Mechanistic insights into p53-promoted RNA-RNA annealing1

J. Mol. Biol. (1997) 266, 677±687

Mechanistic Insights into p53-promoted RNA-RNA Annealing Wolfgang Nedbal, Manfred Frey, Bernhard Willemann Hanswalter Zentgraf and Georg Sczakiel* Forschungsschwerpunkt Angewandte Tumorvirologie Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242 D-69120, Heidelberg, Germany

The tumour suppressor protein p53 promotes the annealing of complementary nucleic acids in vitro. We observed an up to 1600-fold increase of RNA-RNA annealing by recombinant p53 protein which was shown to bind to RNA in a sequence-independent way. Nuclease mapping experiments suggest that p53 binds to intramolecular duplex portions and only marginally changes the overall secondary structure of RNA at conditions of increased annealing. Thus, the mechanism of p53-promoted RNA-RNA annealing does not seem to be dependent on an activity that melts or changes RNA structure. The activation enthalpy of RNA-RNA annealing is decreased in the presence of p53, i.e. the p53 protein could stabilize the transition state whereas the activation entropy is unfavourable. A comparison with thermodynamic data measured for other facilitators strongly suggests that the mechanism of p53-promoted RNA-RNA annealing is distinct from the mechanism by which other facilitators work. The annealing activity of p53 is almost abolished in the presence of magnesium indicating that it can be sharply regulated in vitro and, in principle, could also be regulated in vivo. # 1997 Academic Press Limited

*Corresponding author

Keywords: p53 protein; RNA-RNA annealing; protein-RNA interactions; macromolecular assembly; p53-RNA interaction

Introduction Interactions of complementary ribonucleic acids (RNA) are essential in biological processes such as the regulation of mRNA translation, the splicing of pre-mRNA (Gesteland & Atkins, 1993; Madhani & Guthrie, 1994), and pathways involving RNA (Wagner & Simons, 1994). Consequently, the possibility of ef®cient and regulatable interactions between complementary RNA is a prerequisite for the metabolism in living cells. The association of complementary RNA has been described in thermodynamic and kinetic terms in many cases (Uhlenbeck, 1972; Yoon et al., 1975; Labuda & PoÈrschke, 1980; Wagner & Simons, 1994). In the case of naturally occuring prokaryotic antisense RNA as well as arti®cial antisense RNA in mammalian cells, kinetic models seem to be appropriate to understand the relationship between ef®cacy in vivo and biochemical parameters determined in vitro (Wagner & Simons, 1994; Rittner et al., 1993). For this reason, facilitators of RNA-RNA anAbbreviation used: CTAB, cetyltrimethylammonium bromide. 0022±2836/97/090677±11 $25.00/0/mb960813

nealing can be described by their in¯uence on the association rate constant (kass) for a given pair of complementary RNA. This view of RNA-RNA annealing in vivo is consistent with the ®nding that a series of eukaryotic cellular proteins, including several hnRNP proteins (Pontius & Berg, 1990; Portman & Dreyfuss, 1994), the nucleocapsid protein (NC) of the human immunode®ciancy virus type 1 (Dib-Haij et al., 1993; Tsuchihashi et al., 1993), the yeast translational initiation factor TifIII (Altmann et al., 1995) and the tumour suppressor protein p53 (Oberosler et al., 1993) perform a signi®cant annealing activity in vitro. The annealing activity for most of these proteins was not reported to be sequence speci®c. The p53 protein has been shown to be a potent facilitator of RNA-RNA annealing (Oberosler et al., 1993; Wu et al., 1995) and most of its structural and functional domains have been described (for a review see Soussi & May, 1996). For these reasons, the p53 protein is a suitable candidate to investigate the molecular mechanism of non-sequence-speci®c protein-promoted RNA-RNA annealing in more detail. Here, we address the mechanism of p53-promoted RNA-RNA annealing # 1997 Academic Press Limited

678

p53-promoted RNA-RNA Annealing

Table 1. p53-promoted annealing of different pairs of complementary RNA Sense RNA (nucleotides) SR6b (645) SR6b (645) SR6b (645) SR4c (420) SR4c (420) se150c (150) se150c (150)

Antisense RNA (nucleotides) aY69b (69) Rz60c (100) aY150d (150) aY69b (69) Rz60c (100) Rz60c (100) aY69b (69)

Maximal increase of k (x-fold)a 580 900 490 1600 730 1230 720

a

[p53]  1 mM. Reference: Homann et al. (1993). c Reference: Homann (1995). d Reference: Sczakiel et al. (1992). b

in terms of thermodynamic and structural parameters as well as in terms of possibilities for its regulation. Recently, a structural model for the antisense RNA aY69 (Eckardt, Romby & G. S., unpublished) and a scheme for duplex formation between aY69 (69 nt) and its complementary RNA SR6 (645 nt) had been proposed (Eckardt & G. S., unpublished). For this reason, major parts of the experiments of this study focused on the p53-promoted annealing of this pair of complementary RNA.

