Eva Brázdová Jagelská

Václav Brázda

Petr Pečinka

Emil Paleček

Miroslav Fojta

DNA TOPOLOGY INFLUENCES P53 SEQUENCE-SPECIFIC DNA BINDING THROUGH STRUCTURAL TRANSITIONS WITHIN THE TARGET SITES

Keywords: scDNA, supercoiled DNA, lin, linear DNA, o, oligodeoxynucleotide, SK, plasmid pBluescript p53 protein / DNA binding / protein-DNA complex

Tumor suppressor protein p53 is one of the most important factors regulating cell proliferation, differentiation and programmed cell death in response to a variety of cellular stress signals. The p53 is a nuclear phosphoprotein, and its biochemical function is closely associated with its ability to bind DNA in a sequence-specific manner and operate as a transcription factor. Using a competition assay, we investigated the effect of DNA topology on the DNA binding of human wild type p53 protein. We prepared sets of topoisomers of plasmid DNA with and without p53 target sequences differing in their internal symmetry. Binding of p53 to any DNA increased with increasing negative superhelix density (-σ). At -σ ≤ 0.03, the relative effect of DNA supercoiling on protein-DNA binding was similar for DNAs bearing both symmetrical and non-symmetrical target sites. On the other hand, at higher -σ target sites with a perfect inverted repeat sequence exhibited a more significant enhancement of p53 binding due to increasing levels of negative DNA supercoiling.

For -σ = 0.07, about a three-fold additional increase of binding was observed for a symmetrical target site, compared to a non-symmetrical target. The p53 target sequences possessing the inverted-repeat symmetry were shown to form cruciform structure in sufficiently negative scDNA. We show that formation of the cruciforms in DNA topoisomers at -σ ≥ 0.05 correlates with the extra enhancement of p53-DNA binding.

INTRODUCTION

The p53 tumor suppressor protein is an important transcription factor that induces cell cycle arrest or apoptosis in response to oncogenic transformation and DNA damage. Point mutations and deletions in the p53 gene are the most frequently observed alterations in human cancers [START_REF] Hainaut | IARC Database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualisation tools[END_REF]. The central role of p53 as a tumor suppressor protein has led several studies to explore the possibility of restoring p53 function for therapeutic benefit [START_REF] Hupp | Strategies for manipulating the p53 pathway in the treatment of human cancer[END_REF][START_REF] Baroni | A global suppressor motif for p53 cancer mutants[END_REF][START_REF] Brazda | Restoring wild-type conformation and DNA-binding activity of mutant p53 is insufficient for restoration of transcriptional activity[END_REF]. Interaction with DNA in a sequence-specific manner is crucial for its function. The consensus sequence for p53 binding sites consists of two copies of the motif 5`-RRRC(A/T)(T/A)GYYY-3`, separated by 0 to 13 bp [START_REF] El-Deiry | Definition of a consensus binding site for p53[END_REF]. Surprisingly, some sequences perfectly matching the p53 consensus were found not to bind p53. A comprehensive list of known p53 targets can be found in [START_REF] Qian | Groups of p53 target genes involved in specific p53 downstream effects cluster into different classes of DNA binding sites[END_REF][START_REF] Wei | A global map of p53 transcription-factor binding sites in the human genome[END_REF]. While the basic features of p53-DNA binding are known, it is still poorly understood which parameters determine the selectivity of these interactions. Wildtype p53 possesses the ability to linearly diffuse along DNA to find the target sequence [START_REF] Mckinney | p53 linear diffusion along DNA requires its C terminus[END_REF][START_REF] Brazda | Searching for target sequences by p53 protein is influenced by DNA length[END_REF]. It was proposed that non sequence-specific binding of the p53 protein plays an important role in activation of sequence-specific binding. A family of specific structural elements recognized by p53 has been established, including base mismatches [START_REF] Degtyareva | Analysis of the binding of p53 to DNAs containing mismatched and bulged bases[END_REF], Holliday junction and cruciform structures [11], bent DNA [12,[START_REF] Nagaich | DNA bending is essential for the site-specific recognition of DNA response elements by the DNA binding domain of the tumor suppressor protein p53[END_REF],

and structurally flexible MAR/SAR DNA [START_REF] Deppert | Binding of MAR-DNA elements by mutant p53: possible implications for its oncogenic functions[END_REF]. The p53 protein binds preferentially to supercoiled (sc) DNA even in the absence of the target sequence in the scDNA molecule [15][START_REF] Palecek | Binding of p53 and its core domain to supercoiled DNA[END_REF][START_REF] Brazda | Specific modulation of p53 binding to consensus sequence within supercoiled DNA by monoclonal antibodies[END_REF]. Many p53 target sequences display an internal symmetry, being capable of assuming non-B DNA conformation [START_REF] Kim | DNA-conformation is an important determinant of sequence-specific DNA binding by tumor suppressor p53[END_REF]. These structures are known to be stabilized by negative DNA supercoiling [START_REF] Palecek | Local supercoil-stabilized DNA structures[END_REF]. Contemporary analysis of the symmetry of natural p53 target sequences showed a strong palindrome coupling between the p53 half-sites [START_REF] Ma | Sequence analysis of p53 response-elements suggests multiple binding modes of the p53 tetramer to DNA targets[END_REF]. The target sites can be very often described as two inverted repeats and the two half-sites are highly repetitive. The symmetric nature of the p53 target sequence corresponds to the symmetric binding of the p53 tetramer [START_REF] Kitayner | Structural basis of DNA recognition by p53 tetramers[END_REF]. Recent data provide strong evidence that the DNA binding activity of p53 is remarkably dependent on structural features within the target. In addition to DNA bending [START_REF] Nagaich | DNA bending is essential for the site-specific recognition of DNA response elements by the DNA binding domain of the tumor suppressor protein p53[END_REF], formation of stem-loop or cruciform structure can enhance p53 DNA binding [START_REF] Gohler | Specific interaction of p53 with target binding sites is determined by DNA Biochemical Journal Immediate Publication[END_REF]23]. Importantly, p53 had a dramatically greater affinity for a DNA microcircle bearing a target site than for the same but linear DNA [12]. The p53 protein has also been shown to co-operate in DNA binding with other proteins recognizing and/or modulating the DNA structure, such as the cruciform binding protein HMGB1, topoisomerases and various proteins of chromatin remodeling complexes [START_REF] Jayaraman | High mobility group protein-1 (HMG-1) is a unique activator of p53[END_REF][START_REF] Lee | SWI/SNF complex interacts with tumor suppressor p53 and is necessary for the activation of p53-mediated transcription[END_REF]. HMGB1 selectively recognizes cruciform DNA [START_REF] Bianchi | Specific recognition of cruciform DNA by nuclear protein HMG1[END_REF] and is capable of interacting with DNA in a non-sequence-specific manner, causing a significant bending of the DNA double helix and acting as an activator of p53 sequence specific DNA binding [START_REF] Jayaraman | High mobility group protein-1 (HMG-1) is a unique activator of p53[END_REF]. The role of DNA superhelicity-induced cruciform extrusion as an event stimulating p53 sequence-specific DNA binding has been proposed [START_REF] Palecek | Enhancement of p53 sequence-specific binding by DNA supercoiling[END_REF] but has not been clearly demonstrated at the level of structural transitions of a particular target sequence upon changes of the DNA topology.

Here, we studied p53 binding to topologically constrained DNAs containing internally symmetrical or non-symmetrical target sites. Using a competition assay, we demonstrate a considerable correlation between the change of negative superhelix density, cruciform extrusion within the p53 target sites, and a remarkable extra enhancement of p53 DNA binding. The p53 targets having adopted cruciform structures always exhibited stronger p53 binding than p53 target sites not forming the cruciform or a cruciform formed within a p53 non-target sequence. and with the oligo strand of the duplex oCFNO, oPGM2-4, oPEV. Constructs with defined length (84 bp) and orientation were selected. Constructs with cruciform without consensus sequence were denoted as pCFNO, synthetic sequences with target sequence with inverted repeat as pPGM2-4, synthetic sequences with target sequence without inverted repeat as pPEV. pPGM1 was prepared as described [15].

p53 protein

Wt human full-length (fl) p53 protein expressed in insect cells was prepared and characterized as described [15]. A molecular weight of 53kD for this postranslationally modified protein was used in all calculations.

Competition experiments

0.6 µg of the competitor, 0.4 µg of the indicator pPGM1/PvuII fragments and p53 protein were mixed at 1/5 molar ratio (the ratio was calculated from the concentrations of DNA and p53 tetramers) in 20 µl of DNA binding buffer (5 mM Tris-HCl, pH 7.0, 1 mM EDTA, 50 mM KCl and 0,01% Triton X-100). The samples were incubated for 30 min on ice and loaded on a 1% agarose gel containing 0.33x TBE buffer, pH 8.0. Electrophoresis was performed for 4h at 100V (usually 4 V/cm) at low temperature (~6°C). The gels were stained with ethidium bromide, photographed, scanned and rendered digitally. The band intensities were quantified by Image QuaNT software. Standard deviations of these determinations, obtained with different competitors were calculated from 6 determinations of one competitor.

