTs) at that location. ESTs are sequences at the 5' ends of cDNA clones: now that the genomic sequence of Drosophila has been completed they have been mapped to their genomic locations. The sequences of these cDNA clones have been arranged in groups (clots) and care must be taken to try to identify the longest EST as many are truncated at either the 5' or 3' end. Usually, the homology between ribonucleases is such that the full-length clone can be identified from sequence comparison with, for example, the human homolog. This cDNA can then be obtained from Research Genetics (Huntsville, AL; http://www.resgen.com). If the full-length EST is not available then it may be necessary to experimentally identify the 5' end of the gene by primer extension or similar techniques. E x p r e s s i o n a n d P u r i f i c a t i o n of P a c m a n

Subcloning and Expression In vitro characterization of the S. cerevisiae 5' --+ 3'-exoribonuclease Xrnlp and the mouse homolog mXrnlp has usually been performed by overexpression of the protein in yeast and then purification by biochemical methods.15-17 However, we wished to express the entire Drosophila 5' --+ 3'-exoribonuclease Pacman in the bacteria E. coli and purify it by affinity chromatography. This method has the 14 |. S. Milan, Nucleic Acids Res. 25, 3187 (1997). 15 A. Johnson and R. D. Kolodner, J. Biol. Chem. 266, 14046 (1991). 16 A. Stevens, J. Biol. Chem. 255, 3080 (1980). 17 V. 1. Bashkirov, J. A. Solinger, and W. D. Heyer, Chromosoma 104, 215 (1995).

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advantage that it allows rapid purification of protein and that the same method can be applied to the preparation of truncated or otherwise mutated proteins for further analysis or for raising antibodies. A number of plasmid vectors have been constructed to express eukaryotic proteins of interest in bacteria. The vector used should be a multicopy plasmid and include a selectable marker, a "tag" for purification [typically a hexahistidine (His6) tag], and a promoter that can be induced when required. The latter feature is particularly useful when the overexpression of the protein of interest is toxic to the bacterial cell. We have used the His6 tag vector pET28a (Novagen, Madison, WI), which has a T71ac promoter that can be induced by isopropyl/%D-thiogalactopyranoside (IPTG). The vector also carries the natural promoter and coding sequence of the lac repressor (lacl), orientated so that the T7lac and lacI promoters diverge. When this type of promoter is used in DE3 lysogens to express target genes, the lac repressor acts both at the lacUV5 promoter in the host chromosome to repress transcription of the T7 RNA polymerase gene by the host polymerase and at the T7lac promoter in the vector to block transcription of the target gene by any T7 RNA polymerase that may be made. Therefore, in the absence of IPTG, expression from the plasmid is repressed. In the pET series of vectors, it is typically arranged so that the cDNA is subcloned into the vector, in-frame, as an NdeI-NotI fragment. It is usually necessary to introduce a suitable NdeI site at the start codon by polymerase chain reaction (PCR). Because of the large size of the pacman cDNA, we cloned pacman into pET28b by a three-way ligation (i.e., a 1300-nucleotide NdeI-NotI fragment generated by PCR, a 3400-bp HindIII-NotI fragment, and the vector cut with NdeI-NotI) to reduce the possibility of introducing mutations by PCR. In our experience, inserts of this size in an E. coli plasmid can be unstable and it is prudent to check the plasmid by sequencing and restriction enzyme analysis before proceeding further. It is also easier to transform the initial ligation mix into a host strain such as JM109, DH5ct, or HB 101 before proceeding to transform the correct construct into the expression host BL21 (DE3).

Expression of Protein in Escherichia coli BL21(DE3 ) The best way to determine the fight conditions for protein expression is to perform all the procedures in mini- or midiscale. We found that full-length Pacman protein expression could be sensitive to conditions such as the time for culturing cells before and after induction, and the concentration of the inducer. The more truncated (smaller) forms of protein occurred along with protein degradation when the cell culture was grown for grown too long or with an increased concentration of IPTG. 1. Inoculate a 5-ml culture of LB selective medium from a BL21(DE3) colony carrying the correct construct and grow at 37 ° overnight. 2. Inoculate 500 ml of LB containing the appropriate antibiotic with 5 ml of the overnight culture. Incubate for 3 hr at 37 ° on an orbital shaker.