Results The p53 protein increases RNA-RNA annealing in a sequence-independent way The recombinant p53 protein used in this study was expressed in Escherichia coli and puri®ed by af®nity chromatography via its amino-terminal histidine tag (see Materials and Methods). The in¯uence of the p53 protein on RNA-RNA annealing was ®rst measured with six pairs of complementary RNA with varying length (Table 1). To distinguish double-stranded RNA formed during the annealing reaction from complexes between p53 protein and RNA, samples were treated with proteinase K prior to further analysis. The time course of the annealing reaction was followed by separation of single-stranded RNA and duplex RNA on semi-denaturing polyacrylamide gels (Figure 1A) and second order rate constants were calculated as described (Homann et al., 1993). The p53-promoted annealing reaction resulted in the completely formed RNA-RNA double strand as was indicated by the accumulation of RNase T1-resistent RNA (Figure 1B). By treatment of samples with phenol or proteinase K, it was excluded that the product of the reaction is an aggregate between singlestranded or double-stranded RNA and the p53 protein (data not shown). For all pairs of complementary RNA, annealing was increased in the presence of p53 protein by a factor ranging between 480 and 1600 (Table 1) and increased annealing was not dependent on the length of the RNA used. These results strongly suggest that the annealing activity of p53 is not sequence speci®c.

Figure 1. Effect of p53 on the rate of RNA-RNA annealing. A, Time course of the association reaction between the HIV-1-derived RNA SR6 (unlabelled, 645 nt, 200 nM left panel and 20 nM right panel) and the complementary RNA aY69 (32P-labelled, 0.5 nM, 69 nt) in the absence (left panel) or presence (right panel) of p53 protein (1.3 mM). Note that the rate limiting RNA concentrations (SR6) are different in the presence or absence of p53 protein. B, The product of the p53-promoted anealing reaction is the completely formed RNA-RNA duplex. Aliquots of the reaction mixture were treated extensively with RNase T1 and products were analysed on 5% (w/v) polyacrylamide gels. Left-hand side: nondenatured samples; right-hand side: heat-denatured samples.

The p53 protein binds to RNA To test the relationship between binding of p53 to RNA and increased annealing, we performed gel shift experiments with a set of different proteins at the same buffer conditions and temperature that were used for the annealing reaction. Binding of p53 was measured for RNA ranging in size between 69 and 645 nucleotides (Table 1). Binding studies with a hydrolysate of a 150-mer (aY150; Sczakiel et al., 1992) indicated that also chain lengths as short as 20 nucleotides were bound by p53. Further, p53 bound to partially doublestranded RNA (data not shown). RNA binding was also measured with a set of control proteins including a p53 protein with an amino acid exchange at position 267 termed p53mt267 (Arg267Trp) as well as with the unrelated p78Rep

679

p53-promoted RNA-RNA Annealing Table 2. Binding and annealing activity of the p53 protein and a set of control proteins

p53 (wt) p53-
30 p53mt267 p78Rep BSA Lysozyme Proteinase K

Bindinga

Annealing activitya

100 <5 100 100 0 <7 0

100 <1 31 0 0 n.d.b 0

a Binding was measured with aY69 in the absence of magnesium. Annealing was measured with aY69 and SR6. b n.d., not determined.

protein encoded by the adeno-associated virus type 2 (AAV-2; V. Wollscheid et al., unpublished results) which were expressed, puri®ed and renatured according to the protocol used for p53. The relatively basic lysozyme bound weakly to RNA (Table 2) whereas neither the deletion mutant p53-
30 nor the unrelated proteinase K and bovine serum albumine (BSA), respectively, showed binding (Table 2). The comparison of the binding properties of p53(wt), p53-mt267 and p78Rep with their ability to enhance RNA-RNA annealing (Table 2) is not consistent with a simple correlation between binding and increased annealing. It is noteworthy that the p53-mt276 protein which was identi®ed in a human tumour of the liver (Volkmann et al., 1994) showed a reduced annealing activity despite its unaffected ability for binding. This ®nding indicates that binding of p53 with RNA per se is not related to the annealing activity and could indicate a difference between the wild-type p53 protein and the p53-mt276 mutant that could be linked with their phenotype.

