Detailed knowledge of the structure of the ribosomal particles during their assembly on mRNA is a prerequisite for understanding the intricate translation initiation process. In vitro preparation of eukaryotic translation initiation complexes is limited by the rather tricky assembly from individually purified ribosomal subunits, initiation factors and initiator tRNA. In order to directly isolate functional complexes from living cells, methods based on affinity tags have been developed which, however, often suffer from non-specific binding of proteins and/or RNAs. In the present study we present a novel method designed for the purification of high-quality ribosome/mRNA particles assembled in RRL (rabbit reticulocyte lysate). Chimaerical mRNA–DNA molecules, consisting of the full-length mRNA ligated to a biotinylated desoxy-oligonucleotide, are immobilized on streptavidin-coated beads and incubated with RRL to form initiation complexes. After a washing step, the complexes are eluted by specific DNase I digestion of the DNA moiety of the chimaera, releasing initiation complexes in native conditions. Using this simple and robust purification setup, 80S particles properly programmed with full-length histone H4 mRNA were isolated with the expected ribosome/mRNA molar ratio of close to 1. We show that by using this novel approach purified ribosomal particles can be obtained that are suitable for biochemical and structural studies, in particular single-particle cryo-EM (cryo-electron microscopy). This purification method thus is a versatile tool for the isolation of fully functional RNA-binding proteins and macromolecular RNPs.

INTRODUCTION

Initiation of protein synthesis in eukaryotic cells is a complex process which requires participation of numerous protein factors and which involves various transient functional complexes. A major goal for understanding the molecular basis of cellular processes performed by ribonucleoprotein particles is to identify the RNA–protein interactions involved. This requires efficient purification methods for the isolation, characterization and identification of the components of these RNA–protein complexes. The heterogeneous character of eukaryotic ribosome complexes and the release of cellular proteases and nucleases concomitantly with cellular disruption impose considerable barriers to researchers in the field, particularly with regard to structure–function analyses.

There is a variety of affinity purification approaches for the isolation of in vitro-assembled RNA–protein complexes, including the use of poly-U Sepharose [1], tobramycin aptamers [2], streptavidin beads [3], streptavidin or Sephadex aptamers [4,5], and Strepto-Tag aptamers [6]. Although the isolated ribosomal quantities are low, affinity-purification methods present other undisputable advantages over classical density-gradient centrifugation processes, and are more appropriate for a certain number of applications requiring a high degree of sample homogeneity and purity such as structural analysis. Cell lysis and purification of eukaryotic ribosomes by conventional methods leads to a long exposure time to a wide range of co-purifying proteases and nucleases [7].

For the affinity purification of eukaryotic ribosomal complexes stalled at different stages, two main strategies to tag the RNA molecules have been used. mRNAs of interest are first in vitro transcribed, either (i) fused with the aptamer sequences or (ii) biotinylated on allylamine-UTP [uridine5′-(tetrahydrogen triphosphate), 5-(2-propen-1-ylamino)], and then incubated with unfractionated cytosolic extract. The retained proteins are eluted with the respective antagonist, and often a second round of purification yields an essentially homogeneous complex. The aptamer strategy (i) is the most widely applied. Boehringer et al. [8] isolated the HCV (hepatitis C virus) IRES (internal ribosome entry site) in complex with the human 80S ribosome for cryo-EM (cryo-electron microscopy) studies using an aptamer-tagged IRES RNA which binds to the aminoglycoside antibiotic tobramycin. HCV IRES RNAs containing three tandem MS2-recognition hairpins at the 5′-end were also employed to study 48S and 80S assembly defects in HeLa cells [9]. The Strepto-Tag aptamers were applied to prepare canonical and IRES 48S complexes from RRLs (rabbit reticulocyte lysates) [1013]. The second strategy (ii) is using a biotin molecule instead of an RNA aptamer to purify the complex. Namy et al. [14] used a minimal IBV (infectious bronchitis virus) pseudoknot annealed to chimaeric biotinylated DNA/2′-O-allyl-modified RNA oligonucleotides. The stalled 80S complexes purified from RRL on an avidin-coated matrix were used for cryo-EM studies.

In the present study we show an alternative approach which allows rapid isolation of eukaryotic ribosomal initiating complexes from RRL without the need for sucrose density-gradient fractionation. For this purpose, in vitro-transcribed full-size eukaryotic mRNA is linked to a biotinylated DNA oligonucleotide with the use of bacteriophage T4 DNA ligase [15,16]. The biotinylated ligated product binds irreversibly to streptavidin-coated beads, and the macromolecular complexes attached to the mRNA are released from the beads by enzymatic DNase I cleavage of the DNA moiety.

Using this protocol, we successfully isolated 80S monosomes stalled on the AUG codon of histone H4 mRNA. Histone H4 mRNA was chosen since it is a remarkably small mRNA of 375-nt long encoding the 103-amino-acid long histone H4 protein. The ORF (open reading frame) is preceded by a very short 5′-UTR (untranslated region) of 9 nt and followed by a 54-nt long 3′-UTR deprived of a poly(A) tail, but instead ending with a short hairpin involved in processing of the 3′-end [17]. These non-canonical features of histone H4 mRNA prevent conventional scanning/translation initiation, and we have recently shown that the ORF of histone mRNA contains two critical structural elements that drive initiation through a unique cap-dependent IRES-like mechanism [18]. The two RNA structures act in concert in order to load 43S initiation complexes directly on to the AUG start codon without any ribosomal scanning [18]. Using the versatile affinity purification method described in the present study, we have assembled and purified ribosomal particles using a chimaeric H4 mRNA–DNA bait. Sample characterization by cryo-EM imaging, sucrose-gradient ultracentrifugation, agarose gel analysis and MS revealed that the purification protocol was efficient and the eluted particles homogenous. This method is a significant technical improvement for the study of eukaryotic ribosome complexes.