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3. Collect a 10-ml aliquot of cells (noninduced sample). 4. A d d IPTG at concentration of 0.5 m M (chosen experimentally), add 0.1 m M phenylmethylsulfonyl fluoride (PMSF), and incubate the culture for a further 2 hr. 5. Centrifuge the cells at 5000 rpm for 5 min at 4 ° and wash the pellet with phosphate-buffered saline (PBS) containing 1 m M P M S E 6. Freeze the cell pellet at - 2 0 ° . 7. Lyse the cells by adding buffer A [20 m M HEPES-KOH (pH 7.0), 3 M urea, 0.5 M NaCI, 10 m M 2-mercaptoethanol, 10-20 m M imidazole (concentration determined experimentally), cooled to 4 ° ] directly to the frozen pellet and vortex it until the cells are completely resuspended. Freeze the lysate and store overnight at - 2 0 ° . 8. Check an aliquot of the cell lysate samples collected from induced and noninduced cultures for protein expression by electrophoreses on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and staining with Coomassie blue or by Western blotting (see below) with an antibody against Pacman protein or a monoclonal antibody against the His6 tag portion of the protein (Sigma, St. Louis, MO) (see Fig. 1A).

C 10 11

FIG. 1. (A) Detection of Pacman protein in bacterial and Drosophila extracts. Lane 1, molecular mass marker (in kilodaltons); lane 2, total bacterial lysate from E. coli expressing full-length Pacman, stained with Coomassie blue. Full-length protein (184 kDa) is marked with an arrow; lane 3, Western blot of total bacterial lysate, probed with the Pacman antibody. Control experiments show that other bands are cleavage products (Pacman protein is susceptibleto cleavage by endogenous proteases). (B) Lanes 4-8, fractions collected from an $200 gel-filtration column. The eluted proteins were stained with Coomassie blue. Pacman protein is marked with an arrow; lane 9, molecular mass marker (in kilodaltons); (C) gel showing the purified Pacman that was used in the band-shift assays; lane 10, Purified protein stained with Coomassie blue; lane 11, molecular mass marker (in kilodaltons).

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Protein Purification on Hexahistidine Tag Column 1. Charge the His6 tag medium (Novagen) with Ni 2+ according to the manufacturer instructions and prepare a 2 x 3 cm column. 2. Equilibrate the column with buffer A [20 mM HEPES-KOH (pH 7.0), 3 M urea, 0.5 M NaC1, 10 mM 2-mercaptoethanol, 10-20 mM imidazole (optimum concentration determined experimentally)]. Apply the cell lysate to the column at a flow rate of 1 ml/min. Wash the column with buffer A untill no protein can be detected in the flowthrough. 3. Elute the affinity-bound proteins with a gradient of 0.02-0.5 M imidazole. Collect the fractions and take aliquots. Check for the presence of Pacman protein by electrophoresis on a SDS-polyacrylamide gel with subsequent staining with Coomassie blue or by Western blotting.

S-200 gel Filtration Further purification of the protein is performed by Sephacryl S-200 HR gel filtration on a 75 × 2.5 cm column equilibrated with buffer B [20 mM HEPES-KOH (pH 7.0), 0.5 M KC1, 10% (v/v) glycerol, 20 mM 2 mercaptoethanol, 2 mM EDTA, 1 mM PMSF]. 1. After Ni 2+ chromatography, load the fraction containing Pacman protein onto the column. 2. Perform the column chromatography at a flow rate of 0.5 ml/min. Collect 5-ml fractions and analyze aliquots of these for the presence of protein, as described for His6 tag chromatography (see Fig. 1B and C). 3. Dialyze the fractions containing Pacman protein against storage buffer [20 mM HEPES-KOH (pH 7.0), 0.3 M KC1, 40% (v/v) glycerol, 2 mM EDTA, 20 rnM 2-mercaptoethanol]. If necessary, concentrate the protein by standard procedures. 4. Make small aliquots of the dialyzed protein solution. The protein can be stored at - 2 0 ° for short-term storage or at - 7 0 ° for long-term storage. In our hands, full-length Pacman protein is stable and functional after purification on the His6 tag column. However, after subesequent purification it tends to become cleaved to give a 115-kDa protein that is, however, functionally active. It is possible that Pacman protein is complexed during its initial purification to cellular factors that stabilize Pacman against cleavage activity and that these are removed in subsequent purification steps. D e t e c t i o n of N u c l e i c Acid B i n d i n g a n d E x o n u c l e a s e Activity The advantage of this method is that it gives relatively quick and easy purification of the protein using an organism (E. coli) that is familiar to most molecular