Proof of the cruciform structure by modification by Osmium tetroxide 2,2' bipyridine

For detection of the cruciform in the p53 target of the pPGM2, we took advantage of the fact that the CTAG tetranucleotide in the center of the insert is recognized and cleaved by XspI restriction endonuclease. If osmium tetroxide complexes modify pyrimidines in the tetranucleotide, the cleavage is inhibited and a new band on the agarose gel occurs. Because two other restriction sites located in the vicinity of the CON sequence could give false results, we mutated A to G and G to A corresponding to 728 and 735 positions in the SK plasmid by means of a site-directed mutagenesis kit (Stratagene). For the modification reaction, 2 µg of plasmid were incubated with 2 mM Os,bipy complex in the p53-binding buffer at 37°C for 30min in the volume of 50ul. After the modification, the samples were precipitated by ethanol, dissolved in the buffer and treated with XspI endonuclease for 90min at 37°C.

Proof of the cruciform structure by 2D electrophoresis

For the cruciform analysis by means of two-dimensional electrophoresis [28],

prepared topoisomers [29] were mixed, loaded on the 1.2% TAE-agarose gel and run in the first dimension at 40 V for 20 hours. After electrophoresis, the gel was soaked in the TAE buffer containing chloroquine at the concentration 2.5 µg/ml for 8 hours.

For the second dimension, the gel was turned by 90° and electrophoresis was run upright to the first dimension in the TAE-chloroquine (2,5 µg/ml) buffer at 40 V for another 20 hours. During electrophoresis, the buffer was circulated between the anodic and cathodic spaces.

Preparation of plasmid DNA topoisomers

RESULTS

As shown previously, the p53 protein binds preferentially to scDNA regardless of the presence or absence of the target sequence [15] and DNA supercoiling can enhance the p53 binding to certain target sites [START_REF] Palecek | Enhancement of p53 sequence-specific binding by DNA supercoiling[END_REF]. The positive effect of negative DNA supercoiling on the sequence-specific DNA binding was well pronounced, especially with targets exhibiting internal symmetry, being able to adopt cruciform structure under the negative superhelical stress (such as in pPGM1) but not those incapable of forming such structure due to the lack of inverted repeat symmetry (such as in pRGC). Here we focused on the effects connected with changes of the DNA negative superhelix density (the driving force of structural transition giving rise to the cruciform structures in inverted repeat DNA segments [START_REF] Palecek | Local supercoil-stabilized DNA structures[END_REF]29]) on p53 binding to various plasmid DNAs.

We prepared plasmid DNA constructs involving (a) synthetic p53 target sequences with a perfect inverted repeat symmetry (pPGM2-4); (b) a perfect inverted-repeat but p53 non-target sequence (pCFNO) (c) an asymmetrical p53 target sequence (pPEV); (d) pBluescriptSK-(SK) vector as control. In agreement with previous data, all of these scDNAs bound p53 protein, forming in agarose gel ladders of bands due to p53-scDNA complexes migrating more slowly than DNA alone (Figure 1A). No significant differences between DNAs containing (Figure 1A, lanes 3-7) or not containing p53 target sequence (Figure 1A, lanes 1-2) or between DNAs containing internally symmetrical (Figure 1A, lanes 1, 4-7) or non-symmetrical (Figure 1A, lanes 2-3) target sites were observed using this simple, non-competitive gel shift assay, showing efficient p53 binding to any scDNA. With PvuII digests of the same plasmid DNAs we observed specific p53 binding to the shorter (474-bp) fragments provided that these fragments involved a p53 target site (Figure 1B, lanes 1-4). No retardation of analogous fragments of SK and CFNO was detected in agreement with absence of the p53 target site.

To probe differences in relative p53 binding affinity to various scDNAs we used a competition assay proposed previously [START_REF] Palecek | Enhancement of p53 sequence-specific binding by DNA supercoiling[END_REF]. Briefly, the 474-bp fragment resulting from PvuII cleavage of the pPGM1 DNA, containing a p53 target site, was used as an indicator substrate in the competition experiments. Binding of the protein to this fragment yielded a well resolved retarded band R (Figure 2, lane 2). Intensity The intensity of the indicator R band decreased by about 70-85% on addition of scDNA (of native -σ) with p53 target sequences possessing inverted repeat symmetry (pPGM1, pPGM2, pPGM3 and pPGM4), but only by approximately 50% on addition of a scDNA involving a p53 non-target inverted repeat (pCFNO). There was also a clear difference between DNAs lacking the inverted repeats. The intensity of the indicator R band was decreased by about 62% (pPEV, containing a nonsymmetrical target site) and about 40% (SK containing neither target site nor a perfect inverted repeat) respectively. Taken together, the p53 target sequences with the inverted repeat symmetry were the best competitors, followed by a nonsymmetrical p53-target and by DNA without any p53 target site with an inverted repeat.

Effect of DNA superhelix density variations on the p53 DNA binding

To study effects of the DNA topological state on p53 binding to various DNA substrates, we prepared sets of topoisomers of the plasmids SK, CFNO, pPEV and pPGM2 and employed them in the competitor assay (Figure 3). All DNAs exhibited increasing competitiveness towards the indicator pPGM1 fragment but courses of the dependences of p53 affinity to individual plasmid DNAs on -σ differed remarkably.

Relaxed (-σ = 0) circular duplex DNAs SK or CFNO (both not containing the p53 target site) bound p53 with similar affinities, causing about a 30 % decrease of the indicator band intensity (Figure 3). Apparent affinities of p53 to relaxed DNA's containing a target site (both pPGM2 and pPEV) were higher that those observed for the non-target plasmids but also similar to one another (50-55 % competition).

Binding of p53 to SK increased slightly with increasing -σ, following an almost linear dependence and reaching 40 % competition for -σ = 0.07. The pPEV plasmid also displayed a linearly increasing dependence of p53 binding on the -σ, showing 60-65 % competition at -σ = 0.07. On the other hand, the behavior of the pPGM2 DNA was different. While between -σ = 0 and 0.03 affinity of p53 to this plasmid followed practically the same trend as observed for pPEV, at more negative superhelix density a considerably steeper increase of binding with -σ was detected. At -σ = 0.05 the pPGM2 DNA caused almost 70 % competition (while pPEV 60 %) and at -σ = 0.07 even 85 % competition (60 -65 % for pPEV). Interestingly, a break on the apparent binding affinity dependence on -σ was observed also for pCFNO which thus became a better substrate for p53 starting from -σ = 0.05 (but remained a substantially worse competitor than pPEV even at -σ = 0.07). The ability of all plasmids in their linearized forms to compete for p53 (not shown) was similar as observed for the respective DNA's in their relaxed (covalently closed circular) forms (Figure 3A).

Cruciform extrusion in plasmids involving inverted-repeat inserts

The above results showed that differences in p53 binding (competition ability) within the groups of plasmid DNA substrates either containing (pPGM2 or pPEV) or lacking (SK or pCFNO) the p53 target sites were not statistically significant (Figure 3A) when the DNAs were relaxed or slightly negatively supercoiled (-σ ≤ 0.03).

However, after the negative superhelix density reached a certain critical value (-0.05), the pPGM2 plasmid (with the internally symmetrical target site) became a significantly "better" competitor than pPEV (with non-symmetrical target site). Such behavior suggests that formation of the cruciform structure within the symmetrical p53 target site of pPGM2 DNA at sufficient -σ may be responsible for the observed extra enhancement of p53 binding. To test this assumption we performed an analysis of structural transitions in the pPGM2 based on S1 nuclease cleavage (Figure 4A), on 2D agarose gel electrophoresis (Figure 4B) [START_REF] Palecek | Local supercoil-stabilized DNA structures[END_REF], and by modification by Osmium tetroxide 2,2' bipyridine. The S1 nuclease cleaves selectively single-stranded DNA and base-unpaired sited in dsDNA (such as loop parts of hairpins and cruciforms). where the major intrinsic inverted repeat is located). We obtained the same results using a single-strand selective chemical probe osmium tetroxide, 2,2' bipyridine [START_REF] Palecek | Local supercoil-stabilized DNA structures[END_REF][START_REF] Palecek | Probing of DNA structure with osmium tetroxide complexes in vitro[END_REF] combined with a restriction cleavage inhibition assay (not shown). We detected extrusion of the cruciform structure in pPGM2 also by DNA 2D gel electrophoresis.

Presence of the supercoil-stabilized cruciform was indicated by the shift of the DNA bands curve [28] approximately at -σ=0.05 (Figure 4B, arrow), thus confirming the S1 cleavage experiments. The superhelix density level critical for the cruciform appearance (-σ=0.05) correlated well with the -σ value at which the pPGM2 (and also pCFNO) displayed the break on the superhelix density-relative p53 binding dependences (Figure 3A).

