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The fragile X mental retardation protein has nucleic acid chaperone properties

The fragile X mental retardation protein has nucleic acid chaperone properties Published online April 19, 2004 Nucleic Acids Research, 2004, Vol. 32, No. 7 2129±2137 DOI: 10.1093/nar/gkh535 The fragile X mental retardation protein has nucleic acid chaperone properties 1 1 1 Caroline Gabus, Rachid Mazroui , Sandra Tremblay , Edouard W. Khandjian and Jean-Luc Darlix* LaboRetro, Unite  INSERM de Virologie Humaine (412), ENS, 46 alle  e d'Italie, 69364 Lyon cedex 07, France and Unite  de recherche en Ge  ne  tique Humaine et Mole  culaire, Centre de recherche Ho à pital St. Franc Ë ois d'Assise, CHUQ, 10 rue de l'Espinay, Que  bec G1L 3L5, PQ Canada Received February 9, 2004; Revised and Accepted March 22, 2004 ABSTRACT expressed, but most abundant in testes and brain, which are the organs strongly affected by the syndrome (4,5). The fragile X syndrome is the most common cause The cellular role of FMRP remains poorly understood and of inherited mental retardation resulting from the the current view is that it regulates mRNA transport and absence of the fragile X mental retardation protein translation in a manner critical for the development of neurons (FMRP). FMRP contains two K-homology (KH) (6,7). To achieve its function, FMRP is thought to be engaged domains and one RGG box that are landmarks char- in a number of interactions with cytoplasmic nucleic acid and protein partners (1,8±15) in association with messenger acteristic of RNA-binding proteins. In agreement ribonucleoparticles (mRNPs) in actively translating ribosomes with this, FMRP associates with messenger ribo- (16,17), however, the exact role of FMRP in translation nucleoparticles (mRNPs) within actively translating remains elusive. Several studies have shown that FMRP can ribosomes, and is thought to regulate translation of act as a negative regulator of translation in vitro and in vivo target mRNAs, including its own transcript. To (18±20) and it has been proposed that in neurons a small investigate whether FMRP might chaperone nucleic fraction of FMRP acts as a repressor for the RNA to be acid folding and hybridization, we analysed the transported and to be delivered at the budding dendrites (20). annealing and strand exchange activities of DNA Indeed, while the great majority of FMRP has been observed oligonucleotides and the enhancement of ribozyme- in the neuron cell body, a small fraction was detected at distal directed RNA substrate cleavage by FMRP and locations such as neurites, dendrites and synaptosomes deleted variants relative to canonical nucleic acid (5,21,22). A series of neuronal mRNAs have been isolated either by immunoprecipitation approaches (8), or by the use of chaperones, such as the cellular YB-1/p50 protein a new technique called `APRA' for antibody positioned RNA and the retroviral nucleocapsid protein HIV-1 NCp7. ampli®cation (24). In addition, several target mRNAs have FMRP was found to possess all the properties of a been isolated by differential display, by oligonucleotide potent nucleic acid chaperone, requiring the KH (ODN)-based and by cDNA-SELEX (9,25,26). Also it has motifs and RGG box for optimal activity. These ®nd- been reported that FMRP interacts with small noncoding ings suggest that FMRP may regulate translation by RNAs such as BC1 and BC200, which in turn mediates their acting on RNA±RNA interactions and thus on the binding to speci®c mRNAs (27). These observations clearly structural status of mRNAs. indicate that FMRP has af®nity to RNAs, however, it is not clear whether all these target mRNAs bind directly to FMRP or if FMRP protein interactors are required. In view of the binding of FMRP to mRNAs and small non- INTRODUCTION coding RNAs, we addressed the question as to whether FMRP The fragile X syndrome is the most common cause of inherited might be a nucleic acid chaperone protein. So-called nucleic mental retardation in humans resulting from the absence of the acid chaperones bind in a cooperative manner to one or more FMR1 gene product, the FMR1 protein [fragile X mental nucleic acid molecules and promote the formation of the most retardation protein (FMRP)] (1,2). FMRP is a protein of 632 stable structure while at the same time preventing folding traps amino acids composed, in N-terminus to C-terminus order, of that may preclude function (28,29). Importantly, nucleic acid a protein-interacting domain (PPId), two K-homology (KH) chaperones do not require ATP to function and once the most motifs, a phosphorylation sequence and an RNA recognition stable nucleic acid structure is achieved, their continued sequence called the RGG box (1±3). FMRP is widely binding is no longer required to maintain the structure (28,29). *To whom correspondence should be addressed. Tel: +33 4 72 72 81 69; Fax: +33 4 72 72 87 77; Email: [email protected] Present address: Rachid Mazroui, Department of Biochemistry, McGill University, Montre Âal, Canada Nucleic Acids Research, Vol. 32 No. 7 ã Oxford University Press 2004; all rights reserved 2130 Nucleic Acids Research, 2004, Vol. 32, No. 7 Canonical nucleic acid chaperones, such as retroviral NC proteins, are able to anneal, under physiological conditions in vitro, a speci®c primer tRNA to a complementary sequence at the 5¢ end of genomic viral RNA; a prerequisite for the initiation of reverse transcription (30,31). In the present study, we examined the ability of FMRP to chaperone the annealing of DNA with complementary sequences as well as strand exchange in a duplex nucleic acid structure in vitro. Furthermore, we investigated whether FMRP is capable of enhancing ribozyme-directed cleavage of an RNA substrate in vitro. Our results show that FMRP is a potent chaperone protein. MATERIALS AND METHODS Recombinant proteins The full-length FMRP and six deleted variants DPPId, DKH1, DKH2, DKHT (DKH1 plus DKH2), DPhD and DRGG FMRP with a C-terminal (His)6-tag were produced in Escherichia coli and puri®ed to homogeneity by af®nity chromatography using Ni-NTA Probond beads (Invitrogen) according to the manufacturer's protocol as previously described (Fig. 1) (3). HIV-1 nucleocapsid protein NCp7 and mutant NC(12±53) were synthesized by the fmoc/opfp chemical method and puri®ed to >98% purity by high-pressure liquid chromato- graphy (30). Proteins were dissolved at 1 mg/ml in buffer Figure 1. Scheme of the FRMP protein and deleted variants. Wild-type re- containing 30 mM HEPES pH 6.5, 30 mM NaCl and 0.1 mM combinant FMRP and its deleted variants used in the present study are sche- ZnCl . matically depicted here. Positions of the deleted regions, namely the protein interacting domain (PPId), the KH1 and KH2 domains, the phosphorylation The YB-1/p50 protein (32) was a kind gift from Lev domain (Phd) and the RGG box are shown. HIV-1 NCp7(1±72) and mutant Ovchinnikov (Moscow, Russia) (31). NC(12±53) with the two CCHC zinc ®ngers (ZF) are shown. The complete YP-1/p50 protein is schematically shown with the N-terminal alanine- and DNA substrates proline-rich (A/P) domain, the cold shock domain (CSD) and basic and acidic amino acids (B/A) clusters domain. Amino acid positions are indi- DNA ODNs corresponding to the HIV-1 TAR and the cated. repeated R sequences (Mal isolate), in the sense and anti-sense orientation were purchased from Eurogentec (Belgium). TAR and R ODNs are 56 and 96 nt in length, Plasmid DNAs respectively. TAR(+) (sense) (30): 5¢-GGTCTCTCTTGTTAGACC- Plasmid DNAs