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biologists. The potential disadvantage is that the purified protein may not have exonuclease activity because it is not correctly modified posttranslationally. The activity of the homologous protein S. cerevisiae Xrulp is often measured by filter binding assays. 15,18However, we chose to measure activity by band-shift analysis, as binding and exonuclease digestion of nucleic acids can then be readily visualised and quantified. Because the active 5' --+ 3'-exoribonuclease will rapidly degrade labeled nucleic acids, it is necessary to stall the processive nuclease so that the product can be easily detected. In a number of experiments, it has been shown that yeast Xrn lp is stalled by poly(G) tracts in vivo and in vitro.19'2° In addition, mouse Xrnlp and S. cerevisiae Xrnlp have been shown to apparently bind to G4 tetraplex, probably because the nuclease is stalled by the stable G-quartet s t r u c t u r e . 21'22 Because mouse Xrnlp is highly homologous to Pacman, we reasoned that G4 tetraplex would provide a suitable target nucleic acid for the activity assays. It is likely that this target would be a suitable substrate for analysis of the activity of many eukaryotic processive ribonucleases. Preparation of G4 Tetraplex 1. Use DNA or RNA oligonucleotides that have previously been shown to form a G4-tetraplex structure in vitro. 21-23 We have used the oligonucleotide 5'-TATGGGGGAGCTGGGGAAGGTGGGATTT-3' and the oligonucleotide 5'TGGACCAGACCTAGCA-3' as competitors. 2. Dissolve the G4 oligonucleotides in 10/zl of TE to 0.5-100/zM. Heat to 90 ° for 60 sec, and then chill on ice and spin briefly. Add 10/zl of TE plus 2 M KC1. In our experience the G4 tetraplex forms spontaneously on the addition of this buffer. 3. Electrophorese the labeled G4 tetraplex on a 1× TBE (50 mM Tris, 50 mM boric acid, 1 mMEDTA)-6% (w/v) polyacrylamide gel. Detect the labeled tetraplex by staining with ethidium bromide and remove the slice containing the tetraplex with a scalpel blade. Crush the gel slice in 3 volumes of elution buffer (0.5 M ammonium acetate, 1 mM EDTA, pH 8.0) and incubate overnight at 37 °. Centrifuge at 10,000g for 10 min at 4 °, remove the supernatant, chloroform extract the DNA, and then concentrate it by ethanol precipitation. 4. Label the oligonucleotide at the 5' end. Add the following to a sterile microcentrifuge tube: Nuclease-free water Kinase buffer (10x; New England BioLabs, Beverly, MA)

~32/zl 5 #1

18 j. A. Solinger, D. Pascolini, and W.-D. Heyer, Mol. Cell. Biol. 19, 5930 (1999). 19 D. Muhlrad, C. J. Decker, and R. Parker, Genes Dev. 8, 855 (1994). 20 T. L. Poole and A. Stevens, Biochem. Biophys. Res. Commun. 235, 799 (1997). 21 V. I. Bashkirov, H. Scherthan, J. A. Solinger, J.-M. Buerstedde, and W. D. Heyer, J. Cell BioL 136, 761 (1997). 22 Z. Liu and W. Gilbert, Cell 77, 1083 (1994).

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G4 tetraplex

2

3

4

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5

P

ss oligo

m

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FIG.2. Purificationof the G4tetraplex. Lanes 1 and 2, annealed 5'-end-labeled G4oligonucleotides prior to gel purification; lanes 4 and 5, 5'-end-labeled G4 tetraplex after gel purification; lanes 5, molecular weight markers (in nucleotides). G4 oligonucleotides [y_32p) ATE 6000 Ci/mmol, 10 Ci/#l (Amersham Pharmacia, Piscataway, NJ) T4 polynucleotide kinase (New England BioLabs) Final volume

10 ng-1 # g ) 5/zl 3/zl 50/zl

5. Vortex the solution gently before incubation at 37 ° for 30 min. Remove the unincorporative radionucleotides from the labeled nucleic acids with a Sephadex G-50 spin column (Amersham Pharmacia) or similar system according to the manufacturer instructions. Check that the G4 oligonucleotides have formed a higher order structure by gel electrophoresis (Fig. 2). Alternatively, the G4 oligonucleotides may be labeled first and then annealed to give the G-quartet structure. 23 23 D. Sen and W. Gilbert, Methods Enzymol. 211, 191 (1992).