Figure 2. Annealing activity of p53 (monitored by the second order rate constant kobs) as a function of the p53/RNA ratio for the association of the complementary RNA aY69 and SR6.

not lack the a-helical domain which is thought to be suf®cient for dimer formation (StuÈrzbecher et al., 1992), one could conclude that dimer formation of p53 can take place whereas the formation of tetramers or higher oligomeric forms, that make use of ionic interactions via the basic domain cannot occur. When a mixture of the wild-type p53 protein and p53-
30 was applied, a dramatic loss of the annealing activity was observed that was reminescent of dominant negative interference (data not shown) suggesting that p53 does not perform its annealing activity as a monomer.

Domains of the p53 protein involved in its annealing activity

The stoichiometry of p53 protein and RNA

The recombinant p53 protein used in this work contains six histidine residues at its amino terminus. This stretch of histidine residues, however, does not seem to be involved in the annealing activity since neither the p53 derivative p53-
30 nor the unrelated protein p78Rep showed annealing activity although both proteins had been expressed by the same system that was used for the wild-type p53 protein. Unspeci®c RNA-protein interactions can be related to interactions between basic portions of a protein with the phosphate backbone of RNA (Draper, 1995). A basic region is located next to an a-helical domain at the carboxy terminus of the p53 protein (StuÈrzbecher et al., 1992) which is not present in the carboxy terminally truncated derivative p53-
30 lacking 30 amino acids. This p53-
30 derivative failed to increase RNA-RNA annealing (Table 2) suggesting that the basic domain of p53 is involved in its annealing activity which is consistent with a recent study (Wu et al., 1995). Since p53-
30 does

The RNA-RNA annealing was measured as a function of the ratio between p53 and RNA. Half maximal enhancement of annealing occurred at a ratio of p53 to SR6 RNA (645 nt in length) of approximately 65 (Figure 2), i.e. one p53 molecule is bound per ®ve phosphates or even more. Assuming that p53 forms a tetramer in solution (McCormick et al., 1981; Stenger et al., 1992), this means that approximately one p53 tetramer is bound per 20 or more phosphates at conditions of maximal enhancement of RNA-RNA annealing. It is reasonable to assume that only a fraction of the renatured recombinant p53 protein was in an active form. Therefore, the results can only be used to derive a lower limit for the ratio between p53 and RNA. The relationship between the ratio of p53/RNA and the association kinetics (Figure 2) indicates a cooperative step in p53-promoted RNA-RNA annealing. Cooperativity could either re¯ect oligomer formation of p53 in solution prior to binding to the RNA or, secondly, cooperative

680

p53-promoted RNA-RNA Annealing

Figure 3. Probing of the RNA structures of aY150 (1ng/ml) in the presence of p53 (400 ng/ml) with the single strandspeci®c RNase A, RNase T1, and RNase Cl3. Arrows with ®lled arrow heads indicate cleavage at positions that are not in¯uenced by p53 protein. Arrows with open arrow heads indicate cleavage at positions that are strongly decreased by p53 protein (RNase A). Note that beside the indicated disappearing cleavage products with RNase A weaker bands disappear as well in the presence of p53 protein (middle panel).

binding of forms of p53 to the RNA. It is noteworthy, however, that the p53 protein can multimerize in solution (McCormick et al., 1981) and that the recombinant p53 protein used in this work forms aggregates of approximately 700 kDa as was observed by electronmicroscopy (M. F. et al., unpublished results) The binding of p53 protein does not cause major changes of the overall secondary structure of RNA For a number of proteins with nucleic acid annealing activity including the p53 protein it was speculated that changes of RNA structure could be important (Kumar & Wilson, 1990; Oberosler et al., 1993; Portman & Dreyfus, 1994). A sensitive method to detect structural changes of RNA is chemical or enzymatic probing. To test whether p53 in¯uences the secondary structure of RNA in the p53RNA complex, we performed enzymatic probing. To test whether p53 in¯uences the secondary structure of RNA in the p53-RNA complex, we performed enzymatic probing of RNA molecules in the presence of p53 protein at the same temperature and buffer conditions that were used in the annealing experiments before. In the case of the RNA aY69, only very few sites are accessible for RNase A, RNase T1, and RNase Cl3, however, no