Similarly, we detected the negative superhelicity-dependent structural transitions in the scDNAs involving the (p53 non-target) inverted repeat (pCFNO Fig 4A, lane 10) as well as in plasmids containing other symmetrical p53 target sites (pPGM3-4, not shown). S1 cleavage occurred in all cases at sites of anticipated cruciform extrusion (i.e., within the inserted inverted repeats). On the other hand, no indication of a distinct supercoil-dependent structural transition was detected under the same conditions with SK and pPEV (Figure 4, lane 11, 9) plasmids not containing sequence motifs prerequisite for such transitions (Figure 3B). Numerous recent data have provided evidence that DNA binding of p53 is strongly dependent on structural features of the target DNA [START_REF] Nagaich | DNA bending is essential for the site-specific recognition of DNA response elements by the DNA binding domain of the tumor suppressor protein p53[END_REF][START_REF] Brazda | Specific modulation of p53 binding to consensus sequence within supercoiled DNA by monoclonal antibodies[END_REF][START_REF] Kitayner | Structural basis of DNA recognition by p53 tetramers[END_REF][START_REF] Kim | The complex interactions of p53 with target DNA: we learn as we go[END_REF]. It has been shown that full length (fl) p53 was able to bind sequence-specifically to target oligonucleotides adopting stem-loop structures in the absence of "activating" antibodies such as PAb421 [START_REF] Kim | DNA-conformation is an important determinant of sequence-specific DNA binding by tumor suppressor p53[END_REF]. Later Prives et al. [12] showed that a p53 target site within a topologically constrained DNA minicircle was bound by the fl p53 with a higher affinity than the same target site in non-constrained linear DNA. Our observations [START_REF] Palecek | Enhancement of p53 sequence-specific binding by DNA supercoiling[END_REF] revealed enhanced binding of the fl p53 protein to certain target sites within large (~3 kB) plasmid DNAs, compared to the same target sites in 20-mer oligonucleotides or in the same but linearized plasmids. Strikingly, effects of negative DNA superhelicity were especially well pronounced for symmetrical target sites able to adopt cruciform structures under topological constraint in the negatively scDNA.

Hence, it has been tentatively concluded that cruciform extrusion was the cause for the enhanced sequence-specific p53-DNA binding.

In this paper we demonstrate for the first time that relative affinity of the p53 protein to the same (internally symmetrical) p53 target site is remarkably changed upon extrusion of a cruciform structure within the inverted repeat featuring the target site.

Thus, the structural transition in the binding site upon topological stress represents a switch between two apparently distinct states differing in the affinity of p53-DNA recognition (Figure 3B). Change from the low-affinity to the high-affinity binding state correlated well with the jump of the topoisomer spots in the 2D agarose gel electrophoresis and the cruciform extrusion starting at -σ=0.05 (Figure 4). On the other hand, relative binding of p53 to the pPEV scDNA (in which the p53 target site was non-symmetrical and thus unable of forming the cruciform structure, Figure 3B) had a monophasic, approximately linear dependence on -σ (Figure 3A). This trend reflected the known ability of fl p53 to bind selectively scDNA with native superhelix density regardless of the presence or absence of a target site. The SK vector, containing neither a p53 target site nor a perfect palindromic DNA segment, exhibited a dependence that was parallel to that observed for the pPEV but was shifted to lower relative binding affinities (Figure 3). Hence, in the pPEV the overall p53 binding affinity appears to be a simple sum of the sequence-specific binding to the target site (not undergoing a distinct structural transition upon changes of -σ), and the supercoil-selective (but sequence non-specific) p53 DNA binding. Interestingly, the pCFNO displayed the same behavior as the SK for -σ≤0.03 but exhibited certain extra enhancement of p53 binding at superhelix densities sufficient for the cruciform extrusion. This suggests that the cruciform structure itself (lacking features of the sequence-specific p53 binding site) can be bound by the protein with certain selectivity, in agreement with results obtained previously by scanning force microscopy [11]. The difference in competition abilities of plasmids pCFNO and SK at -σ=0.07 was about 10%, in contrast to at least 20 % difference between plasmids pPGM2 (or pPGM3-4) and pPEV. The best substrate for p53 protein binding is therefore a p53 target sequence with the extruded cruciform structure. Natural p53 responsive elements often possess more or less perfect inverted repeat symmetry [START_REF] Kim | DNA-conformation is an important determinant of sequence-specific DNA binding by tumor suppressor p53[END_REF], and our preliminary experiments with some of them (such as mdm2 or gaad45) revealed similar effects of negative DNA superhelicity as reported here for the synthetic p53 targets (not shown). A combination of the structural and sequence features of the DNA substrate can thus be important for fine tuning the protein DNA interaction.

The binding of p53 to its target DNA presents an apparent steric problem: p53 target sites may but need not involve insertions of variable DNA segments between the half-site. The question arises as to how p53 is able to bind such a broad spectrum of targets differing in both nucleotide sequence and conformation. It is unclear just how p53 can recognize specific DNA sequences, and at the same time how it can recognize different geometries with sequence variability as cruciform structure.

Cruciform extrusion itself might solve the possible problem with the intervening sequences that, in the cruciform structure, are displaced into the single-stranded loops while the target half-sites are located in the hairpin stems. It simultaneously renders the binding site a considerable flexibility at the four-way junction. For target sites without the intervening stretches, central parts of the binding sequence are inherently located within the cruciform apical loops, which may cause another [START_REF] Kim | DNA-conformation is an important determinant of sequence-specific DNA binding by tumor suppressor p53[END_REF] sequence-specific p53 binding to various hairpin oligonucleotide substrates, including those mimicking the p53 target (half) sites having adopted the cruciform (stem-loop) structure.

In more complex DNA, such as scDNA or DNA in the chromatin environment, specific structural profiles may be influenced by DNA supercoiling and/or by chromatin remodeling activities maintaining the structural fluidity of the chromatin [START_REF] Kingston | A novel type of interaction between cruciform DNA and a cruciform binding protein from HeLa cells[END_REF].It seems that flexibility of the p53 target sequences in long DNA targets can be one of the crucial factors in p53-DNA binding. Determination of the p53 protein bound to DNA by X-ray crystallography showed that the p53 protein bends the DNA by about 20° [START_REF] Nagaich | DNA bending is essential for the site-specific recognition of DNA response elements by the DNA binding domain of the tumor suppressor protein p53[END_REF]36]. Our results are in good agreement with the new view of p53 regulatory tasks in the context of chromatin assembly in eukaryotic cells. In this view, the p53 protein will not bind simultaneously to all p53 targets sequences, but only to those p53 targets that exhibit a structural architecture that is compatible with p53-preferred DNA structure. Cruciform target sites stabilized by DNA supercoiling can be one of the favorite structures for effective p53/DNA binding in vivo. The correlation between p53 binding affinity and the presence of cruciform structures in topologically constricted DNAs reflects the complexity of protein-DNA interaction. Transient supercoils occurring in the eukaryotic genome during DNA replication, transcription (processes that involve a local separation of DNA strands [37,[START_REF] Pearson | Inverted repeats, stem-loops, and cruciforms: significance for initiation of DNA replication[END_REF]) as well as in result of proteins binding [39,[START_REF] Van Holde | Unusual DNA structures, chromatin and transcription[END_REF] may facilitate formation of secondary structures in DNA. Moreover, active chromatin remodeling is a typical feature for many promotors and is essential for gene transcription [START_REF] Brettingham-Moore | GM-CSF promoter chromatin remodelling and gene transcription display distinct signal and transcription factor requirements[END_REF]. The strong impact of topological conformation of DNA on p53 sequence specific binding in vitro predicts that the ability of p53 to bind its target sites will be strongly influenced by factors capable of modifying chromatin structure in a living cell. Changes in DNA supercoiling and stabilization of the non-B DNA structures may thus up-and down-regulates p53-DNA binding abilities.

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T MATERIALS AND METHODS DNA Duplex oligonucleotides oCFNO (CATGATGTGATCACATCATG), oPGM2 (AGACATGCCTAGGCATGTCT), oPGM3 (AGGCATGCCTAGGCATGCCT), oPGM4 (AAACATGTTTAAACATGTTT), oPEV (AGGCTAGTCTGAGCATGTTC) with HindIII adapters were cut by HindIII and inserted into the HindIII site of SK. The ligated DNA was then used to transform electrocompetent cells of the Top10 strain of E.coli (Invitrogen). DNA from single colonies selected for Amp resistance were digested with PvuII and electrophoresed. Clones with a PvuII fragment longer than 448 bp were subjected to amplification with M13-20 primer, (5'GTAAAACGACGGCCAGT3')

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T Proof of the cruciform structure by S1 nuclease cleavage For the cruciform detection, 2 µg of plasmid were digested by S1 nuclease (2 U/µg DNA) for 2h at 37°C in the nuclease S1 buffer (30 mM sodium acetate pH 4.6, 280 mM NaCl, 1 mM ZnSO 4 ). After the cleavage, samples were precipitated by ethanol, dissolved in water and digested by the restriction endonuclease ScaI for 90 min.