pS14, pS20 and pR3 were kindly provided by AGGTCGAGCCCGGGAGCTCTCTGGCTAGCAAGGA- E. Bertrand (33). All plasmid DNAs were ampli®ed in E.coli ACCC-3¢; TAR(±) (anti-sense): 5¢-GGGTTCCTTGCTAGC- 1035 (RecA-) and puri®ed by af®nity chromatography CAGAGAGCTCCCGGGCTCGACCTGGTCTAACAAG- (Qiagen protocol). AGAGACC-3¢; R(+).wt (sense): 5¢-GGTCTCTCTTGTT- In vitro RNA synthesis AGACCAGGTCGAGCCCGGGAGCTCTCTGGCTAGC- AAGGAACCCACTGCTTAAGCCTCAATAAAGCTTGC- RNAs were labelled with [a- P]UTP during in vitro tran- CTTGAGTGCCTCCC-3¢; R(-).wt (anti-sense): 5¢-GGGAG- scription, as previously described (31), using T7 RNA GCACTCAAGGCAAGCTTTATTGAGGCTTAAGCAGT- polymerase. Template DNAs pS14, pS20 and pR3 were GGGTTCCTTGCTAGCCAGAGAGCTCCCGGGCTCGA- digested with PstI, ®rst treated by Klenow to remove the 3¢ CCTGGTCTAACAAGAGAGACC-3¢. strand overhang and substrate RNA and the ribozyme DNA ODNs corresponding to R(±) in which 7 nt were generated by in vitro transcription with the following modi- mutated at the 3¢ end (underlined nucleotide): R(±).3¢- ®cations: for substrate RNA (transcription of pS14 and pS20 modi®ed: 5¢.GGGAGGCACTCAAGGCAAGCTTTATTG- DNA) the concentration of UTP was 10 mM and 50 mCi AGGCTTAAGCAGTGGGTTCCTTGCTAGCCAGAGAG- [a- P]UTP (Amersham, UK) were added. For the ribozyme CTCCCGGGCTCGACCTGGTCTAACATCAGTCTCTA-3¢; (pR3 DNA template), the concentration of UTP was 100 mM TAR(±) and R(+) ODNs were P-labelled with 50 mCi of and 10 mCi of [a- P]UTP were added. 32 32 [g- P]ATP using T4 polynucleotide kinase. P-labelled Following in vitro RNA synthesis, the DNA template was DNAs TAR(±) and R(+) were puri®ed by 10% PAGE, 7 M removed by treatment with RNase-free DNase I (Promega) for urea in 50 mM Tris-borate, 1 mM EDTA, pH 8.3 (0.53 TBE) 20 min at 37°C, followed by phenol and chloroform extrac- before use. tions, and ethanol precipitation. All P-labelled RNAs were Nucleic Acids Research, 2004, Vol. 32, No. 7 2131 Figure 2. FMRP has nucleic acid annealing activity. (A) Schematic representation of the assay. HIV-1 TAR (+) and (±) DNA sequences are shown. 5¢ P-la- belled TAR(±) DNA is hybridized to TAR(+) upon heating at 65°C or following addition of a nucleic acid chaperone such as HIV-1 NCp7. (B) Annealing as- says. Conditions were as described in Materials and Methods. Control hybridization was conducted for 30 min at 65 (lane 1) or 37°C (lane 2). Protein to ±8 nucleotide molar ratios were 1:48, 1:24 and 1:12, corresponding to a concentration of 0.35, 0.7 and 1.4 3 10 M, respectively. The vertical arrow shows the direction of electrophoresis. P-labelled TAR(±) DNA and double-stranded TAR DNA are indicated on the right. (1) NCp7, lanes 3±5; NC(12±53), lanes 6± 8; p50, lanes 9±11. (2) FMRP, lanes 1±3; FMRP DPPId, lanes 4±6; FMRP DKH1, lanes 7±9; FMRP DKH2, lanes 10±12; FMRP DKHT, lanes 13±15. (3) FMRP, lanes 3±5; FMRP DPhd, lanes 6±8; FMRP DRGG, lanes 9±11. Curves shown on the right are quantitative assessments of the percentage of double- stranded DNA formed. Note that FMRP is as active as the canonical chaperone HIV-1 NCp7. Only FMRP mutant DKH1 was as active as wild-type protein, whereas mutants DRGG, DKH2, DPPId and DPhd were ~2.5±5 times less active and mutant DKHT was poorly active (see at molar ratio of 1:48). 2132 Nucleic Acids Research, 2004, Vol. 32, No. 7 puri®ed by 8% PAGE, 7 M urea in 0.53 TBE. RNAs were stop solution (0.3% SDS, 15 mM EDTA), extracted with 30 ml recovered by elution in 0.3 M sodium acetate, 0.1% SDS, for of phenol and 15 ml of chloroform. The aqueous phase was 4hat37°C and ethanol precipitated. RNAs were dissolved in precipitated with ethanol and the pellet resuspended in 45% sterile H O. formamide, 0.53 TBE, and 0.1% dye. P-labelled RNAs were analysed on 8% PAGE in 0.53 TBE and, subsequently, TAR(±)/TAR(+) annealing assays gels were autoradiographed. Quantitation was by PhosphorImaging. HIV-1 TAR(+) and P-labelled TAR(±) ODNs were incu- bated (0.015 pmol, each equivalent to a DNA concentration of ±9 3 3 10 M) with or without protein in 10 ml containing 20 mM RESULTS Tris±HCl (pH 7.0), 30 mM NaCl, 0.1 mM MgCl 10 mM 2, ZnCl and 5 mM DTT. Reactions were performed at 37°C for FMRP and the deleted versions (denoted DPPId, DKH1, 10 min except for the positive control which was kept at 65°C DKH2, DKHT, DPhd and DRGG) were expressed in E.coli for 30 min under oil. Reactions were stopped with 5 ml of 20% with a C-terminal (His)6-tag and puri®ed by Ni-chelate glycerol, 20 mM EDTA pH 8.0, 0.2% SDS, 0.25% chromatography (see Materials and Methods). HIV-1 NCp7 bromophenol blue and 0.4 mg/ml calf liver tRNA. Samples and its truncated version denoted DNC(12±53) were synthe- were analysed by 8% native PAGE in 0.53 TBE and, sized and puri®ed according to a published procedure (30). subsequently, gels were autoradiographed. Quantitation was YB-1/p50 was provided by Lev Ovchinnikov (32) (Fig. 1). To by PhosphorImaging. con®rm that FMRP stably binds nucleic acids and RNA in particular, we examined by native gel electrophoresis the DNA strand transfer and exchange assays formation of nucleoprotein complexes between FMRP and Lys,3 ±8 0.03 pmol of P-labelled R(+).wt, 0.03 pmol of R(±). 3¢- either tRNA , BC1 or BC200, at concentrations of 10 M. modi®ed and 0.03 pmol of R(±).wt were separately heat Results of the gel shift experiments show that FMRP binds denatured for 2 min at 90°C and chilled on ice. All RNA (Supplementary ®g. 1) and clearly has a high af®nity for Lys,3 components were kept at 4°C. 0.03 pmol each of P-labelled structured RNA molecules such as tRNA (lanes 2±4 A), ±8 R(+).wt and R(±).3¢-modi®ed, at a concentration of 6 3 10 BC1 RNA (lanes 6±8 A) and BC200 RNA (lanes 10±12 A) in a M, were mixed with reaction buffer to a ®nal concentration of manner similar to YB-1/p50 (Supplementary ®g. 1E). Under 20 mM Tris±HCl, pH 7.0, 30 mM NaCl, 0.1 mM MgCl , the present ionic conditions, K was estimated to be 25 nM. 10 mM ZnCl and 5 mM DTT in 5 ml ®nal volume, incubated Deletion of the KH motifs caused a strong reduction of FMRP for 30 min at 62°C under oil and chilled on ice. Then, RNA-binding activity (Supplementary ®g. 1B). Similar results 0.03 pmol of R(±).wt was added together with the protein were obtained with single-stranded DNA ODNs of 56±98 nt in using a protein to nucleotide molar ratio as indicated in the length (data not shown and see below). legends. Assays were for 5 min at 37°C, they were then chilled FMRP has DNA annealing activity on ice and stopped with 2.5 ml of 20% glycerol, 20 mM EDTA pH 8.0, 0.2% SDS, 0.25% bromophenol blue and 0.4 mg/ml To assay for nucleic acid chaperone activity, FMRP was calf liver tRNA. Samples were resolved by 6% native PAGE examined for its ability to enhance the annealing of comple- in 0.53 TBE at 4°C and, subsequently, gels were autoradio- mentary DNA ODNs (Fig. 2A). To this end, complementary graphed. Quantitation was by PhosphorImaging. P-Tar(±) and Tar(+) ODNs (56 nt) were incubated in the presence of increasing concentrations of FMRP, subsequently Hammerhead ribozyme cleavage assays chased with a tRNA excess. Duplex formation was analysed Ribozyme and substrate RNA were independently heated for by native gel electrophoresis. 