in¯uence of p53 on the cleavage pattern was observed (data not shown). To substantiate further this ®nding, we also used the 150 nt long RNA aY150 for enzymatic probing in the presence or absence of the wild-type p53 protein. For RNase T1 and RNase CL3, no signi®cant changes of cleavage positions and cleavage intensities were observed (Figure 3). In the case of RNase A, loss of cleavage at sites located within certain subregions of aY150 can be seen (Figure 3). Loss of cleavage could be explained by interference between binding of p53 with the RNA and nuclease-mediated cleavage. The remaining RNase A-mediated cleavages occur at the same sites that were cleaved in the absence of p53 and show that the nuclease is active. The in¯uence of p53 on the cleavage pattern of the double strand-speci®c RNase V1 is shown in Figure 4. A partial protection of p53 against cleavage by RNase V1 indicates that either doublestranded portions of the RNA are bound by p53 or, theoretically, that p53 alters the structure of the double-stranded portions such that they are not cleavable by RNase V1. The ®ndings in the use of RNase A and RNase V1 together with the unaffected cleavage by RNase T1 and RNase CL3 are consistent with binding of p53 to the RNA with minor but not overall structural changes. Thus, we assume that the arrangement of secondary struc-

681

p53-promoted RNA-RNA Annealing

The annealing activity of p53 is magnesiumdependent in vitro

Figure 4. Probing of aY150 with the double strandspeci®c RNase V1 in the presence or absence of p53 protein. Filled bars on the left indicate cleavage products that do not occur in the presence of p53 protein.

ture elements assembling the overall structure of RNa is not affected signi®cantly by p53. However, one cannot exclude that p53 in¯uences the threedimensional arrangement of structural elements such that annealing is promoted.

Annealing studies of naturally occuring complementary RNA in vitro are usually performed in the presence of magnesium (e.g. Persson et al., 1990; Tomizawa, 1984). Conversely, the proteinpromoted RNA-RNA annealing of nucleic acids had been studied at low magnesium concentrations (43 mM; Portman & Dreyfus, 1994; Altmann et al., 1995) or in the absence of magnesium (Kumar & Wilson, 1990; Oberosler et al., 1993; Tsuchihashi & Brown, 1994; Wu et al., 1995). The annealing activity of the HIV-1 NC protein was reported to be in¯uenced by magnesium (Lapadat-Topolsky et al., 1995). Thus, we tested the in¯uence of magnesium on p53-promoted RNA-RNA annealing. Magnesium by itself increased the association rate of aY69 and SR6 from kass ˆ 3  103 Mÿ1sÿ1 to k ˆ 2.7  104 Mÿ1sÿ1 in the absence of p53 protein though at 100 mM NaCl. When p53 was added in the absence of magnesium, the annealing rate was increased 570-fold. Conversely, the p53-mediated increase in the presence of magnesium (10 mM) was only two to fourfold (Table 3). At approximately 2 mM magnesium we observed a half reduction of the annealing activity of p53 (Figure 5). The p53 protein used in this work was renatured in a buffer that contained Zn2‡. Assuming that Zn2‡ bound to the p53 protein was necessary for its annealing activity, one could hypothesize that increased concentrations of magnesium could displace Zn2‡ from p53 protein and, thereby, decrease its annealing activity. However, when the RNA association kinetics were measured at increasing concentrations of Zn2‡ (0 to 10 mM) and in the presence of 3 mM magnesium, no increase of RNA-RNA annealing was observed. Conversely, Zn2‡ alone, i.e. in the absence of Mg2‡ had the same inhibitory effect on p53-promoted RNA-RNA annealing as was measured for magnesium. Influence of p53 on the activation energy of RNA-RNA annealing The association rate constant k and the Gibbs free activation energy (
G6ˆ) are linked via the

Table 3. Effect of p53 on thermodynamic parameters of RNA-RNA annealing at 37 C p53 (1.3 mM) ÿ ‡ ÿ ‡ a

Mg2‡ (10 mM)

kass (Mÿ1 sÿ1)

Eaa (kJ/mol)


G6ˆb (kJ/mol)


H6ˆc (kJ/mol)


S6ˆd (eue)

‡ ‡ ÿ ÿ

2.7  104 8.0  104 3.0  103 1.7  106

43  3 46  4 100  5 79  4

49 47 57 43

40  3 43  4 97  5 76  4

ÿ30  10 ÿ12  13 131  16 108  13

Ea was calculated from an Arrhenius plot (lnk versus 1/T).
G6ˆ was calculated from the relationship:
G6ˆ ˆ ÿ RTln(kh/kBT) where R is the gas constant, k is the rate constant at the given temperature T, h is the Planck's constant, and kB is the Boltzmann's constant. c
H6ˆ is given by:
H6ˆ ˆ Ea ÿ RT. d
S6ˆ was calculated from the relationship:
G6ˆ ˆ
H6ˆ ÿ T
S6ˆ. e Entropy values are given in eu (cal molÿ1 Kÿ1). 1 eu is equivalent to 4.184  10ÿ3 kJ molÿ1 Kÿ1. b