  Topoisomeric samples were prepared according to [29]. Briefly, 40 µg of DNA per ml was incubated in a solution containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.1 Biochemical Journal Immediate Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T mM EDTA, 1 mM DTT and 20% glycerol (Promega). Appropriate concentrations of ethidium bromide (-σ=0.01 -1.69µM, -σ=0.03 -3.8µM, -σ=0.05 -6.13µM, -σ=0.07 -9.1µM) and DNA topoisomerase I (Promega) were added and samples were incubated at 37°C for 3 h. After complete relaxation, ethidium bromide and topoisomerase were removed by two-phenol extraction followed by three chloroform extractions and ethanol precipitation. The relaxed (covalently closed circular) DNA was prepared in the same way without ethidium bromide addition. Determination of DNA superhelix density Superhelix densities (-σ) of the plasmid DNA samples were determined by electrophoresis on agarose gels containing different concentrations of chloroquine (0, 1, 5, 10 µg per ml) [29, 30]. The electrophoresis was performed for 6 h at 100 V. The gels were stained with ethidium bromide, photographed, scanned and evaluated digitally. The mean superhelix densities of topoisomers were determined from the strongest band in the distribution. Biochemical Journal Immediate Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T

  Biochemical Journal Immediate Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T of this band was affected by the additions of the tested scDNAs. The scDNAs represented the competitors (Figure 2, lanes 3-9). Decrease of the R band intensity relative to the intensity detected in the absence of the competitors reflected relative affinity of p53 to the given competitor. Complete absence of the band R was taken as 100%.

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T

  Cleavage of sc pPGM2 (at native -σ) by S1 followed by restrictase ScaI produced two specific DNA fragments (1720bp, 1267bp), indicating presence of the cruciform structure within the p53 target site of this scDNA (see scheme in Fig 4C). Upon the same treatment of relaxed pPGM2 we obtained only full-length linear DNA (2987bp), suggesting absence of the cruciform structure representing the specific cleavage site for the S1 nuclease (Fig 4C, lane 1). The same experiments were performed with Biochemical Journal Immediate Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T pPGM2 topoisomers differing in the -σ levels. We did not observe any indication of the specific S1 cleavage at -σ=0.01 or 0.03, but we detected the cruciform in sc pPGM2 at superhelical densities -0.05 or -0.07 (Figure 4A, lanes 4,5). A comparison of the band intensities resulting from the S1 cleavage suggested that the cruciform content in pPGM2 increased strongly from -σ=0,05 to 0.07. (The pBluescript vector, used for construction of all plasmid substrates in this work, contains a few imperfect inverted repeats [31, 32] that may undergo local structural transitions in the scDNA. The weaker bands in lanes 4-6, 8-11) of Figure 4, corresponding to about 2211 and 776 bp DNA fragments, may have arisen from S1 cleavage close to replication origin

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T DISCUSSION Presence of cruciform structure in p53 target sequences is an important attribute in p53-DNA binding

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T problem for the p53-DNA recognition. Nevertheless, Kim et al. have demonstrated

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1 (

 1 Figure 1 (A) Binding of the p53 protein to supercoiled DNAs. 0.4 µg sc pCFNO (lane 1), sc SK (lane 2), sc pPEV (lane 3), sc pPGM1 (lane 4), sc pPGM2 (lane 5), sc pPGM3 (lane 6), sc pPGM4 (lane 7) DNAs were incubated with 0.35 µg of wt p53 (lanes 1-9) in 50 mM KCl, 5 mM Tris-HCl (pH 7.6) and 0.01% Triton X-100 on ice for 30 min. Samples were electrophoresed on 1% agarose gel at 100 V and 4°C for 3-4 h. Lane 8 contains DNAs of sc SK without p53 protein. (B)Binding of the p53 protein to DNA fragments. Plasmids pPEV (lane 1), pPGM1 (lane 2), pPGM2 (lanes 3, 7), pPGM3 (lane 4), pCFNO (lane 5), SK (lane6) were cleaved by PvuII into two fragments; shorter one (474bp) contains the p53 target site except SK and pCFNO (lanes 5 and 6). The fragments were incubated with 0.35 µg of wt p53 (lanes 1-6) in 50mM KCl, 5 mM Tris-HCl (pH 7.6) and 0.01% Triton X-100 on ice for 30 min. There is a control without p53 protein in lane 7.

Figure 2 (

 2 Figure 2 (A) Competition of superhelical DNAs for p53 SSDB to p53 target in lin 474bp fragment. 0.4 µg of indicator pPGM1/PvuII (lanes 1-9), with 0.6 µg of sc DNA of SK (lane 3), pPGM1 (lane 4), pPGM2 (lane 5), pPGM3 (lane 6), pPGM4 (lane 7), pPEV (lane 8) and pCFNO (lane 0) were incubated with 0.55 µg of wt p53 (lanes 2-9) in 50mM KCl, 5 mM Tris-HCl (pH 7.6) and 0.01% Triton X-100 on ice for 30 min. (B) Bar graph of increasing p53 binding to competitor DNAs expressed as the inverted percentage of band R without competitor DNAs (lane 2).

Figure 3 (

 3 Figure 3 (A) Competition of linear DNA, relaxed DNA and topoisomers for p53 SSDB to p53 target in lin 474bp fragment. 0.4 µg of indicator pPGM1/PvuII with 0.6 µg of competitor DNAs were incubated with 0.55 µg of wt p53 in 50mM KCl, 5 mM Tris-HCl (pH 7.6) and 0.01% Triton X-100 on ice for 30 min. The graph of increasing p53 binding to competitor DNAs expressed as the inverted percentage of band R without competitor DNAs (pPGM2 -full, black,

Figure 4

 4 Figure 4 Evidence of cruciform structure in topoisomers of pPGM2 (A) Evidence of cruciform in topoisomers of pPGM2 by S1 nuclease cleavage. The plasmids: pPGM2 relaxed (lane 1), pPGM2 -σ=0.01 (lane 2), pPGM2 -σ=0.03 (lane 3), pPGM2 -σ=0.05 (lane 4), pPGM2 -σ=0.07 (lane 5), sc pPGM2 (lane 6), sc pCFNO (lane 8), sc pPEV (lane 9), sc pPGM2 (lane 10), sc SK (lane 11) undergone cleavage by S1 nuclease and then by treatment by ScaI restriction endonuclease. SK digested by BglΙ (lane 7) was used as a control. Bands resulting from the nuclease S1 cleavage at the cruciform site in pPGM2 are about 1820 and 1167 bp. (B) Evidence of cruciform in topoisomers of pPGM2 by 2D electrophoresis. Arrow shows -σ=0.05 (for details see Materials and Methods). (C) Proof of the cruciform structure by S1 nuclease cleavage -scheme (a) The result of S1/ScaI cleavage without presence of cruciform. (b) The result of the S1/ScaI cleavage in presence of cruciform. (c) The control cleavage by BglI restriction enzyme.

  Fig.2

  Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

We thank M. Kotlyar for critical reading of the manuscript. This work was supported by grants of GACR 301/07/P160 and 301/05/0416, GA ASCR IAA500040701, IAA500040513 and B500040502, IGS UO 1074 and by the Academy of Sciences of the Czech Republic, grants AV0Z50040507, AV0Z50040702. Biochemical Journal Immediate Publication. Published on 14 Feb 2008 as manuscript BJ20071648 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071648 S t a g e 2 ( a ) P O S T -P R I N T


Maria V Zabolotskaya

Dominic P Grima

Ming-Der Lin

Tze-Bin Chou

Sarah F Newbury
email: s.newbury@bsms.ac.uk

The 5' -3' exoribonuclease Pacman is required for normal male fertility and is dynamically localised in cytoplasmic particles in Drosophila testis cells

Keywords: P-bodies, Xrn1, RNA stability, spermatogenesis, RNA degradation

published or not. The documents may come

The 5' -3' exoribonuclease Pacman is required for normal male fertility and is dynamically localised in cytoplasmic particles in Drosophila testis cells

Introduction

The control of RNA stability is a crucial element of gene regulation in all organisms. There is increasing evidence that specific, timed transcript degradation is critical for regulation of many cellular processes, including early development and RNA interference. The importance of RNA stability in developmental processes is revealed by the phenotypes of animals or plants where the expression of genes involved in RNA stability has been disrupted. For example, mice deficient for TTP, an RNA binding protein regulating stability of RNAs such as GMCSF and TNF, develop a systemic inflammatory syndrome with autoimmunity and myeloid hyperplasia (bone marrow overgrowth) [START_REF] Carballo | Roles of tumor necrosis factor-alpha receptor subtypes in the pathogenesis of the tristetraprolin-deficiency syndrome[END_REF]. In Drosophila, mutations in the 3' -5' deadenylase ccr4 (twin) result in a level of sterility and maternal effect lethality in females [START_REF] Zaessinger | Oskar allows nanos mRNA translation in Drosophila embryos by preventing its deadenylation by Smaug/CCR4[END_REF]. Furthermore, C. elegans adults carrying mutations in dicer (dcr-1), a double-stranded endoribonuclease which plays a key role in the first steps of the RNA interference pathway, are sterile and have defects in developmental timing [START_REF] Grishok | Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing[END_REF][START_REF] Knight | A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans[END_REF]. Finally, Arabidopsis mutant for DST1, which regulates the stability of RNAs such as CCL and SEN1, have defects in their circadian rhythm [START_REF] Gutierrez | Identification of unstable transcripts in Arabidopsis by cDNA microarray analysis: rapid decay is associated with a group of touch-and specific clock-controlled genes[END_REF]. Therefore transcript degradation during development can be selective and also modulated suggesting a little studied layer of control of gene expression during development.