1 min at 90°CinH O. The reaction buffer was added to yield Results show that addition of FMRP caused nearly ®nal concentrations of 5 mM MgCl , 100 mM NaCl, 20 mM complete duplex formation [Fig. 2B, compare lanes 1±3 in Tris±HCl, pH 7.5. After slow cooling to 37°C, RNAs were (2) with lane 2 in (1)]. This effect is not simply due to further incubated for 5 min at 20°C. 0.1 pmol of ribozyme and enhanced molecular crowding, since at the same protein 0.02±2 pmol of RNA substrate were then combined in a ®nal concentration, BSA did not enhance duplex formation (data volume of 10 ml, each protein was added to the ®nal ratio not shown). Also, the propensity of FMRP to anneal the TAR indicated in the legend of the ®gures and incubation was for substrates is not simply a general feature of DNA binding 25 min at 37°C. Reactions were terminated by adding 20 mlof proteins, since at similar concentrations, the single-stranded Figure 3. FMRP activates DNA strand exchange. (A) Schematic representation of the assay. DNA sequences corresponding to the HIV-1 R region of 96 nt in length. 5¢ P-labelled R(+).wt was hybridized to R(±).mut to generate a double-stranded DNA with seven mismatches at one end (step 1). R(±).wt completely complementary to R(+).wt is added together with HIV-1 NCp7 or FMRP (step 2). Strand exchange is visualized by native 6% PAGE in 0.5 TBE. (B) Strand exchange assays. Conditions were as described in Materials and Methods. Proteins are indicated at the top of the ®gure. *R(+) is the P-labelled DNA. Double-stranded [*R(+):R(±).wt] and [*R(+):R(±).mut] are indicated on the right. Percentages of strand exchange were assessed by PhorphorImaging and are indicated in parentheses (see below). The vertical arrow shows the direction of electrophoresis. Lanes 1 and 2, *R(+).wt alone and annealed to R(±).wt. Lane 3, *R(+) hybridized to R(±).mut. Lane 4, [*R(+):R(±).mut] incubated with R(±).wt at 37°C for 30 min. Protein to nucleotide molar ratios were 1:8, 1:4 and ±7 1:2 corresponding to a protein concentration of 1, 2 and 4 3 10 M, respectively. NCp7, lanes 5±7 (64, 75 and 78%); NC(12±53), lanes 8±10 (4, 5 and 6%); p50, lanes 11±13 (22, 32 and 50%); FMRP wild type, lanes 14±16 (53, 70 and 78%); FMRP DPPId, lanes 17±19 (19, 31 and 62%); FMRP DKH1, lanes 20± 22 (44, 61 and 73%); FMRP DKH2, lanes 23±25 (40, 54 and 65%); FMRP DKHT, lanes 26±28 (24, 39 and 53%); FMRP DPhd, lanes 29±31 (26, 32 and 60%); FMRP DRGG, lanes 32±34 (39, 53 and 60%). Note that FMRP displays greater DNA strand exchange activity than YB-1/p50 but is similar to HIV-1 NCp7. FMRP variants DPPId, DKHT and DPhd display a DNA exchange activity ~2.5-fold lower than wild-type FMRP. Nucleic Acids Research, 2004, Vol. 32, No. 7 2133 DNA binding protein T4gp32 was unable to enhance duplex lanes 13±15 in Fig. 2B (2) and right panel]. FMRP was found to be as active as the canonical chaperone proteins HIV-1 formation (33±37 and data not shown). FMRP DKH1 was as active as FMRP wild type (compare lanes 1±3 and 7±9; see NCp7 and YB-1/p50 [Fig. 2B (1), lanes 3±5 and 9±11 in also right panel), whereas other deleted variants were either comparison with lanes 1±3 in (2); see also right panels], while ~3-fold less active (DPPId, DKH2, DPhd and DRGG) [Fig. 2B mutant NC(12±53) was inactive [lanes 6±8 in (1)] in (2) and (3); see also right panels] or poorly active [DKHT; agreement with previous reports (31). 2134 Nucleic Acids Research, 2004, Vol. 32, No. 7 Nucleic Acids Research, 2004, Vol. 32, No. 7 2135 We also examined the ability of FMRP to promote the Enhancement of ribozyme cleavage of an RNA substrate Lys,3 by FMRP hybridization of replication primer tRNA to a comple- mentary sequence at the 5¢ end of the HIV-1 genomic RNA, Cleavage of an RNA substrate by a hammerhead ribozyme is a called the primer binding site. Indeed, FMRP was able to model system which allows examination of both the RNA hybridize the cellular tRNA to the viral RNA (Supplementary annealing and unwinding activities of nucleic acid chaperone ®g. 2; lanes 2±5). proteins. Chaperones can enhance the rate of ribozyme cleavage by activating the annealing of the substrate RNA to FMRP has DNA strand exchange activity the hammerhead ribozyme (Fig. 4A, step 1) and the release of Another property of nucleic acid chaperones is to direct the the RNA products (Fig. 4A, step 3), thus allowing cyclic reuse formation of the most stable nucleic acid conformation. This of the ribozyme (31,38). The ribozyme cleavage assay intends can be assayed by examining the strand exchange activity of to investigate whether FMRP enhances ribozyme cleavage of the protein (Fig. 3A). An imperfect DNA duplex with two an RNA substrate in a manner similar to NCp7 and hnRNP A1 partially complementary ODNs is formed by heating at 62°C. chaperone proteins (see Fig. 4A) (33,38). Then, the chaperone is added together with another ODN with We selected the R3 hammerhead ribozyme and two RNA the potential to form a perfect duplex with one of the two substrates, namely S14, with a 14 nt substrate ribozyme duplex initial ODNs. After incubation under physiological conditions, length (7 nt either side of the cleavage site) and S20, with 10 nt the protein is removed and the ratio of the two DNA duplexes, either side of the cleavage site. The above RNA substrates the perfect duplex and the one containing seven mismatches, is were selected due to their likely biological relevance, as assessed by native gel electrophoresis. As shown in Figure 3B indicated by the similarity of data obtained in vitro and in (lanes 14±16), FMRP exchanged the matched strand [R(±).wt] cultured cells (33). P-labelled RNA S14, the hammerhead for the mismatched strand [R(±).mut] as observed with HIV-1 ribozyme and FMRP were mixed, incubated at 37°C for 25 NCp7 (lanes 5±7). FMRP was more active than p50 (lanes 11± min, protein removed by phenol extraction and RNA products 13). In contrast, the level of the mismatched duplex remained recovered and analysed by PAGE under denaturing condi- unaffected by BSA, T4gp32 (30,31,34 and data not shown) or tions. In the absence of a nucleic acid chaperone, ribozyme- mutant NC(12±53) (compare lanes 8±10 and 14±16). The directed cleavage of the RNA substrate occurred slowly at FMRP domains, such as PPId, KH and Phd, appeared to be 37°C (Fig. 4B, top, lanes 1 at 4°Cand 2at37°C; P-RNA critical for full activity (lanes 17±34). substrate is S14 and upon cleavage it is DS14). In agreement Taken together, these results indicate that FMRP does with previous reports (33), HIV-1 NCp7 caused extensive indeed possess DNA annealing and strand exchange ribozyme-directed cleavage of S14 RNA (lanes 3±5). On the activities, which are properties expected for a nucleic acid other hand, NC(12±53) was poorly active (lanes 6±8). chaperone (28,29), and that several domains, including the Interestingly, FMRP showed a clear enhancement of ribo- PPId, KH and RGG motifs, are important determinants for zyme-directed cleavage of S14 RNA (lanes 9±11). Deleted these activities. versions of FMRP, namely DPPId and DKHT were found to Figure 4. Enhancement of ribozyme cleavage by FMRP. (A) Assay schematic. RNA substrate and hammerhead ribozyme, both P-labelled, were generated by in vitro transcription and puri®ed by PAGE. Cleavage of the P-labelled RNA substrate by the hammerhead ribozyme ®rst necessitates hybridization of the ribozyme to the substrate (step 1). After substrate cleavage (step 2, see arrow head), the RNA products must be released to allow recycling of the ribo- zyme (step 3). At the end of the reaction, protein is removed by phenol extraction and P-labelled RNAs analysed by PAGE under denaturing conditions to visualize the P-labelled RNA products. In the absence of a nucleic acid chaperone, annealing of the substrate to the ribozyme and release of the RNA pro- ducts appear to be slow. Addition of a nucleic acid chaperone will accelerate hybridization of the substrate to the ribozyme and dissociation of the RNA pro- ducts, and thus ribozyme turnover. Base pairing between the RNA substrate and ribozyme R3 are underlined on the substrate sequence. Ribozyme-mediated cleavage occurs on the 3¢ side of A (space) for RNA S14, ...GAUUAAGUAGUA AGAGUGUCUGCA-3¢, and for RNA S20, ...GAUUAAGUAGUA AGAGUGUCUGCA-3¢.