682

p53-promoted RNA-RNA Annealing

Figure 5. Magnesium dependence of the rate constant (kobs) of p53-promoted annealing of aY69 and SR6. The upper dotted line represents kobs in magnesium-free buffer. The lower dotted line represents kobs in the absence of p53 protein and in the presence of 10 mM magnesium.

equation
G6ˆ ˆ ÿ RTlnk and increased annealing is re¯ected by a decrease of
G6ˆ. However,
G6ˆ is composed of a heat term (
H6ˆ) and an entropy term (
S6ˆ). The in¯uence of p53 on both parameters provides further information on the mechanisms of p53-promoted RNA-RNA annealing. The activation energies were calculated from the temperature dependence of the annealing reaction between aY69 and SR6 in the presence or absence of p53 or magnesium, respectively (Figure 6 and Table 3). In the presence of magnesium, there was a slight p53-mediated increase of RNA-RNA annealing (approximately threefold) which is re¯ected by a decrease of
G6ˆ (Table 3). In this case, the activation entropy
S6ˆ is increased by p53 from ÿ30 to ÿ12 kJ/mol at 37 C whereas there is an unfavourable effect of p53 on Ea and
H6ˆ, respectively (Table 3). Conversely, in case of the approximately 570-fold increase of the annealing reaction in the absence of magnesium, the Arrhenius activation energy (Ea) and, hence,
H6ˆ was substantially lowered from 97 to 76 kJ/mol whereas
S6ˆ was decreased from 131 kJ/mol to 108 kJ/mol at 37 C which is not in favour of increased annealing.

Figure 6. Arrhenius plots for the annealing of aY69 and SR6 in the absence or presence of magnesium (10 mM) or p53 protein (1.3 mM). The Arrhenius activation energy (Ea) was calculated from the relationship: ÿdlnkobs/ dTÿ1 ˆ Ea/R. A, Arrhenius plots for annealing reactions in the absence of magnesium. B, Same reactions in the presence of 10 mM magnesium.

Discussion Characteristics of the annealing activity of the p53 protein The annealing between several pairs of complementary RNA was increased by p53 by a factor ranging between 490 and 1600 (Table 1). This increase is larger than that reported for other proteins that promote the annealing of nucleic acids including the hnRNP protein A1 (>300-fold for DNA-DNA annealing; Pontius & Berg, 1990) and the HIV-1-encoded capsid protein NC (20-fold for RNA-RNA annealing; Herschlag et al., 1994). In a

683

p53-promoted RNA-RNA Annealing

recent study, a factor for p53-promoted RNA-RNA annealing of approximately 200-fold was reported (Oberosler et al., 1993). The difference between that study and this work in quantitative terms could re¯ect different experimental conditions. However, the annealing activity of p53 seems to be stronger than that of many other protein facilitators reported so far, indicating that the annealing activity of p53 could be biologically signi®cant. Domains of the p53 protein involved in the annealing activity The p53 protein has been dissected into structural and functional domains. The amino-terminal domain (positions 1 to 42) is involved in trans activation (Raycroft et al., 1990; Fields & Jang, 1990; Unger et al., 1992; Lin et al., 1994), the central domain (positions 90 to 300) facilitates the sequencespeci®c binding of DNA (Bargonetti et al., 1991; Pavletich et al., 1993; Wang et al., 1993) and the carboxy-terminal domain (positions 311 to 393) is involved in p53 protein oligomerization (StuÈrzbecher et al., 1992) and contains the nuclear localization signal (Shaulsky et al., 1991) as well as phosphorylation sites for casein kinase II (Ser392; Meek et al., 1990) and for cdc-2 (Ser315; Bischoff et al., 1990; StuÈrzbecher et al., 1990). Recently, the ability of p53 to promote the annealing of complementary DNA and RNA was assigned to the carboxy-terminal domain of an alternative splicing product of p53 (Wu et al., 1995). Interestingly, the carboxy-terminal domain was found to be suf®cient for cellular transformation, presumably by interfering with the formation of functionally intact p53 protein oligomers (Shaulian et al., 1992). A mechanistic view of p53-promoted RNARNA annealing The p53-promoted annealing of complementary RNA is accompanied by a decrease of the Arrhenius activation energy (Ea) which is equivalent to a favourable change of the activation enthalpy
H6ˆ. The lowering of Ea (
H6ˆ) indicates that a high energy state of the annealing between aY69 and SR6 is stabilized by the p53 protein. Conversely, the activation entropy
S6ˆ is not signi®cantly in favour of annealing. A favourable in¯uence of a faciliator on
S6ˆ, as found for example for CTAB-promoted RNA-RNA annealing (Nedbal & Sczakiel, 1996), could indicate increased collision frequency between RNA strands or steering; however, this does not seem to be the case for p53-promoted RNARNA annealing. Even though it is impossible to describe the nature of the high energy state (``transition state'') of this pairing reaction some information can be derived from a model for the annealing between aY69 and SR6 that was proposed recently on the basis of structural and kinetic data (S. Eckardt & G. S., unpublished results). According to this model, interactions of approximately three complementary bases located within