One of the key ribonucleases in the 5' -3' mRNA degradation pathway is the exoribonuclease Xrn1. This enzyme is known to be a processive 5' -3' exoribonuclease which degrades mRNA after they have been de-capped [START_REF] Decker | Mechanisms of mRNA degradation in eukaryotes[END_REF][START_REF] Johnson | Rat1 and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm respectively[END_REF][START_REF] Muhlrad | Deadenylation of the unstable mRNA encoded by the yeast MFA2 gene leads to decapping followed by 5' -3' digestion of the transcript[END_REF]. Xrn1 is extremely well conserved in all eukaryotes, with homologues in humans (xrn1), Drosophila (pacman) and C. elegans (xrn-1) [START_REF] Newbury | The 5' -3' exoribonuclease xrn-1 is essential for ventral epithelial enclosure during C. elegans embryogenesis[END_REF][START_REF] Till | Identification and developmental expression of a 5'-3' exoribonuclease from Drosophila melanogaster[END_REF]. Indeed we have previously shown that Drosophila Pacman has exonuclease activity in-vitro and that it can complement a null XRN1 mutation [START_REF] Newbury | The 5' -3' exoribonuclease xrn-1 is essential for ventral epithelial enclosure during C. elegans embryogenesis[END_REF][START_REF] Till | Identification and developmental expression of a 5'-3' exoribonuclease from Drosophila melanogaster[END_REF][11]. Xrn1 is not only involved in the normal decay of mRNA but is required the major pathway in nonsense-mediated decay [START_REF] Gatfield | Nonsense-mediated mRNA decay in Drosophila: at the intersection of the yeast and mammalian pathways[END_REF], RNA interference [START_REF] Orban | Decay of mRNas targeted by RISC requires XRN1, the Ski complex and the exosome[END_REF] and degradation via microRNAs [START_REF] Souret | AtXRN4 degrades mRNA in Arabidopsis and its substrates include selected miRNA targets[END_REF]. Mutations in xrn1 in yeast or silencing of xrn-1 in C. elegans leads to phenotypic defects suggesting that this enzyme is required for normal cellular processes [START_REF] Decker | Mechanisms of mRNA degradation in eukaryotes[END_REF][START_REF] Muhlrad | Deadenylation of the unstable mRNA encoded by the yeast MFA2 gene leads to decapping followed by 5' -3' digestion of the transcript[END_REF][START_REF] Newbury | The 5' -3' exoribonuclease xrn-1 is essential for ventral epithelial enclosure during C. elegans embryogenesis[END_REF][START_REF] Johnson | Strand exchange protein 1 from Saccharomyces cerevisiae[END_REF]. Our recent work has also shown that mutations in pacman in Drosophila result in defects in dorsal and thorax closure and reduced survival after wound healing [START_REF] Grima | The 5' -3' exoribonuclease pacman is required for epithelial sheet sealing in Drosophila and genetically interacts with the phosphatase puckered[END_REF].

Recent exciting work has shown that 5' -3' degradation of cytoplasmic mRNAs seems to take place in specific particles known as processing bodies (P-bodies) [START_REF] Sheth | Decapping and decay of messenger RNA occur in cytoplasmic processing bodies[END_REF][START_REF] Parker | P-bodies and the control of mRNA degradation and translation[END_REF][START_REF] Eulalio | P bodies: at the crossroads of post-transcriptional pathways[END_REF]. In both yeast and human cells, P-bodies have been shown to harbour many enzymes of the basic decay machinery including the decapping complex and the exoribonuclease Xrn1. Strong evidence that P-bodies are sites of normal mRNA decay rather than merely storage sites for RNA decay factors is that they accumulate mRNA decay intermediates [START_REF] Sheth | Decapping and decay of messenger RNA occur in cytoplasmic processing bodies[END_REF]20] although mRNA degradation may also take place in sub-microscopic particles [START_REF] Eulalio | P-body formation is a consequence, not the cause of RNA-mediated gene silencing[END_REF]. In mammalian cells P-bodies are not only involved in normal mRNA turnover but have also been reported to be the sites of nonsense-mediated decay and miRNA-induced translational silencing [22,23]. P-bodies are dynamic structures that can vary in size and number according to stress conditions or the availability of untranslated RNAs [START_REF] Kedersha | Stress granules and processing bodies are dynamically linked sites of mRNP remodeling[END_REF][START_REF] Teixeira | Processing bodies require RNA for assembly and contain nontranslating mRNAs[END_REF]. Although P-bodies have been well studied in yeast and human tissue culture cells, they have rarely been observed in natural tissues. Their relevance to developmental processes is also unclear.

In this paper, we have examined the function of the exoribonuclease pacman (pcm) in Drosophila testis. We have chosen testis as a model system because they undergo a well defined differentiation pathway, remarkably similar to spermatogenesis in mammals [START_REF] Fuller | Genetic control of cell proliferation and differentiation in Drosophila spermatogenesis[END_REF]. In both flies and mammals, cells (spermatogonia) produced from self-renewing germ cells first mitotically divide to form spermatocytes and then, after a period of growth and transcription, shut off the transcriptional programme, enter meiotic divisions and differentiate into spermatids. Post-transcriptional pathways are highly regulated during the spermatogenesis process with many transcripts being synthesised in primary spermatocytes and then translationally repressed until long after meiotic divisions [START_REF] White-Cooper | Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila[END_REF]. The contribution of mRNA stability to spermatogenesis is as yet unclear.

This paper analyses the effect of the 5' -3' exoribonuclease pacman on male fertility in the fruit-fly Drosophila melanogaster. We show that Pacman is required for normal testis development and male fertility. We also demonstrate that Pacman is located in cytoplasmic particles akin to P-bodies in both mitotically dividing spermatogonia and transcriptionally active spermatocytes. These particles also include the decapping activator dDcp1 and the helicase Me31b. These P-bodies, like those in human tissue culture cells, increase in size and number upon stress. Therefore we show, for the first time, that an exoribonuclease is crucial to normal male spermatogensis.

Materials and Methods

Fly stocks were cultivated on standard media at 25 o C in uncrowded conditions. The wildtype stock was Oregon R (Bloomington Stock Centre). The transgenic stock P{w+; YFP-dDcp1} was provided by M.D. Lin [START_REF] Lin | Drosophila decapping protein 1, dDcp1, is a component of the oskar mRNP complex and directs its posterior localization in the oocyte[END_REF]. The pcm mutants were generated by P-element excision and have been described previously [START_REF] Grima | The 5' -3' exoribonuclease pacman is required for epithelial sheet sealing in Drosophila and genetically interacts with the phosphatase puckered[END_REF]. Molecular characterisation of the pcm 3 and pcm 5 alleles showed that the pcm 3 allele comprises a deletion of 1522 bp resulting in a frame-shift mutation. The resulting mutant protein is a C-terminal truncation of 197 aminoacids relative to wild-type plus an extra 46 amino-acids encoded from intergenic sequences; the 3' UTR was also deleted. The pcm 5 allele is an intragenic deletion which removes 516 base pairs, resulting in a frame-shift. The pcm 5 protein is C-terminally truncated by 348 amino-acids compared to wild-type, with a 29 amino-acid extension encoded from out-of-frame pcm sequences. The 3' UTR is intact in this mutant. The wildtype protein is 1612 amino-acids.

Measurement of male fertility.

The fertility of pcm mutant males was tested by crossing (at 25 ºC) individual virgin males (control, pcm 3 and pcm 5 ) to individual wild-type virgin females. 25 vials per strain were set up. The flies were left to mate for 2 days and then removed from the vials. F1 generation offspring were counted for 8 days after the first offspring hatched. The observed differences between the control and pcm 3 and control and pcm 5 were statistically significant in both cases (Mann-Whitney test, W=415.5, P<0.0001 and W=389, P<0.0001, respectively).

Measurement of sperm number.

Seminal vesicles were dissected from 3 day old virgin males (n=15, at 25 ºC) in a drop of phosphate buffered saline (PBS) and transferred to a fresh drop of PBS. The seminal vesicles were then ruptured and the sperm inside allowed to dissociate. The preparations were left for 10 min and then allowed to dry. The time allowed for dissociation and drying ensured that the sperm were well spread out. The slides then fixed with 20 l 4% paraformaldehyde solution in HEPES buffer and stained with DAPI. Samples were mounted in 85% glycerol with 2.5% n-propyl gallate and cover slips were sealed with nail varnish. Sperm were then viewed at X40 power with a Chroma long pass DAPI filter using a Leica fluorescent microscope. A video camera and monitor were used to view the slides and the sperm were counted. The observed differences between the control and pcm 3 and also the control and pcm 5 mutants were statistically significant in both cases (Mann-Whitney test, W=0, P<0.0001 and W=0, P<0.0001, respectively).