(B) Ribozyme-directed cleavage of RNA substrate. Percentages of ribozyme-directed RNA cleavage at 25 min were assessed by PhorphorImaging and are indicated in parentheses (see below). The vertical arrow shows the direction of electrophoresis. Ribozyme R3 (0.3 pmol) and RNA S14 (0.1 pmol) were incubated as described in Materials and Methods. P-labelled RNA substrate (S14) and product (DS14) were analysed by denaturing 8% PAGE in 0.53 TBE. Lanes 1±2, R3 and S14 at 4 or 37°C for 30 min (5% and 20%). Reactions with proteins were for 15 min at 37°C. Lanes 3±5, HIV-1 NCp7 at protein to nucleotide molar ratios of 1:20, 1:10 and 1:5, respectively (60, 73 and 75%), corresponding to a protein concentration of 0.5, 1 and 2 3 ±7 10 M, respectively. Lanes 6±8, NC(12±53) at molar ratios of 1:2.5, 1:1.2 and 1:0.6, respectively (25% for all ratios), corresponding to a protein concentra- ±7 tion of 4, 8 and 16 3 10 M, respectively. Lanes 9±11, FMRP at protein to nucleotide molar ratios of 1:20, 1:10 and 1:5, respectively (33, 45 and 66%), cor- ±7 responding to a protein concentration of 0.5, 1 and 2 3 10 M, respectively. Respective concentrations of the FMRP variants (lanes 12±20) were the same (see below). Lanes 12±14, FMRP DPPId at molar ratios of 1:20, 1:10 and 1:5, respectively (30, 35 and 39%). Lanes 15±17, FMRP DKHT at molar ratios of 1:20, 1:10 and 1:5, respectively (25, 28 and 32%). Lanes 18±20, FMRP DRGG at molar ratios of 1:20, 1:10 and 1:5, respectively (30, 40 and 57%). R3, S14 and the 5¢ sequences of S14 (DS14) are identi®ed on the right. Markers are on the left. Note that the RNA products rapidly accumulate in the presence of a chaperone like NCp7 (lanes 3±5) or FMRP (lanes 9±11), whereas they do so only slowly in the absence of a chaperone (lane 2). (C) Ribozyme-directed cleavage of RNA substrate S20. Ribozyme R3 (0.3 pmol) and RNA S20 (0.1 pmol) were incubated as described in Materials and Methods. P-labelled RNA substrate (S20) and product (DS20) were analysed by denaturing 8% PAGE in 0.53 TBE. Lanes 1±2, R3 and S20 at 4 or 37°C for 30 min (5 and 25%). Lanes 3±4, HIV-1 NCp7 at protein to nucleotide molar ratios of 1:10 and 1:5, respectively (15%). Lanes 5±6, NC(12±53) at molar ratios of 1:1.2 and 1:0.6, respectively (10%). Lanes 7±8, FMRP at protein to nucleotide molar ratios of 1:10 and 1:5, respectively (16%), corresponding to a protein concentration of 1 ±7 and 2 3 10 M, respectively. Respective concentrations of the FMRP variants (lanes 9±14) were the same (see below). Lanes 9±10, FMRP DPPId at molar ratios of 1:10 and 1:5, respectively (16%). Lanes 11±12, FMRP DKHT at molar ratios of 1:10 and 1:5, respectively (17%). Lanes 13±14, FMRP DRGG at molar ratios of 1:10 and 1:5, respectively (20%). 2136 Nucleic Acids Research, 2004, Vol. 32, No. 7 poorly enhance ribozyme-directed cleavage of S14 RNA FMRP is associated with mRNPs complexes, which shuttle (lanes 12±14 and 15±17, respectively). The DRGG deleted between translating ribosomes and cytoplasmic granules in version of FMRP proved to be ~3-fold less active than cells (20). The emerging picture proposes that FMRP modu- complete FMRP (lanes 18±20). lates translation through a network of protein±RNA and Next, we examined the effects of FMRP using RNA protein±protein interactions. According to our ®ndings, FMRP substrate S20, which is able to form an extended duplex of 20 should also act on mRNA metabolism and translation by nt with the hammerhead ribozyme (Fig. 4A) and which means of RNA±RNA interactions. These interactions might be prevents activation of ribozyme cleavage by HIV-1 NCp7 or under the control of non-coding RNA, as proposed for BC1 hnRNP A1 (33,38). In the absence of a chaperone, only RNA (27), or by causing mRNA dimerization by intermole- minimal ribozyme-directed cleavage of RNA S20 was cular G-quartet formation (9,10,15). By analogy with YB-1/ observed at 37°C (Fig. 4B, bottom, lanes 1 and 2) in contrast p50, a major cellular chaperone associated with mRNPs to what was seen with RNA S14 (Fig. 4B, top). NCp7 did not (32,39), it can also be envisioned that a low level of FMRP enhance ribozyme cleavage of RNA S20, in agreement with should stimulate translation, whereas a high level of FMRP previous data (lanes 3±4) (31). FMRP and deleted versions should mask the mRNA and hence inhibit its translation were also found to exhibit very little, if any, enhancing activity (20,23,29). In support of this notion, recruitment of FMRP into using RNA S20 (bottom, lanes 7±14). mRNPs and polyribosomes in vivo is abolished upon deletion of either the PPId domain or the KH motifs (3). Interestingly, these very same domains are important determinants of the DISCUSSION nucleic acid chaperoning activities of FMRP in vitro, sug- FMRP is not only an RNA-binding protein (Supplementary gesting that FMRP needs all its nucleic acid binding and chaperoning activities to exert its chaperone function in vivo. ®g. 1), but is also a nucleic acid chaperone, thus providing Our results open new perspectives on the functional role(s) of nucleic acid remodelling properties for this cellular protein. FMRP in RNA metabolism. Further analyses are required to This conclusion is supported by several lines of evidence. investigate these functional aspects. FMRP promotes the hybridization of complementary DNAs under low ionic strength conditions (Fig. 2), directs formation of the most stable duplex structure by achieving strand SUPPLEMENTARY MATERIAL exchange and, once nucleic acid molecules have been refolded Supplementary Material is available at NAR Online. into their most stable structure, the protein is no longer required to maintain the structure (Fig. 3). FMRP enhances ribozyme-directed cleavage of an RNA substrate, most ACKNOWLEDGEMENTS probably by activating substrate RNA annealing to the Thanks are due to Lev Ovchinnikov for the YB-1/p50 protein, ribozyme in physiological relevant conditions (Fig. 4). In Edouard Bertrand for the hammerhead ribozyme system, agreement with these ®ndings, FMRP can promote the Lys3 Ju È rgen Brosius for the BC1 and BC200 plasmids and Mike hybridization of replication primer tRNA to a comple- Rau (UK) for corrections. This study was supported by funds mentary sequence at the 5¢ end of the HIV-1 genomic RNA, from ANRS (to J.-L.D.), the Canadian Institutes of Health (to called the primer binding site (Supplementary ®g. 2) (34,35). E.W.K.) and a FRSQ/INSERM exchange program. R.M. was According to these criteria, FMRP resembles the YB-1/p50 a recipient of a postdoctoral fellowship from the Fragile X protein, which is a ubiquitous cellular chaperone (32) bound to Research Foundation of Canada/Canadian Institutes of Health mRNPs, involved in mRNA metabolism and in the regulation Research Partnership Challenge Fund programme. of translation (39) including its own transcript (40). Deleted variants of FMRP, namely, DPPId, DKH1, DKH2, DKH1/KH2 (DKHT), DPhd and DRGG (Fig. 1), were exam- REFERENCES ined for their ability to activate the annealing of complemen- 1. Bardoni,B. and Mandel,J.L. 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The fragile X mental retardation protein has nucleic acid chaperone properties