a central portion on either strand form critical (rate-limiting) interactions. Assuming that p53 can bind to intramolecular as well as intermolecular double-stranded portions, one could imagine that the p53 protein stabilizes an initial base-speci®c complex between aY69 and SR6. Binding could occur at the contact site, however, it could occur at other sites of either RNA strand as well. In the latter case, p53-p53 interactions could additionally stabilize a ®rst and weak complex formed between both RNA strands. According to this model, major structural changes of the RNA are not required. This model is consistent with the earlier ®nding that duplex formation between oliog(A) and oligo(U) requires the formation of two to three basepairs before elongation of duplex formation proceeds without any further consumption of energy (PoÈrschke & Eigen, 1971). However, when considering that RNase CL3 cleavage is speci®c for C (no in¯uence of p53) and RNase A cleaves beside C as well as U (partial in¯uence of p53; Figure 3, middle panel) one could conclude that p53 has a base-preferential in¯uence on RNA. It remains unclear whether there is any U-speci®c binding of p53 which, eventually, could be related to the fact that the annealing activity with RNA (approximately 1000-fold) is greater than in the case of DNA (<200-fold) which was measured at even tenfold higher p53 concentrations (Oberosler et al., 1993). It is noteworthy that the mechanism underlying p53-promoted RNA-RNA annealing seems to be distinct from the mechanism of other facilitators. For example, in case of cetyltrimethylammonium bromide (CTAB), a model substance used for the hnRNP protein A1 (Pontius & Berg, 1991), the activation entropy of RNA-RNA annealing is positive whereas it is negative in the presence of p53 at a comparable increase of annealing (Nedbal & Sczakiel, 1996). For CTAB, a stabilization of a nonbase-speci®c pre-complex formed in solution between both complementary strands was suggested to explain its annealing activity. For the annealing activity of p53 a similar effect is not indicated by the thermodynamic data described in this work (Table 3). There is further experimental evidence that not all of the known facilitators share the same molecular mechanism. For example, the ribosomal protein S12 from E. coli protein seems to preferentially bind to single-stranded RNA portions and is though to act as ``RNA chaperones'', i.e. it resolves biologically inactive RNA structures and converts them into active ones (Coetzee et al., 1994). Role of magnesium The strong p53-mediated increase of RNA-RNA annealing was measured in the absence of magnesium which is similar to the experimental conditions that were used to study the facilitatormediated increase of nucleic acid annealing in a variety of cases (Oberosler et al., 1993; Wu et al., 1995; Pontius & Berg, 1990; Dib-Hajj et al., 1993). If