Measurements of testes.

Testes were dissected from 2 day old virgin males (n=20, at 25 ºC) in Ringer's solution (130 mM NaCl, 4.7 mM KCl, 1.9 mM CaCl 2 , 10 mM HEPES buffer, pH 6.9) and mounted in Ringer's solution on a microscope slide without a coverslip. The testes were visualized with light microscopy at X20 power and the length and width (200 m from the tip of the testes) of the testes were measured with Zeiss AxioVision imaging system software. The observed differences for the length and width of the testes between the control and pcm 3 and also the control and pcm 5 were statistically significant in both cases (for the length of the testes: Mann-Whitney test, W=255, P<0.025 and W=361, P<0.0001, respectively; for the width of the testes: Mann-Whitney test, W=445, P<0.0001 and W=283, P<0.0001, respectively).

Western blotting and co-immunoprecipitation from testes extract. SDS-PAGE and Western blotting was performed essentially as described [START_REF] Sambrook | Molecular Cloning[END_REF] with binding of antibodies being detected using a ECL Western blot reagent kit (Amersham). Primary polyclonal anti-PCM antibody (raised in rabbit) [START_REF] Grima | The 5' -3' exoribonuclease pacman is required for epithelial sheet sealing in Drosophila and genetically interacts with the phosphatase puckered[END_REF] was used at 1:2000. We are confident that this antibody (raised to the 54kD C-terminal portion of the protein) can detect the truncated mutant pcm proteins because it can pull down PCM from testis extracts (Figure 4, panel L). The monoclonal anti-actin antibody (Sigma) was used at 1:10000 and primary antibody anti-dDcp1 (raised in rabbit), provided by J. Wilhelm, was used at a dilution of 1:2000. The secondary antibodies were monoclonal anti-rabbit HRP conjugated antibody (Sigma) and monoclonal anti-mouse HRP conjugated antibody (Sigma), both used at 1:80000. Testes dissected from 0-1 day old males were homogenized in lysis buffer (50 mM Tris-HCl pH 7.5-8.0, 0.5% Triton X-100, 150 mM NaCl, protease inhibitors) (140 testes, 500 l buffer used), then cleared by centrifugation (10.000 rpm for 10 min at 4°C). 40 l was retained as the "input" sample, the remainder was pre-cleared with protein G-sepharose (Roche), then incubated with rabbit anti-PCM and precipitated with protein G-sepharose (at 4°C). Beads were washed, then bound proteins were eluted by boiling in SDS sample buffer. For the negative control wild type testes were immunoprecipitated without any anti-PCM antibody.

Immunofluorescence

For immunofluorescence, testes were dissected from 0-1 day old males in Ringer's solution, with testes from about ten males being used per slide. The testes were transferred to a 20 l drop of Ringer's solution on a poly-l-lysine-treated slide and cut open with tungsten needles. Paraformaldehyde (20 l of 4% solution) in HEPES buffer was added and the testes were left to fix for 15 minutes at room temperature. Testes were squashed by adding a cover slip, the slide was dipped in liquid nitrogen and the cover slip was removed with a scalpel. Testes were then stored in PBS+0.1% Tween-20 (PBS-T) until all the samples had been prepared. Samples were blocked for 30 minutes with PBS-T+10% FCS, then incubated with primary antibody diluted in blocking solution overnight at 4°C. Testes were rinsed once then washed four times for 10 minutes each in PBS-T. Testes were incubated with secondary antibodies diluted in blocking solution for 2 hours at room temperature. Samples were washed as before and mounted in 85% glycerol with 2.5% n-propyl gallate. Cover slips were sealed with nail varnish and the samples were imaged using a Leica TCS SP 2UV confocal microscope. Primary antibodies used were rabbit anti-PCM, rabbit anti-dDcp1 (provided by J. Wilhelm) and mouse anti-Me31B (provided by A. Nakamura). For immunohistochemistry, primary antibodies were used at a dilution of 1:500, 1:1000, and 1:1000, respectively. Secondary antibodies used were Cy-3-conjugated monoclonal mouse anti-rabbit IgG, Cy-5-conjugated goat anti-mouse IgG, FITC-conjugated goat anti-rabbit IgG (Jackson Immuno Research). The secondary antibodies were used at a dilution of 1:200. The size of the cytoplasmic particles were measured using ImageJ software [START_REF] Abramoff | Image processing with ImageJ[END_REF]. In each case the size of particles were measured for 5 different areas (35 m x 35 m) to ensure repeatable results. The observed differences between the cases were statistically significant according to Mann-Whitney test. The difference between the sizes of the cytoplasmic particles in pcm 5 mutants under normal and stress conditions as detected using the dDcp1 antibody were rather low and the results of the Mann-Whitney test were inconclusive (W=236, P=0.091, which is >0.05 but <0.1); therefore we also used the exact permutation test. The percentage of co-localisation was calculated using ImageJ software. In each case the percentage of co-localisation was determined for 3 areas (25 m x 25 m) to ensure repeatable results.

Results

The 5' -3' exoribonuclease pacman affects male fertility in Drosophila In order to study the effect of the 5' -3' exoribonuclease pacman on male fertility we examined males carrying mutations in the pacman gene. These mutations had been previously constructed using P-element excision of a P-element transposon located 584nt downstream of the pcm polyA site, between pcm and the convergent gene Nat1 on the Xchromosome at position 18D [START_REF] Grima | The 5' -3' exoribonuclease pacman is required for epithelial sheet sealing in Drosophila and genetically interacts with the phosphatase puckered[END_REF]. Two of the strongest alleles (pcm 3 and pcm 5 ) were used in this study (Figure 1, panel A). Molecular characterisation of these alleles showed that both encoded truncated proteins. The pcm 3 allele was the result of a deletion from the P-element insertion site into the 3' end of the pcm gene and resulted in a C-terminal truncation of 197 amino-acids relative to wild-type. The pcm 5 allele results from an intragenic deletion and encodes a protein C-terminally truncated by 348 amino-acids (see Methods for further details). Using a polyclonal antibody which specifically detects PCM [START_REF] Grima | The 5' -3' exoribonuclease pacman is required for epithelial sheet sealing in Drosophila and genetically interacts with the phosphatase puckered[END_REF] in Western blotting experiments we showed that testes dissected from hemizygous pcm 3 males produced truncated protein at levels significantly lower than wild-type whereas pcm 5 animals expressed very low levels levels of pcm protein (Figure 1, panel B and C).

We next tested the effect of these pcm mutant alleles on male fertility by mating individual males to 3-5 day old OregonR wild type virgin females and counting the offspring. The pcm 5 males were seriously compromised in their fertility producing only 47% of the progeny of control males at 25°C. (Figure 2, panel A). Examination of testes from these mutant males showed that the mutant testes from 1 day-old pcm 5 males were much shorter and thinner than controls (Figure 2, panels B and D). Measurements of the length and width of these testes also showed that they were smaller than controls (Figure 2, panels C, D and G). Male pcm 3 flies were intermediate in numbers of offspring and size of testes between pcm 5 and controls (Figure 2, panels A, C, D andF). These smaller testes produced fewer sperm (68% of wild-type in the case of pcm 3 ; 52% of wild-type for pcm 5 , (Figure 2, panel B) which would account for the lower fertility of the pcm mutants.

Pacman is localised in cytoplasmic particles in wild-type testes

To start to understand how the exoribonuclease pcm might affect male fertility, we first examined the expression and localisation of pcm protein in Drosophila testis. Using our anti-PCM antibody, we showed that PCM is well expressed at the tip of the testis, in the spermatagonia where the initial mitotic divisions take place (Figure 3, panel A) [START_REF] Fuller | Genetic control of cell proliferation and differentiation in Drosophila spermatogenesis[END_REF]. Closer examination revealed that PCM is located in foci in these cells (Figure 3, panel B). To confirm that PCM is located in cytoplasmic granules in living cells, we generated a UAS-GFP-PCM transgenic which expresses GFP fused in frame to the N-terminus of PCM under the control of the UAS enhancer. When this GFP-PCM is expressed in testes using the bam-GAL4 driver, GFP-PCM also accumulates in particles (Figure 3, panel C). However in bam-Gal4, UAS-GFP-PCM flies the GFP-PCM expression also appears in the fusome, a membranous organelle that allows endoplasmic reticulum connectivity between cells [START_REF] Snapp | The fusome mediates intercellular endoplasmic reticulum connectivity in Drosophila ovarian cysts[END_REF]. This is most likely to be because it is over-expressed from the foreign promoter. The expression of PCM in spermatogonia suggests that it is required for the amplification or maintenance of these dividing spermatogonia prior to their entry into pre-meiotic Sphase as primary spermatocytes.