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Oxford University Press
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10.1093/nar/gkh535
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Abstract

Published online April 19, 2004 Nucleic Acids Research, 2004, Vol. 32, No. 7 2129±2137 DOI: 10.1093/nar/gkh535 The fragile X mental retardation protein has nucleic acid chaperone properties 1 1 1 Caroline Gabus, Rachid Mazroui , Sandra Tremblay , Edouard W. Khandjian and Jean-Luc Darlix* LaboRetro, Unite  INSERM de Virologie Humaine (412), ENS, 46 alle  e d'Italie, 69364 Lyon cedex 07, France and Unite  de recherche en Ge  ne  tique Humaine et Mole  culaire, Centre de recherche Ho à pital St. Franc Ë ois d'Assise, CHUQ, 10 rue de l'Espinay, Que  bec G1L 3L5, PQ Canada Received February 9, 2004; Revised and Accepted March 22, 2004 ABSTRACT expressed, but most abundant in testes and brain, which are the organs strongly affected by the syndrome (4,5). The fragile X syndrome is the most common cause The cellular role of FMRP remains poorly understood and of inherited mental retardation resulting from the the current view is that it regulates mRNA transport and absence of the fragile X mental retardation protein translation in a manner critical for the development of neurons (FMRP). FMRP contains two K-homology (KH) (6,7). To achieve its function, FMRP is thought to be engaged domains and one RGG box that are landmarks char- in a number of interactions with cytoplasmic nucleic acid and protein partners (1,8±15) in association with messenger acteristic of RNA-binding proteins. In agreement ribonucleoparticles (mRNPs) in actively translating ribosomes with this, FMRP associates with messenger ribo- (16,17), however, the exact role of FMRP in translation nucleoparticles (mRNPs) within actively translating remains elusive. Several studies have shown that FMRP can ribosomes, and is thought to regulate translation of act as a negative regulator of translation in vitro and in vivo target mRNAs, including its own transcript. To (18±20) and it has been proposed that in neurons a small investigate whether FMRP might chaperone nucleic fraction of FMRP acts as a repressor for the RNA to be acid folding and hybridization, we analysed the transported and to be delivered at the budding dendrites (20). annealing and strand exchange activities of DNA Indeed, while the great majority of FMRP has been observed oligonucleotides and the enhancement of ribozyme- in the neuron cell body, a small fraction was detected at distal directed RNA substrate cleavage by FMRP and locations such as neurites, dendrites and synaptosomes deleted variants relative to canonical nucleic acid (5,21,22). A series of neuronal mRNAs have been isolated either by immunoprecipitation approaches (8), or by the use of chaperones, such as the cellular YB-1/p50 protein a new technique called `APRA' for antibody positioned RNA and the retroviral nucleocapsid protein HIV-1 NCp7. ampli®cation (24). In addition, several target mRNAs have FMRP was found to possess all the properties of a been isolated by differential display, by oligonucleotide potent nucleic acid chaperone, requiring the KH (ODN)-based and by cDNA-SELEX (9,25,26). Also it has motifs and RGG box for optimal activity. These ®nd- been reported that FMRP interacts with small noncoding ings suggest that FMRP may regulate translation by RNAs such as BC1 and BC200, which in turn mediates their acting on RNA±RNA interactions and thus on the binding to speci®c mRNAs (27). These observations clearly structural status of mRNAs. indicate that FMRP has af®nity to RNAs, however, it is not clear whether all these target mRNAs bind directly to FMRP or if FMRP protein interactors are required. In view of the binding of FMRP to mRNAs and small non- INTRODUCTION coding RNAs, we addressed the question as to whether FMRP The fragile X syndrome is the most common cause of inherited might be a nucleic acid chaperone protein. So-called nucleic mental retardation in humans resulting from the absence of the acid chaperones bind in a cooperative manner to one or more FMR1 gene product, the FMR1 protein [fragile X mental nucleic acid molecules and promote the formation of the most retardation protein (FMRP)] (1,2). FMRP is a protein of 632 stable structure while at the same time preventing folding traps amino acids composed, in N-terminus to C-terminus order, of that may preclude function (28,29). Importantly, nucleic acid a protein-interacting domain (PPId), two K-homology (KH) chaperones do not require ATP to function and once the most motifs, a phosphorylation sequence and an RNA recognition stable nucleic acid structure is achieved, their continued sequence called the RGG box (1±3). FMRP is widely binding is no longer required to maintain the structure (28,29). *To whom correspondence should be addressed. Tel: +33 4 72 72 81 69; Fax: +33 4 72 72 87 77; Email: [email protected] Present address: Rachid Mazroui, Department of Biochemistry, McGill University, Montre Âal, Canada Nucleic Acids Research, Vol. 32 No. 7 ã Oxford University Press 2004; all rights reserved 2130 Nucleic Acids Research, 2004, Vol. 32, No. 7 Canonical nucleic acid chaperones, such as retroviral NC proteins, are able to anneal, under physiological conditions in vitro, a speci®c primer tRNA to a complementary sequence at the 5¢ end of genomic viral RNA; a prerequisite for the initiation of reverse transcription (30,31). In the present study, we examined the ability of FMRP to chaperone the annealing of DNA with complementary sequences as well as strand exchange in a duplex nucleic acid structure in vitro. Furthermore, we investigated whether FMRP is capable of enhancing ribozyme-directed cleavage of an RNA substrate in vitro. Our results show that FMRP is a potent chaperone protein. MATERIALS AND METHODS Recombinant proteins The full-length FMRP and six deleted variants DPPId, DKH1, DKH2, DKHT (DKH1 plus DKH2), DPhD and DRGG FMRP with a C-terminal (His)6-tag were produced in Escherichia coli and puri®ed to homogeneity by af®nity chromatography using Ni-NTA Probond beads (Invitrogen) according to the manufacturer's protocol as previously described (Fig. 1) (3). HIV-1 nucleocapsid protein NCp7 and mutant NC(12±53) were synthesized by the fmoc/opfp chemical method and puri®ed to >98% purity by high-pressure liquid chromato- graphy (30). Proteins were dissolved at 1 mg/ml in buffer Figure 1. Scheme of the FRMP protein and deleted variants. Wild-type re- containing 30 mM HEPES pH 6.5, 30 mM NaCl and 0.1 mM combinant FMRP and its deleted variants used in the present study are sche- ZnCl . matically depicted here. Positions of the deleted regions, namely the protein interacting domain (PPId), the KH1 and KH2 domains, the phosphorylation The YB-1/p50 protein (32) was a kind gift from Lev domain (Phd) and the RGG box are shown. HIV-1 NCp7(1±72) and mutant Ovchinnikov (Moscow, Russia) (31). NC(12±53) with the two CCHC zinc ®ngers (ZF) are shown. The complete YP-1/p50 protein is schematically shown with the N-terminal alanine- and DNA substrates proline-rich (A/P) domain, the cold shock domain (CSD) and basic and acidic amino acids (B/A) clusters domain. Amino acid positions are indi- DNA ODNs corresponding to the HIV-1 TAR and the cated. repeated R sequences (Mal isolate), in the sense and anti-sense orientation were purchased from Eurogentec (Belgium). TAR and R ODNs are 56 and 96 nt in length, Plasmid DNAs respectively. TAR(+) (sense) (30): 5¢-GGTCTCTCTTGTTAGACC- Plasmid DNAs pS14, pS20 and pR3 were