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G40ligonucleotide-Binding Reaction

1. The binding reaction is performed in a total volume of 20/~1. Add purified Pacman protein in a volume of 1-5/zl (~5-25 ng) to a mixture containing binding buffer [20 mM HEPES (pH 7.5) 100 mM KC1, 10% (v/v) [glycerol] 1 pM competitor oligonucleotide (5'-TGGACCAGACCTAGCA-3'), 1 /zg of poly(dI-dC), and 200 cpm of Y-end-labeled G4 tetraplex oligonucleotides. 2. Mix the reagents gently and then centrifuge briefly at 10,000g for 5 sec at 4 °. 3. Incubate at 4 ° for 30 min. 4. Electrophorese the reaction products on a 6% (w/v) polyacrylamide gel buffered with lx TBE. 5. After electrophoresis is complete, dry the gel on a gel dryer and visualize the band shifts by autoradiography. Nuclease Assay

Incubate the G4 tetraplex with the enzyme as described as above, except that the binding buffer is supplemented with 3 mM MgC12 and the reaction is carried out at 25 ° for 30 min. We have used the above-described method to show the full-length 184-kDa Pacman protein, purified from E. coli as described above, has both binding and exonuclease activity in vitro (Fig. 3). We have also used Ni 2+ chelate and ion-exchange chromatography to purify the N-terminal and C-terminal portions of the Pacman protein to 90% homogeneity for use in our functional assays. Conclusions and Prospects This chapter has focused on a method for rapid purification and analysis of the Drosophila ribonuclease pacman. The method given is likely to be generally applicable to the purification of other ribonucleases. By analysis of the activity of "wild-type" and "mutant" proteins in vitro, it should be possible to determine their mechanisms of action, their active domains, and perhaps to reconstitute an active "RNA degradation" complex in vitro. It will also be possible to determine the specificity of these ribonucleases for particular RNA sequences or secondary structures. In addition, it is now thought that RNA interference (RNAi), where double-stranded RNAs introduced into the cell promote repression of the homologous RNA, is achieved through specific degradation events, 24 perhaps involving one or more of the 5' --> 3'- or 3'---> 5'-ribonuclease complexes. An understanding of the mechanisms whereby interfering RNA promotes specific degradation of its target RNA may be possible by biochemical analysis in vitro. Biochemical work

24j. M. Bosherand M. Labouesse,Nat. Cell Biol. 2, E31 (2000).

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' - Mg2+ " + Mg2+'

pacrnan protein

G4 tetraplex

cleavage product

FIG. 3. Pacman protein binds to, and cleaves, G4 tetraplex in the presence of magnesium ions. Full-length Pacman protein (184 kDa) was expressed in E. coli and purified with the His6 tag system. Increasing amounts of protein were then incubated with (34 tetraplex that had been labeled at the 5' end. C, Control with no protein.

will also facilitate future structural analysis of the protein. In a few cases, progress has already been made in defining the catalytic domains of these RNA-binding proteins and their interactions with particular RNAs. For example, the yeast protein Xrnlp is known to have acidic N-terminal domains that are likely to contain the exonuclease, and Xmlp has also been shown to be stalled by stable stem-loop s t r u c t u r e s . 17,18,20, 25,26

While the biochemical analysis of ribonucleases is likely to provide new information about their mechanisms of action, it is a particularly powerful approach when carried out in combination with genetic experiments. For example, analysis of mutant proteins generated in vivo can provide valuable information about the functions of these proteins. 26 Therefore these experiments, particularly in combination with genetic experiments, are likely to shed light on one of the least understood mechanisms of gene expression in eukaryotes. 25 A. Holler, I. Bashkirov, J. A. Solinger, U. Reinhart, and W.-D. Heyer, Eur. J. Biochem. 231, 329 (1995). 26 A. M. Page, K. Davis, C. Molineux, R. D. Kolodner, and A. W. Johnson, Nucleic Acids Res. 26, 3707 (1998).