We then examined the localisation of PCM in primary spermotocytes, which are cells that do not divide but undergo massive transcription and growth. In these cells, PCM is located in discrete, cytoplasmic foci as detected in both GFP-PCM flies (Figure 3, panel D) and immunocytochemistry experiments (Figure 3, panel E). The staining of granules in testis from transgenic flies expressing GFP-PCM co-localises with PCM antibody staining (Figure 3, panel F). These experiments show that PCM is also required at this stage of differentiation. This is the first time that cytoplasmic foci that may be related to P-bodies have been seen in Drosophila testis. These particles are reminiscent of Xrn1 particles in mouse cells under conditions where the cells are not stressed, and native levels of protein (rather than protein expressed from a foreign promoter) are observed. In human tissue culture cells such as HEK293 cells, P-bodies can also be present in unstressed cells [START_REF] Kedersha | Stress granules and processing bodies are dynamically linked sites of mRNP remodeling[END_REF]. This data shows that PCM is dynamically localised in testis cells and suggests that these particles could be Drosophila P-bodies.

Pacman is co-localised with dDcp1 and Me31B in testis cells

In both yeast and human tissue culture cells P-bodies have been shown to contain enzymes required for decapping of mRNA (dDcp1 and dDcp2) as well as the helicase Me31B/Dhh1/p54 [17-20, 32, 33]. In both human and yeast cells, Dcp1 is generally regarded as a marker for P-bodies [START_REF] Anderson | RNA granules[END_REF]. To determine whether the PCM foci seen in testis cells might be P-bodies, we investigated whether dDcp1 is located in cytoplasmic granules and if PCM is co-localised with dDcp1. Use of both an anti-dDcp1 antibody and a dDcp1-YFP transgenic show that dDcp1 is localised in cytoplasmic granules in both spermatogonia and primary spermatocytes (Figure 4, panel A and B). Co-localisation experiments showed that dDcp1 is largely, but not completely co-localised with PCM in spermatocytes in that 95% of PCM protein visualised is co-localised with dDcp1 (Figure 4, panels C-E).

Another protein known to be critically important in P-body formation in S. cerevisiae is the RNA helicase Dhh1. This enzyme is highly conserved with the known homologue in Drosophila being Me31B [START_REF] Nakamura | Development[END_REF]. Using an antibodies to Me31B, dDcp1, and PCM we showed that Me31B co-localises with dDcp1 (Figure 4, panels I-K) and Me31B co-localises with PCM (Figure 4, panels F-H) in spermatocytes (95% in both cases). Close examination of these particles revealed that co-localisation of Me31b and dDcp1 was almost complete, whereas both of these were partially co-localised with PCM (see insets in panels E, H and K). This suggests a structure to these particles which has not previously been observed. The co-localisation between PCM, dDcp1 and Me31B strongly suggest that these particles are akin to P-bodies.

To determine whether PCM physically interacts with dDcp1 we carried out immunoprecipitation experiments. Figure 4 panel L shows that our PCM antibody can coprecipitate dDcp1 protein from both wild-type and mutant testes extracts (no cross-linking procedures were used). Therefore PCM can bind to dDcp1, suggesting molecular and functional interactions between the two proteins.

Pacman mutations affect the size and numbers of testis P-bodies

In the yeast S. cerevisiae, cells mutant for Xrn1 are blocked in 5' -3' exonuclease degradation (xrn1 ) show dramatic increases in size and number of P-bodies [START_REF] Sheth | Decapping and decay of messenger RNA occur in cytoplasmic processing bodies[END_REF]. If these testis granules are the sites where mRNA degradation takes place, we would therefore expect the size of P-bodies to increase when the Xrn1 homologue Pacman is depleted.

To test this prediction we first immunostained hand-dissected testes using our PCM antibody. In pcm 5 mutants, only diffuse, low intensity staining was seen with no cytoplasmic foci visible (Figure 5, panel C). This result is consistent with our Western blotting experiments (Figure 1, panel B) where no PCM band was visible. The pcm 3 mutants showed diffuse, faint staining with few obvious cytoplasmic particles (Figure 5, panel B). Note that this staining in panels B and C was visualised using a higher power of the laser than the other panels. Therefore, as expected, these hypomorphic mutations result in a reduction of staining in these mutants. In addition, in the case of the pcm 3 mutation which encodes a C-terminal truncation of PCM (Figure 1, panel A), these experiments also suggest that the C-terminus of PCM may be important in localising PCM to P-bodies. The C-terminus of PCM includes a polyglutamine repeat, which is deleted at the protein level in the pcm 3 mutant. These data suggest that protein motifs within the Cterminus of PCM, possibly including the polyglutamine repeat, are involved in the localisation of PCM to P-bodies.

We then examined the effects of these pcm mutations on P-bodies using the P-body marker dDcp1. In these pcm mutants, P-bodies are dramatically increased in size and number in a similar way to that observed in yeast cells mutant for Xrn1 (Figure 5, panels D-F). Quantitation of the size difference reveals that the particles are increased 2.7 times in pcm 5 mutants compared to controls (Figure 5, panel G). These results provide additional evidence that the testis granules marked by PCM and Dcp1 are equivalent to yeast Pbodies.

The increase is dDcp1 localisation to P-bodies could be due to an increase in dDcp1 expression or recruitment of dDcp1 from the cytoplasm to these RNA granules. To test this, we performed Western blotting experiments on hand-dissected testis from wild-type and pcm mutants using the dDcp1 antibody. These results showed that the amount of dDcp1 decreases in pcm mutants (Figure 5, panels H and I), supporting the notion that the increase in Dcp1 staining in P-bodies in pcm mutants is due to massive recruitment of this protein to P-bodies.

Pacman foci increase in size and number upon heat stress

In yeast and human tissue culture cells, environmental stresses can result in increases in sizes and numbers of P-bodies. Stresses such as glucose deprivation, osmotic stress and UV irradiation increase the size and numbers of P-bodies in yeast cells [START_REF] Kedersha | Stress granules and processing bodies are dynamically linked sites of mRNP remodeling[END_REF]. Although heat stress has not been reported to lead to an increase in size or numbers of P-bodies in yeast [START_REF] Teixeira | Processing bodies require RNA for assembly and contain nontranslating mRNAs[END_REF], the heat stress used was relatively mild (37 o C) so may not have been strong enough to elicit a P-body response [START_REF] Bond | Stressed out! Effects of environmental stress on mRNA metabolism[END_REF]. In human DU145 cells, heat shock (44 o C for 1 hour) can result in an increase in numbers of P-bodies [START_REF] Kedersha | Stress granules and processing bodies are dynamically linked sites of mRNP remodeling[END_REF].

If these testis granules are akin to human P-bodies, we would expect them to increase in size when the male flies are stressed. To test this prediction we subjected male Drosophila to heat stress. We used heat stress as it is easy to administer to live flies and has already been shown to affect male fertility without substantially compromising viability [37]. Our experiments show that, in testes dissected from males subjected to heat shock (37 o C for 1 hour), the numbers and sizes of granules marked by PCM increases 4.1-fold (Figure 6, B). In addition, granules marked by dDcp1 also increase between 1.3 and 2.1-fold in wild-type and mutant testis when males are heat-shocked (Figure 6, panels D -L). The response of these testis granules to heat stress is very similar to that seen in human tissue culture cells, confirming that these granules are likely to be P-bodies [START_REF] Kedersha | Stress granules and processing bodies are dynamically linked sites of mRNP remodeling[END_REF]. These results raise the possibility that P-bodies are important in the response of testis cells to stress and suggest that mRNA turnover and its relationship to translation is similar between Drosophila and humans.

Discussion

Pacman is located in particles akin to P-bodies in Drosophila testes PCM is located in cytoplasmic particules in both spermatogonia and spermatocytes. What is the function of these particles? Data in this paper suggests that they are akin to Pbodies, which are thought to be involved in the degradation of RNAs. The evidence for this is that PCM (an exoribonuclease) is co-localised with the decapping activator Dcp1 and the helicase Me31b (a Dhh1 homologue). In addition, we have shown that PCM is physically associated with dDcp1. Dcp1 is often regarded as a marker for P-bodies [START_REF] Anderson | RNA granules[END_REF]. In addition, these particles behave in the same way as yeast and human P-bodies in that they increase in size upon stress (Figure 6) an also in mutants where members of the 5' -3' pathway is downregulated.

Although elegant work in yeast has shown that P-bodies accumulate partially degraded mRNAs, further work has shown that P-bodies can also store mRNAs. Therefore the function of P-bodies in yeast is not yet clear. However, our recent work examining PCM localisation of PCM in Drosophila egg chambers has shown the existence of two types of particle containing PCM, dDcp1 and dDcp2: one type in the nurse cell cytoplasm which responds to stress and depletion of RNA decay factors and the other type in the oocyte which is resistant to these factors [START_REF] Lin | Drosophila processing bodies in oogenesis development[END_REF]. Since the oocyte particles are also co-localised with known translational repressors such as Oskar, these appear to be storage bodies, rather than particles involved in mRNA degradation [START_REF] Lin | Drosophila processing bodies in oogenesis development[END_REF]. Furthermore, in Drosophila neurones, PCM is co-localised with translation repression factors such as dFMR1, Cup and Pumilio in neuronal granules [39], suggesting that it is also involved in translational repression in these tissues. Although we cannot rule out that the PCM particles we see in testis are storage granules, their response to stresses and depletion of mRNA factors, together with their small size, suggests to us that they are involved in RNA degradation.