kindly provided by AGGTCGAGCCCGGGAGCTCTCTGGCTAGCAAGGA- E. Bertrand (33). All plasmid DNAs were ampli®ed in E.coli ACCC-3¢; TAR(±) (anti-sense): 5¢-GGGTTCCTTGCTAGC- 1035 (RecA-) and puri®ed by af®nity chromatography CAGAGAGCTCCCGGGCTCGACCTGGTCTAACAAG- (Qiagen protocol). AGAGACC-3¢; R(+).wt (sense): 5¢-GGTCTCTCTTGTT- In vitro RNA synthesis AGACCAGGTCGAGCCCGGGAGCTCTCTGGCTAGC- AAGGAACCCACTGCTTAAGCCTCAATAAAGCTTGC- RNAs were labelled with [a- P]UTP during in vitro tran- CTTGAGTGCCTCCC-3¢; R(-).wt (anti-sense): 5¢-GGGAG- scription, as previously described (31), using T7 RNA GCACTCAAGGCAAGCTTTATTGAGGCTTAAGCAGT- polymerase. Template DNAs pS14, pS20 and pR3 were GGGTTCCTTGCTAGCCAGAGAGCTCCCGGGCTCGA- digested with PstI, ®rst treated by Klenow to remove the 3¢ CCTGGTCTAACAAGAGAGACC-3¢. strand overhang and substrate RNA and the ribozyme DNA ODNs corresponding to R(±) in which 7 nt were generated by in vitro transcription with the following modi- mutated at the 3¢ end (underlined nucleotide): R(±).3¢- ®cations: for substrate RNA (transcription of pS14 and pS20 modi®ed: 5¢.GGGAGGCACTCAAGGCAAGCTTTATTG- DNA) the concentration of UTP was 10 mM and 50 mCi AGGCTTAAGCAGTGGGTTCCTTGCTAGCCAGAGAG- [a- P]UTP (Amersham, UK) were added. For the ribozyme CTCCCGGGCTCGACCTGGTCTAACATCAGTCTCTA-3¢; (pR3 DNA template), the concentration of UTP was 100 mM TAR(±) and R(+) ODNs were P-labelled with 50 mCi of and 10 mCi of [a- P]UTP were added. 32 32 [g- P]ATP using T4 polynucleotide kinase. P-labelled Following in vitro RNA synthesis, the DNA template was DNAs TAR(±) and R(+) were puri®ed by 10% PAGE, 7 M removed by treatment with RNase-free DNase I (Promega) for urea in 50 mM Tris-borate, 1 mM EDTA, pH 8.3 (0.53 TBE) 20 min at 37°C, followed by phenol and chloroform extrac- before use. tions, and ethanol precipitation. All P-labelled RNAs were Nucleic Acids Research, 2004, Vol. 32, No. 7 2131 Figure 2. FMRP has nucleic acid annealing activity. (A) Schematic representation of the assay. HIV-1 TAR (+) and (±) DNA sequences are shown. 5¢ P-la- belled TAR(±) DNA is hybridized to TAR(+) upon heating at 65°C or following addition of a nucleic acid chaperone such as HIV-1 NCp7. (B) Annealing as- says. Conditions were as described in Materials and Methods. Control hybridization was conducted for 30 min at 65 (lane 1) or 37°C (lane 2). Protein to ±8 nucleotide molar ratios were 1:48, 1:24 and 1:12, corresponding to a concentration of 0.35, 0.7 and 1.4 3 10 M, respectively. The vertical arrow shows the direction of electrophoresis. P-labelled TAR(±) DNA and double-stranded TAR DNA are indicated on the right. (1) NCp7, lanes 3±5; NC(12±53), lanes 6± 8; p50, lanes 9±11. (2) FMRP, lanes 1±3; FMRP DPPId, lanes 4±6; FMRP DKH1, lanes 7±9; FMRP DKH2, lanes 10±12; FMRP DKHT, lanes 13±15. (3) FMRP, lanes 3±5; FMRP DPhd, lanes 6±8; FMRP DRGG, lanes 9±11. Curves shown on the right are quantitative assessments of the percentage of double- stranded DNA formed. Note that FMRP is as active as the canonical chaperone HIV-1 NCp7. Only FMRP mutant DKH1 was as active as wild-type protein, whereas mutants DRGG, DKH2, DPPId and DPhd were ~2.5±5 times less active and mutant DKHT was poorly active (see at molar ratio of 1:48). 2132 Nucleic Acids Research, 2004, Vol. 32, No. 7 puri®ed by 8% PAGE, 7 M urea in 0.53 TBE. RNAs were stop solution (0.3% SDS, 15 mM EDTA), extracted with 30 ml recovered by elution in 0.3 M sodium acetate, 0.1% SDS, for of phenol and 15 ml of chloroform. The aqueous phase was 4hat37°C and ethanol precipitated. RNAs were dissolved in precipitated with ethanol and the pellet resuspended in 45% sterile H O. formamide, 0.53 TBE, and 0.1% dye. P-labelled RNAs were analysed on 8% PAGE in 0.53 TBE and, subsequently, TAR(±)/TAR(+) annealing assays gels were autoradiographed. Quantitation was by PhosphorImaging. HIV-1 TAR(+) and P-labelled TAR(±) ODNs were incu- bated (0.015 pmol, each equivalent to a DNA concentration of ±9 3 3 10 M) with or without protein in 10 ml containing 20 mM RESULTS Tris±HCl (pH 7.0), 30 mM NaCl, 0.1 mM MgCl 10 mM 2, ZnCl and 5 mM DTT. Reactions were performed at 37°C for FMRP and the deleted versions (denoted DPPId, DKH1, 10 min except for the positive control which was kept at 65°C DKH2, DKHT, DPhd and DRGG) were expressed in E.coli for 30 min under oil. Reactions were stopped with 5 ml of 20% with a C-terminal (His)6-tag and puri®ed by Ni-chelate glycerol, 20 mM EDTA pH 8.0, 0.2% SDS, 0.25% chromatography (see Materials and Methods). HIV-1 NCp7 bromophenol blue and 0.4 mg/ml calf liver tRNA. Samples and its truncated version denoted DNC(12±53) were synthe- were analysed by 8% native PAGE in 0.53 TBE and, sized and puri®ed according to a published procedure (30). subsequently, gels were autoradiographed. Quantitation was YB-1/p50 was provided by Lev Ovchinnikov (32) (Fig. 1). To by PhosphorImaging. con®rm that FMRP stably binds nucleic acids and RNA in particular, we examined by native gel electrophoresis the DNA strand transfer and exchange assays formation of nucleoprotein complexes between FMRP and Lys,3 ±8 0.03 pmol of P-labelled R(+).wt, 0.03 pmol of R(±). 3¢- either tRNA , BC1 or BC200, at concentrations of 10 M. modi®ed and 0.03 pmol of R(±).wt were separately heat Results of the gel shift experiments show that FMRP binds denatured for 2 min at 90°C and chilled on ice. All RNA (Supplementary ®g. 1) and clearly has a high af®nity for Lys,3 components were kept at 4°C. 0.03 pmol each of P-labelled structured RNA molecules such as tRNA (lanes 2±4 A), ±8 R(+).wt and R(±).3¢-modi®ed, at a concentration of 6 3 10 BC1 RNA (lanes 6±8 A) and BC200 RNA (lanes 10±12 A) in a M, were mixed with reaction buffer to a ®nal concentration of manner similar to YB-1/p50 (Supplementary ®g. 1E). Under 20 mM Tris±HCl, pH 7.0, 30 mM NaCl, 0.1 mM MgCl , the present ionic conditions, K was estimated to be 25 nM. 10 mM ZnCl and 5 mM DTT in 5 ml ®nal volume, incubated Deletion of the KH motifs caused a strong reduction of FMRP for 30 min at 62°C under oil and chilled on ice. Then, RNA-binding activity (Supplementary ®g. 1B). Similar results 0.03 pmol of R(±).wt was added together with the protein were obtained with single-stranded DNA ODNs of 56±98 nt in using a protein to nucleotide molar ratio as indicated in the length (data not shown and see below). legends. Assays were for 5 min at 37°C, they were then chilled FMRP has DNA annealing activity on ice and stopped with 2.5 ml of 20% glycerol, 20 mM EDTA pH 8.0, 0.2% SDS, 0.25% bromophenol blue and 0.4 mg/ml To assay for nucleic acid chaperone activity, FMRP was calf liver tRNA. Samples were resolved by 6% native PAGE examined for its ability to enhance the annealing of comple- in 0.53 TBE at 4°C and, subsequently, gels were autoradio- mentary DNA ODNs (Fig. 2A). To this end, complementary graphed. Quantitation was by PhosphorImaging. P-Tar(±) and Tar(+) ODNs (56 nt) were incubated in the presence of increasing concentrations of FMRP, subsequently Hammerhead ribozyme cleavage assays chased with a tRNA excess. Duplex formation was analysed Ribozyme and substrate RNA were independently heated for by native gel electrophoresis. 