684 one considers that the intracellular magnesium concentration was estimated to be in the range of 100 mM to 1 mM (Cowan, 1995; Romani & Scarpa, 1992), then the absence of magnesium is closer to the in vivo situation than 10 mM magnesium as used in this study. When either magnesium or zinc was added, the in¯uence of p53 on RNA-RNA annealing was reduced and above a concentration of 3 mM magnesium or zinc almost abolished (Figure 5). A similar observation was made for the hnRNP protein A1 which also does not promote the annealing reaction at 10 mM magnesium (W. N. et al., unpublished results). One could speculate that magnesium abolishes binding of p53 to RNA due to the neutralisation of negative charges of the phosphate backbone. This view, however, seems to be unlikely since binding of p53 with RNA was measured not only in the absence of magnesium but also, to a similar or even greater extent, in its presence. Alternatively, the ability of p53 protein to form oligomeric complexes (McCormick et al. 1981) could play a role. If tetrameric or higher aggregates of p53 were necessary for increased annealing, one could speculate that magnesium or zinc decrease p53-p53 interactions. It was reported that the carboxy-terminal domain of the p53 protein is suf®cient for dimer formation as well as the formation of larger complexes (StuÈrzbecher et al., 1992). By interference with these ionic interactions, magnesium could mediate its inhibitory effect on p53-promoted RNA-RNA annealing. One could speculate that the regulation of the annealing activity of p53 in vivo could involve divalent ions such as magnesium, however, completely different ways for regulation are conceivable as well. For example, phosphorylation of the hnRNP protein A1 was shown to reduce its annealing activity (Cobianchi et al., 1993). Considering that potential phosphorylation sites are located at the carboxy terminus of the p53 protein which is involved in its annealing activity, one could speculate that the function of magnesium in vitro is substituted by a protein phosphorylation step in vivo. At least two options to control the annealing activity are conceivable. Firstly, the annealing activity of p53 can be directly controlled by factors that act in vivo as magnesium does in vitro which could involve binding of cations to p53 or, alternatively, phosphorylation of p53. Secondly, the annealing activity can be regulated by modulating the available p53 concentration in vivo (Figure 2). Similarly, the annealing activity could be modulated by controlling the p53 concentration in the relevant subcellular compartment. Biological implications The model for p53-promoted RNA-RNA annealing described here could have several biological implications. For example, immediately after DNA damage, p53 protein levels increase posttranscriptionally, Conversely, in cells entering the cell cycle, p53 protein levels are low. Thus, the in-

p53-promoted RNA-RNA Annealing

crease of p53 levels was suggested to be due to a control of p53 expression on the post-transcriptional level, presumably involving binding of p53 to a structured domain located within the 50 -UTR of its mRNA (Mosner et al., 1995). This view is supported by the ®nding that p53 occurs in the cytoplasm of cells of certain tissues (Moll et al., 1992). The p53 protein could perform regulation by in¯uencing dynamic RNA-RNA interactions that involve the 50 -UTR of the p53 mRNA. For example, speci®c intra-strand interactions or interactions between the 50 -UTR of the p53 mRNA and auxilliary factors in trans could be modulated by the p53 protein. According to an earlier ®nding one could hypothesize that the control of p53 gene expression at the post-transcriptional level involves the activity of an antisense RNA (Khochbin & Lawrence, 1989) as well as the p53 protein. In a more general view, the annealing activity of p53 seems to be analogous in functional terms though not in mechanistic terms with the activity of a series of other cellular proteins that promote the annealing of nucleic acids. It is conceivable that an even larger set of cellular proteins, predominantly located in the nucleus but in the cytoplasm as well (Kaslan & Heyer, 1994; Altmann et al., 1995) can promote the annealing of nucleic acids in vivo. The set of such proteins could form sub-cellular environments for more dynamic interactions between complementary nucleic acids in vivo than expected from in vitro studies in the absence of facilitators. Various biological functions have been assigned to the tumour suppressor protein p53. It was shown to bind to DNA in a sequence-speci®c manner (Kern et al., 1991; Bargonetti et al., 1992; El-Deiry et al., 1992) as well as in a mode that is not sequence-speci®c (Steinmeyer & Deppert, 1988). Beside sequence-speci®c interactions of the p53 protein with DNA, biochemical studies have shown that p53 can bind single-stranded DNA ends (Bakalkin et al., 1994) and increase the annealing of complementary DNA (Oberosler et al., 1993; Wu et al., 1994; Bakalkin et al., 1994) as well as RNA (Oberosler et al., 1993; Wu et al., 1995) in a nonsequence-speci®c manner. However, it is important to note that the annealing of complementary DNA is obviously distinct from the annealing of complementary RNA. For example, the pairing process between structured RNA and clearly less folded DNA seem to follow different rules and recombination of complementary DNA strands cannot be compared with RNA-RNA annealing in thermodynamic and kinetic terms. Accordingly, interactions between complementary RNA and DNA could eventually be regulated by different means.

Materials and Methods Synthesis of RNA For RNA run-off transciption in vitro with T7 RNA polymerase, linearized plasmids carrying the T7 promoter