The large size of spermatocytes has allowed us to analyse the internal structure of PCMcontaining granules in a natural tissue in a way that has not previously been possible in yeast or human tissue culture cells. As shown in Figure 4, Me31b and dDcp1 are almost completely co-localised with each other in these particles, whereas PCM is partially colocalised with both. Note that this co-localisation has been seen using antibody staining of wild-type testis, showing that co-localisation is not an artifact arising from over-expression of fusion proteins. These results are similar to that seen in the cytoplasm of nurse cells in Drosophila egg chambers, where PCM is partially colocalised in particles with Me31b and dDcp1 [START_REF] Lin | Drosophila processing bodies in oogenesis development[END_REF]39]. These data suggest a scenario where the helicase Me31b and the decapping activator dDcp1 bind mRNA together and then bring it to the exoribonuclease PCM. Further experiments using GFP-and YFP-tagged proteins in live testis imaging are in progress to understand the dynamic nature of these particles in these natural tissues.

Pacman affects male fertility

Our data show that the exoribonuclease pcm is required for normal male fertility in Drosophila melanogaster. Examination of Drosophila males carrying hypomorphic mutations in pcm show that their testes are small and abnormal and that they produce fewer offspring compared to controls. The expression levels of pcm are correlated with the number of offspring produced as pcm 5 males (which express undetectable levels of truncated protein) produce fewer offspring that pcm 3 males, (expressing higher levels of mutant protein). These data show for the first time, that an exoribonuclease is required for spermatogenesis.

How could a exoribonuclease, which is relatively non-specific in-vitro [11] affect spermatogenesis? PCM is expressed at the tip of the testis, in the spermatogonia, showing that PCM is required in these mitotically dividing cells. In these cells, it is possible that pcm perturbs TGF-beta signalling as the mutant testes strongly resemble those mutant in the TGF-beta ligand Punt [40]. Indeed, in complementary experiments, we have shown that pcm genetically interacts with puckered, a member of the JNK signalling pathway that can regulate TGF-beta signalling [START_REF] Martin-Blanco | Puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila[END_REF]. TGFbeta signalling is required for maintenance of the germline stem cells: lack of the TGF-ligand Glass bottom boat (Gbb) results in a reduction of stem cells with the concomitant reduction of mitotically dividing spermatogonia. In spermatocytes, PCM may disrupt differentiation of spermatocytes by the inappropriate upregulation of target RNAs. Since pcm is highly conserved, and the spermatogenesis process has similarities between Drosophila and humans, it is also likely that human Xrn1 affects human spermatogenesis. Our further work to elucidate the targets of PCM in Drosophila spermatogenesis will therefore promote understanding of gene regulation in human germ cells. Protein extracts were prepared from wild-type (control and wt lanes) or pcm 5 testes. These were used directly (input -labelled 1) or immunoprecipitated (labelled 2) with (wt and pcm 5 ) or without (control) our anti-PCM antibody. No crosslinking procedures were used. Extracts were then electrophoresed on a non-denaturing gel and Western blotted using an anti-dDcp1 antibody. In the pcm mutant the amount of dDcp1 co-precipitated is less than in the wt as expected.

Biochemical

  Journal Immediate Publication. Published on 25 Jul 2008 as manuscript BJ20071720 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071720 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 25 Jul 2008 as manuscript BJ20071720 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071720 S t a g e 2 ( a ) P O S T -P R I N T panels A and

Biochemical

  Journal Immediate Publication. Published on 25 Jul 2008 as manuscript BJ20071720 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071720 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Figure Legends. Figure 1. (A)Diagram showing the genomic structure of pcm. Exons ( ), introns ( ), the "domino" repeat (QQEAQ), the polyglutamine repeat (polyQ) and the poly-adenylation site is shown. The insertion site of the P-element used to generate deletions in the pcm gene is given, as is the downstream gene CG12202 (Nat1). The pcm alleles described in this paper are given below: gaps represent deletions at the genomic DNA level. (B) Western blotting of pcm hemizygous mutant male testes showing that the truncated pcm proteins are not detectable (pcm 5 ) or are poorly expressed (pcm3 ). An actin loading control is shown below. (C) Histogram of normalized expression levels relative to loading controls. Means and standard errors are given.

Figure 2 .

 2 Figure 2. Effects of pcm on spermatogenesis. (A) pcm mutant males produce fewer offspring than controls. Means and standard errors are given. (B) pcm mutant males produce fewer mature sperm than controls. Seminal vesicles were dissected from 3-day old virgin males at 25 o C and the mature sperm counted. Means and standard errors are given. (C), (D) pcm mutant testes are shorter (C) and thinner (D) than controls. Means and standard errors are given.(E-G) pcm mutant testes are smaller and "weedier" than controls. (E), (F), and (G) represent testis dissected from 2-day old wild-type, pcm 3 , and pcm 5 males, respectively.

Figure 3 .

 3 Figure 3. Localization of PCM in testis cells. (A) PCM antibody staining shows that PCM is highly expressed at the tip of the testis. (B) PCM is expressed in cytoplasmic foci in spermatogonia. Scale bar: 10 m. (C) GFP-PCM is expressed in cytoplasmic foci in spermatogonia from transgenic males. (D-F) GFP-PCM expressed in spermatocytes in transgenic males and (D) co-localizes with PCM detected using PCM antibody (E). (F) Merged image. Scale bar: 10 m.

Figure 4 .

 4 Figure 4. PCM is partially co-localized with the mRNA decapping enzyme dDcp1 and the helicase Me31B in P-bodies. (A, B) dDcp1 antibody staining (A) and YFP-dDcp1 expressed in testis cells in transgenic males (B) showing that dDcp1 is expressed in cytoplasmic foci in testis cells. Scale bars: 10 m. (C-E) YFP-dDcp1 expressed in testis cells in transgenic males (C) is partially co-localized with PCM detected using PCM antibody (D). (E) Merged image. Scale bar: 2 m. (F-H) The helicase Me31B (F) is partially co-localized with PCM (G) in cytoplasmic particles in testis cells. (H) Merged image. Scale bar: 2 m. (I-K) The helicase Me31B (I) is partially co-localized with dDcp1 (J) in cytoplasmic particles in testis cells. (K) Merged image. Scale bar: 2 m. (L) PCM and dDcp1 proteins physically interact.Protein extracts were prepared from wild-type (control and wt lanes) or pcm5 testes. These were used directly (input -labelled 1) or immunoprecipitated (labelled 2) with (wt and pcm5 ) or without (control) our anti-PCM antibody. No crosslinking procedures were used. Extracts were then electrophoresed on a non-denaturing gel and Western blotted using an anti-dDcp1 antibody. In the pcm mutant the amount of dDcp1 co-precipitated is less than in the wt as expected.

Figure 5 .

 5 Figure 5. (A-C) PCM antibody staining shows that cytoplasmic particles are more diffuse in pcm mutants than controls. (A), (B), and (C) represent wild-type, pcm 3 , and pcm 5 testis, respectively. Images for pcm mutants (B, C) were taken at a higher power of confocal

Figure 6 .

 6 Figure 6. Under stress conditions the cytoplasmic particles containing PCM and dDcp1 increase in size. (A-C) Particles containing PCM in wild-type spermatogonias increase in size upon stress. (A) Normal condition. (B) Stress condition. (C) PCM cytoplasmic particles increase in size under stress conditions. Means and standard errors are given. The observed difference between the normal and stress conditions was statistically significant (Mann-Whitney test, W=2.5, P<0.0001). (D-L) Particles containing dDcp1 in wild-type (D-F), pcm 3 (G-I), and pcm 5 (J-L) spermatogonias increase in size upon stress. (D, G, and J) Normal condition. (E, H, and K) Stress condition. Scale bar: 5 m.(F, I, and L) dDcp1 cytoplasmic particles in wild-type (F), pcm 3 (I), and pcm 5 (L) increase in size upon stress. Means and standard errors are given. The observed difference between the normal and stress conditions in wild-type, pcm 3 , and pcm 5 was statistically significant (wild-type, Mann-Whitney test, W=49, P<0.0001; pcm 3 , Mann-Whitney test, W=125.5, P=0.0054; pcm5 , exact permutation test, T=262, P=0.049).

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 25 Jul 2008 as manuscript BJ20071720 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071720 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

We would like to thank Helen Glenwright, Carol Roberts and Dawn Field for technical help and Sergey Budaev for help with the statistical analysis. We are grateful to James Wilhelm for his gift of the anti-dDcp1 antibody, and Akira Nakamura for his gift of anti-Me31B antibody. This work was supported by the Leverhulme Trust (F-086398), the UK Biotechnology and Biological Sciences Research Council (G16929-2, and BB-C005163-1), the Nuffield Foundation, the Genetics Society, and the National Science Council (Taiwan, 94-2311-B-002-032).