1 min at 90°CinH O. The reaction buffer was added to yield Results show that addition of FMRP caused nearly ®nal concentrations of 5 mM MgCl , 100 mM NaCl, 20 mM complete duplex formation [Fig. 2B, compare lanes 1±3 in Tris±HCl, pH 7.5. After slow cooling to 37°C, RNAs were (2) with lane 2 in (1)]. This effect is not simply due to further incubated for 5 min at 20°C. 0.1 pmol of ribozyme and enhanced molecular crowding, since at the same protein 0.02±2 pmol of RNA substrate were then combined in a ®nal concentration, BSA did not enhance duplex formation (data volume of 10 ml, each protein was added to the ®nal ratio not shown). Also, the propensity of FMRP to anneal the TAR indicated in the legend of the ®gures and incubation was for substrates is not simply a general feature of DNA binding 25 min at 37°C. Reactions were terminated by adding 20 mlof proteins, since at similar concentrations, the single-stranded Figure 3. FMRP activates DNA strand exchange. (A) Schematic representation of the assay. DNA sequences corresponding to the HIV-1 R region of 96 nt in length. 5¢ P-labelled R(+).wt was hybridized to R(±).mut to generate a double-stranded DNA with seven mismatches at one end (step 1). R(±).wt completely complementary to R(+).wt is added together with HIV-1 NCp7 or FMRP (step 2). Strand exchange is visualized by native 6% PAGE in 0.5 TBE. (B) Strand exchange assays. Conditions were as described in Materials and Methods. Proteins are indicated at the top of the ®gure. *R(+) is the P-labelled DNA. Double-stranded [*R(+):R(±).wt] and [*R(+):R(±).mut] are indicated on the right. Percentages of strand exchange were assessed by PhorphorImaging and are indicated in parentheses (see below). The vertical arrow shows the direction of electrophoresis. Lanes 1 and 2, *R(+).wt alone and annealed to R(±).wt. Lane 3, *R(+) hybridized to R(±).mut. Lane 4, [*R(+):R(±).mut] incubated with R(±).wt at 37°C for 30 min. Protein to nucleotide molar ratios were 1:8, 1:4 and ±7 1:2 corresponding to a protein concentration of 1, 2 and 4 3 10 M, respectively. NCp7, lanes 5±7 (64, 75 and 78%); NC(12±53), lanes 8±10 (4, 5 and 6%); p50, lanes 11±13 (22, 32 and 50%); FMRP wild type, lanes 14±16 (53, 70 and 78%); FMRP DPPId, lanes 17±19 (19, 31 and 62%); FMRP DKH1, lanes 20± 22 (44, 61 and 73%); FMRP DKH2, lanes 23±25 (40, 54 and 65%); FMRP DKHT, lanes 26±28 (24, 39 and 53%); FMRP DPhd, lanes 29±31 (26, 32 and 60%); FMRP DRGG, lanes 32±34 (39, 53 and 60%). Note that FMRP displays greater DNA strand exchange activity than YB-1/p50 but is similar to HIV-1 NCp7. FMRP variants DPPId, DKHT and DPhd display a DNA exchange activity ~2.5-fold lower than wild-type FMRP. Nucleic Acids Research, 2004, Vol. 32, No. 7 2133 DNA binding protein T4gp32 was unable to enhance duplex lanes 13±15 in Fig. 2B (2) and right panel]. FMRP was found to be as active as the canonical chaperone proteins HIV-1 formation (33±37 and data not shown). FMRP DKH1 was as active as FMRP wild type (compare lanes 1±3 and 7±9; see NCp7 and YB-1/p50 [Fig. 2B (1), lanes 3±5 and 9±11 in also right panel), whereas other deleted variants were either comparison with lanes 1±3 in (2); see also right panels], while ~3-fold less active (DPPId, DKH2, DPhd and DRGG) [Fig. 2B mutant NC(12±53) was inactive [lanes 6±8 in (1)] in (2) and (3); see also right panels] or poorly active [DKHT; agreement with previous reports (31). 2134 Nucleic Acids Research, 2004, Vol. 32, No. 7 Nucleic Acids Research, 2004, Vol. 32, No. 7 2135 We also examined the ability of FMRP to promote the Enhancement of ribozyme cleavage of an RNA substrate Lys,3 by FMRP hybridization of replication primer tRNA to a comple- mentary sequence at the 5¢ end of the HIV-1 genomic RNA, Cleavage of an RNA substrate by a hammerhead ribozyme is a called the primer binding site. Indeed, FMRP was able to model system which allows examination of both the RNA hybridize the cellular tRNA to the viral RNA (Supplementary annealing and unwinding activities of nucleic acid chaperone ®g. 2; lanes 2±5). proteins. Chaperones can enhance the rate of ribozyme cleavage by activating the annealing of the substrate RNA to FMRP has DNA strand exchange activity the hammerhead ribozyme (Fig. 4A, step 1) and the release of Another property of nucleic acid chaperones is to direct the the RNA products (Fig. 4A, step 3), thus allowing cyclic reuse formation of the most stable nucleic acid conformation. This of the ribozyme (31,38). The ribozyme cleavage assay intends can be assayed by examining the strand exchange activity of to investigate whether FMRP enhances ribozyme cleavage of the protein (Fig. 3A). An imperfect DNA duplex with two an RNA substrate in a manner similar to NCp7 and hnRNP A1 partially complementary ODNs is formed by heating at 62°C. chaperone proteins (see Fig. 4A) (33,38). Then, the chaperone is added together with another ODN with We selected the R3 hammerhead ribozyme and two RNA the potential to form a perfect duplex with one of the two substrates, namely S14, with a 14 nt substrate ribozyme duplex initial ODNs. After incubation under physiological conditions, length (7 nt either side of the cleavage site) and S20, with 10 nt the protein is removed and the ratio of the two DNA duplexes, either side of the cleavage site. The above RNA substrates the perfect duplex and the one containing seven mismatches, is were selected due to their likely biological relevance, as assessed by native gel electrophoresis. As shown in Figure 3B indicated by the similarity of data obtained in vitro and in (lanes 14±16), FMRP exchanged the matched strand [R(±).wt] cultured cells (33). P-labelled RNA S14, the hammerhead for the mismatched strand [R(±).mut] as observed with HIV-1 ribozyme and FMRP were mixed, incubated at 37°C for 25 NCp7 (lanes 5±7). FMRP was more active than p50 (lanes 11± min, protein removed by phenol extraction and RNA products 13). In contrast, the level of the mismatched duplex remained recovered and analysed by PAGE under denaturing condi- unaffected by BSA, T4gp32 (30,31,34 and data not shown) or tions. In the absence of a nucleic acid chaperone, ribozyme- mutant NC(12±53) (compare lanes 8±10 and 14±16). The directed cleavage of the RNA substrate occurred slowly at FMRP domains, such as PPId, KH and Phd, appeared to be 37°C (Fig. 4B, top, lanes 1 at 4°Cand 2at37°C; P-RNA critical for full activity (lanes 17±34). substrate is S14 and upon cleavage it is DS14). In agreement Taken together, these results indicate that FMRP does with previous reports (33), HIV-1 NCp7 caused extensive indeed possess DNA annealing and strand exchange ribozyme-directed cleavage of S14 RNA (lanes 3±5). On the activities, which are properties expected for a nucleic acid other hand, NC(12±53) was poorly active (lanes 6±8). chaperone (28,29), and that several domains, including the Interestingly, FMRP showed a clear enhancement of ribo- PPId, KH and RGG motifs, are important determinants for zyme-directed cleavage of S14 RNA (lanes 9±11). Deleted these activities. versions of FMRP, namely DPPId and DKHT were found to Figure 4. Enhancement of ribozyme cleavage by FMRP. (A) Assay schematic. RNA substrate and hammerhead ribozyme, both P-labelled, were generated by in vitro transcription and puri®ed by PAGE. Cleavage of the P-labelled RNA substrate by the hammerhead ribozyme ®rst necessitates hybridization of the ribozyme to the substrate (step 1). After substrate cleavage (step 2, see arrow head), the RNA products must be released to allow recycling of the ribo- zyme (step 3). At the end of the reaction, protein is removed by phenol extraction and P-labelled RNAs analysed by PAGE under denaturing conditions to visualize the P-labelled RNA products. In the absence of a nucleic acid chaperone, annealing of the substrate to the ribozyme and release of the RNA pro- ducts appear to be slow. Addition of a nucleic acid chaperone will accelerate hybridization of the substrate to the ribozyme and dissociation of the RNA pro- ducts, and thus ribozyme turnover. Base pairing between the RNA substrate and ribozyme R3 are underlined on the substrate sequence. Ribozyme-mediated cleavage occurs on the 3¢ side of A (space) for RNA S14, ...GAUUAAGUAGUA AGAGUGUCUGCA-3¢, and for RNA S20, ...GAUUAAGUAGUA AGAGUGUCUGCA-3¢.