685

p53-promoted RNA-RNA Annealing were used. To generate the HIV-derived 645 nt long RNA SR6 (positions 5366 to 5929 according to Ratner et al., 1985), the plasmid pRC-SR6 (Homann et al., 1993) was linearized with NotI. The in vitro transcription reaction was stopped by adding ten units DNaseI (RNasefree) and a further incubation for 20 minutes at 37 C to degrade the DNA template. The mixture was extracted with phenol/chloroform (1:1 v/v) and the RNA precipitated with 80% ethanol, redissolved in TE buffer (10 mM Tris-HCl (pH 7.6), 1 mM EDTA) and was puri®ed by gel-®ltration on Sephadex G-50 columns with the same buffer. The complementary 69 nt long aY69 RNA was transcribed from the BglII-linearized paY69 plasmid and the complementary 150 nt long aY150 RNA was transcribed from the XhoI-linearized plasmid paY150 (Homann, 1995) as described above. Uniformly 32Plabelled aY69 RNA was synthesized in 20 ml transcription buffer as described above but with the lack of UTP. Instead of it, 50 mCi [32P]UTP were used and transcription reactions were run for 30 minutes at 37 C. Transcription was stopped by phenol/chloroform (v/v 1:1) extraction and 32P-labelled RNA was puri®ed by preparative 10% (w/v) polyacrylamide gel electrophoresis. Polyacrylamide slides were crushed and soaked and RNA was eluated by adding 400 ml Tris-HCl (pH 7.6), 1 mM EDTA and incubation for 12 hours at 4 C. After futher phenol/chloroform (v/v 1:1) extraction RNA was precipitated with 80% ethanol and resuspended in TrisHCl (pH 7.6), 1 mM EDTA. RNA concentration was calculated by determination of the speci®c radioactivity of incorperated [32P]UTP into RNA transcripts using a liquid scintilation counter. Preparation of 50 end-labelled aY150 RNA The 50 -ends of in vivo transcribed aY150 RNA were dephosphorylated with calf intestine phosphatase for 20 minutes at 37 C and, subsequently, rephosphorylated with [g32P]ATP and T4 polynucleotide kinase. 50 Labelled RNA was puri®ed by preparative polyacrylamide gel electrophoresis as described above. RNA-RNA annealing Annealing reactions were performed at 37 C in 30 ml reaction buffer containing 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, 1 mM DTT, 1 mg/ml BSA and 0.05 units/ ml RNase inhibitor RNasin. 10 nM unlabelled SR6 RNA was mixed with >0.5 nM 32P-labelled aY69 in the reaction buffer and the reaction was started by addition of 1 ml p53 protein (1.7 mg/ml). At different time points, aliquots of the reaction mixture were withdrawn and p53 protein was removed by proteinase K (0.5 mg/ml) digestion in the presence of 1% SDS for two minutes at 37 C. Reactions were stopped by adding a eightfold excess of stop buffer containing 7 M urea, 50 mM Tris-HCl (pH 7.6) and 20 mM EDTA. Reaction products were analysed by electrophoresis on 5% (w/v) polyacrylamide gels. Expression and purification of p53 protein Wild-type human p53 DNA was ampli®ed by PCR of a human p53 cDNA. The PCR was designed to enable cloning via BamHI restriction sites into the expression plasmid pQE-8 (Quiagen GmbH) which adds six histidine residues to the N terminus. The resulting DNA sequence was con®rmed by sequencing. The p53 protein

was overexpressed in E. coli in an unsoluble form and was puri®ed under denaturing conditions in 8 M urea on a NTA resin. The denatured p53 protein was refolded by stepwise dilution of urea in a dialysis procedure. Activation of sequence-speci®c DNA binding of the refolded p53 protein was tested with the monoclonal antibody PAb421 and a 20 bp oligodeoxyribonucleotide 50 -GGACATGCCCGGGCATGTCC-30 (Funk et al., 1992). Structural probing of RNA RNase cleavage reactions were performed in 10 ml of the same buffer as used for RNA annealing reactions. For cleavage reaction of RNase Cl3 10 mM potassium phosphate (pH 7.0) was additionally added. 50 -Labelled RNA was preincubated with 1.3 mM p53 protein and cleavage reaction was started by the addition of RNases at concentrations as indicated in the Figures. At indicated time points aliquots were withdrawn and the reaction was stopped by the addition of a threefold excess of stop buffer containing 7 M urea, 50 mM Tris-HCl (pH 7.6), 100 mM NaCl, 20 mM EDTA and 0.5% SDS. Samples were heated at 95 C for ®ve minutes and cleavage products were analysed on 10% polyacrylamide gels containing 7 M urea. Determination of second order rate constants Vacuum-dried 5% polyacrylamide gels were use to quantify the amounts of single-stranded RNA and duplex RNA using a phosphor imager (Molecular Dynamics). Time-dependent decrease of single-stranded aY69 RNA was computer-®tted and was used to calculate the corresponding second order rate constant.

Acknowledgements We thank H. zur Hausen for continuous support.

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Edited by J. Karn (Received 19 August 1996; received in revised form 5 November 1996; accepted 26 November 1996)

http://www.hbuk.co.uk/jmb Supplementary material, comprising two Figures, is available from JMB Online.