(B) Ribozyme-directed cleavage of RNA substrate. Percentages of ribozyme-directed RNA cleavage at 25 min were assessed by PhorphorImaging and are indicated in parentheses (see below). The vertical arrow shows the direction of electrophoresis. Ribozyme R3 (0.3 pmol) and RNA S14 (0.1 pmol) were incubated as described in Materials and Methods. P-labelled RNA substrate (S14) and product (DS14) were analysed by denaturing 8% PAGE in 0.53 TBE. Lanes 1±2, R3 and S14 at 4 or 37°C for 30 min (5% and 20%). Reactions with proteins were for 15 min at 37°C. Lanes 3±5, HIV-1 NCp7 at protein to nucleotide molar ratios of 1:20, 1:10 and 1:5, respectively (60, 73 and 75%), corresponding to a protein concentration of 0.5, 1 and 2 3 ±7 10 M, respectively. Lanes 6±8, NC(12±53) at molar ratios of 1:2.5, 1:1.2 and 1:0.6, respectively (25% for all ratios), corresponding to a protein concentra- ±7 tion of 4, 8 and 16 3 10 M, respectively. Lanes 9±11, FMRP at protein to nucleotide molar ratios of 1:20, 1:10 and 1:5, respectively (33, 45 and 66%), cor- ±7 responding to a protein concentration of 0.5, 1 and 2 3 10 M, respectively. Respective concentrations of the FMRP variants (lanes 12±20) were the same (see below). Lanes 12±14, FMRP DPPId at molar ratios of 1:20, 1:10 and 1:5, respectively (30, 35 and 39%). Lanes 15±17, FMRP DKHT at molar ratios of 1:20, 1:10 and 1:5, respectively (25, 28 and 32%). Lanes 18±20, FMRP DRGG at molar ratios of 1:20, 1:10 and 1:5, respectively (30, 40 and 57%). R3, S14 and the 5¢ sequences of S14 (DS14) are identi®ed on the right. Markers are on the left. Note that the RNA products rapidly accumulate in the presence of a chaperone like NCp7 (lanes 3±5) or FMRP (lanes 9±11), whereas they do so only slowly in the absence of a chaperone (lane 2). (C) Ribozyme-directed cleavage of RNA substrate S20. Ribozyme R3 (0.3 pmol) and RNA S20 (0.1 pmol) were incubated as described in Materials and Methods. P-labelled RNA substrate (S20) and product (DS20) were analysed by denaturing 8% PAGE in 0.53 TBE. Lanes 1±2, R3 and S20 at 4 or 37°C for 30 min (5 and 25%). Lanes 3±4, HIV-1 NCp7 at protein to nucleotide molar ratios of 1:10 and 1:5, respectively (15%). Lanes 5±6, NC(12±53) at molar ratios of 1:1.2 and 1:0.6, respectively (10%). Lanes 7±8, FMRP at protein to nucleotide molar ratios of 1:10 and 1:5, respectively (16%), corresponding to a protein concentration of 1 ±7 and 2 3 10 M, respectively. Respective concentrations of the FMRP variants (lanes 9±14) were the same (see below). Lanes 9±10, FMRP DPPId at molar ratios of 1:10 and 1:5, respectively (16%). Lanes 11±12, FMRP DKHT at molar ratios of 1:10 and 1:5, respectively (17%). Lanes 13±14, FMRP DRGG at molar ratios of 1:10 and 1:5, respectively (20%). 2136 Nucleic Acids Research, 2004, Vol. 32, No. 7 poorly enhance ribozyme-directed cleavage of S14 RNA FMRP is associated with mRNPs complexes, which shuttle (lanes 12±14 and 15±17, respectively). The DRGG deleted between translating ribosomes and cytoplasmic granules in version of FMRP proved to be ~3-fold less active than cells (20). The emerging picture proposes that FMRP modu- complete FMRP (lanes 18±20). lates translation through a network of protein±RNA and Next, we examined the effects of FMRP using RNA protein±protein interactions. According to our ®ndings, FMRP substrate S20, which is able to form an extended duplex of 20 should also act on mRNA metabolism and translation by nt with the hammerhead ribozyme (Fig. 4A) and which means of RNA±RNA interactions. These interactions might be prevents activation of ribozyme cleavage by HIV-1 NCp7 or under the control of non-coding RNA, as proposed for BC1 hnRNP A1 (33,38). In the absence of a chaperone, only RNA (27), or by causing mRNA dimerization by intermole- minimal ribozyme-directed cleavage of RNA S20 was cular G-quartet formation (9,10,15). By analogy with YB-1/ observed at 37°C (Fig. 4B, bottom, lanes 1 and 2) in contrast p50, a major cellular chaperone associated with mRNPs to what was seen with RNA S14 (Fig. 4B, top). NCp7 did not (32,39), it can also be envisioned that a low level of FMRP enhance ribozyme cleavage of RNA S20, in agreement with should stimulate translation, whereas a high level of FMRP previous data (lanes 3±4) (31). FMRP and deleted versions should mask the mRNA and hence inhibit its translation were also found to exhibit very little, if any, enhancing activity (20,23,29). In support of this notion, recruitment of FMRP into using RNA S20 (bottom, lanes 7±14). mRNPs and polyribosomes in vivo is abolished upon deletion of either the PPId domain or the KH motifs (3). Interestingly, these very same domains are important determinants of the DISCUSSION nucleic acid chaperoning activities of FMRP in vitro, sug- FMRP is not only an RNA-binding protein (Supplementary gesting that FMRP needs all its nucleic acid binding and chaperoning activities to exert its chaperone function in vivo. ®g. 1), but is also a nucleic acid chaperone, thus providing Our results open new perspectives on the functional role(s) of nucleic acid remodelling properties for this cellular protein. FMRP in RNA metabolism. Further analyses are required to This conclusion is supported by several lines of evidence. investigate these functional aspects. FMRP promotes the hybridization of complementary DNAs under low ionic strength conditions (Fig. 2), directs formation of the most stable duplex structure by achieving strand SUPPLEMENTARY MATERIAL exchange and, once nucleic acid molecules have been refolded Supplementary Material is available at NAR Online. into their most stable structure, the protein is no longer required to maintain the structure (Fig. 3). FMRP enhances ribozyme-directed cleavage of an RNA substrate, most ACKNOWLEDGEMENTS probably by activating substrate RNA annealing to the Thanks are due to Lev Ovchinnikov for the YB-1/p50 protein, ribozyme in physiological relevant conditions (Fig. 4). In Edouard Bertrand for the hammerhead ribozyme system, agreement with these ®ndings, FMRP can promote the Lys3 Ju È rgen Brosius for the BC1 and BC200 plasmids and Mike hybridization of replication primer tRNA to a comple- Rau (UK) for corrections. This study was supported by funds mentary sequence at the 5¢ end of the HIV-1 genomic RNA, from ANRS (to J.-L.D.), the Canadian Institutes of Health (to called the primer binding site (Supplementary ®g. 2) (34,35). E.W.K.) and a FRSQ/INSERM exchange program. R.M. was According to these criteria, FMRP resembles the YB-1/p50 a recipient of a postdoctoral fellowship from the Fragile X protein, which is a ubiquitous cellular chaperone (32) bound to Research Foundation of Canada/Canadian Institutes of Health mRNPs, involved in mRNA metabolism and in the regulation Research Partnership Challenge Fund programme. of translation (39) including its own transcript (40). Deleted variants of FMRP, namely, DPPId, DKH1, DKH2, DKH1/KH2 (DKHT), DPhd and DRGG (Fig. 1), were exam- REFERENCES ined for their ability to activate the annealing of complemen- 1. Bardoni,B. and Mandel,J.L. 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Nucleic Acids Research – Oxford University Press

Published: Apr 1, 2004

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