EXPERIMENTAL

RNA transcription and construction of chimaeric mRNA–DNA bait

The template for histone H4 mRNA transcription (375 nt) was generated by PCR amplification from the histone H4 gene cloned in the pUC19 vector [18]. The histone H4 sequence was extended on its 3′-end with a 5′-(CAA)9CAC-3′ tail during the PCR amplification.

The PCR product was phenol-extracted and precipitated with ethanol. In vitro transcription of histone H4 mRNA was performed in a 100 μl volume of TMSDT buffer [40 mM Tris/HCl (pH 8.1), 22 mM MgCl2, 5 mM DTT (dithiothreitol), 0.01% Triton-X100 and 1 mM spermidine] containing 5–8 μg of PCR-template DNA, 5 mM NTP, 40 units of human placenta RNase inhibitor (RNAsin, Promega), and 0.2 mg/ml T7 RNA polymerase. The reaction was incubated at 37°C for 1 h. Then, pyrophosphatase (2 μg, Roche) was added and the mix was further incubated for 30 min, before adding RNase-free DNase I (4 units, New England Biolabs) for 1 h. Transcription products were fractionated on denaturing 6% urea-PAGE and electro-eluted from gel slices using a Biotrap apparatus (Schleicher and Schuell). The pure transcript was capped using the ScriptCap™ m7G Capping System (Epicentre). Radiolabelled transcripts were obtained by substituting the GTP from the kit by [α32P]GTP.

The chimaeric mRNA–DNA bait harbouring a biotin molecule at its 3′-end was constructed in one-step ligation. Histone H4 mRNA (35 μg, final concentration 4.7 μM) was ligated to the biotinylated DNA oligonucleotide [5′-CTCCTCCTCCTC(A)6-3′ with a single biotin molecule bound to the 3′-terminal A, Sigma Genosys], in the presence of two different DNA splints, 5′-(GAG)4GT(GTT)4-3′ and 5′-(GAG)4NGT(GTT)4-3′, where N represents any nucleotide (in order to ligate mRNA transcripts with a 3′ +1 nucleotide extension). Histone H4 mRNA was heated together with the biotinylated DNA and two DNA splints (in equimolar concentrations), at 90°C for 2 min in 30 mM Tris/HCl (pH 7.8), 10 mM MgCl2 and 10 mM DTT and cooled progressively for 10 min at room temperature (20°C). After hybridization, 40 units of T4 DNA ligase (Promega) and 1 mM ATP were added and incubated 4 h at 30°C. Before use, the chimaeric molecule was folded by incubation at 90°C for 2 min and kept on ice.

Purification of ribosomal particles assembled of histone H4 mRNA

Streptavidin MagneSphere Paramagnetic Particles (Promega) were used to isolate ribosomal initiation complexes stalled on histone H4 mRNA. A 50 μl aliquot of biotinylated hybrid mRNA/DNA (35 μg) was incubated with 150 μl of pre-washed streptavidin-coated beads in binding buffer [100 mM potassium phosphate (pH 7.2) and 150 mM NaCl] for 30 min at room temperature, and then washed thoroughly with water.

In parallel, nuclease-treated RRL (100 μl; Promega) was added to 50 mM KAc (potassium acetate) 0.4 units/μl of RNasin (Promega) and 100 μM of each of the 20 amino acids in a total volume of 200 μl. The mix was incubated at 30°C for 5 min and then chilled for 20 min on ice.

The immobilized hybrid histone H4 mRNA was then incubated for 5 min at 30°C with the RRL translation mixture in the presence of 1 mg/ml cycloheximide and 0.5 mg/ml hygromycin. After an additional 3 min incubation on ice in the presence of 8 mM Mg(Ac)2 (magnesium acetate; 10 μl of a 320 mM solution) and 5 min on ice with buffer A [200 μl of 2 mM DTT, 100 mM KAc, 20 mM Tris/HCl (pH 7.6), 2.5 mM Mg(Ac)2, 1 mM ATP, 0.1 mM GMP-PNP (5′-guanylyl imidodiphosphate) and 0.25 mM spermidine], the beads were sequentially washed with ice-cold buffer 250 [2×400 μl of 50 mM Tris/HCl (pH 7.0), 250 mM KAc, 25 mM Mg(Ac)2, 0.1% Igepal, 5 mM 2-mercaptoethanol, 0.1 mg/ml cycloheximide and 250 mM sucrose], buffer 500 [1×400 μl of 50 mM Tris/HCl (pH 7.0), 500 mM KAc, 25 mM Mg(Ac)2, 0.05% Igepal, 5 mM 2-mercaptoethanol, 0.1 mg/ml cycloheximide and 50 mM sucrose] and buffer 50 [3×400 μl of 20 mM Tris/HCl (pH 7.0), 50 mM KAc, 10 mM Mg(Ac)2, 1 mM CaCl2, 1 mM DTT and 0.1 mg/ml cycloheximide] (modified from [19]).

The stalled H4/80S complexes were eluted in 50 μl of buffer 50 supplemented with 87 mM sucrose and 1 μl of RNasin using 10 units of RQ1 RNase-free DNase (Promega), over 30 min at room temperature. The eluted complexes were collected by ultracentrifugation for 1 h at 108000 rev./min in a S140AT rotor (Sorvall-Hitachi) at 4°C. Ribosomal pellets were resuspended in buffer B {20 mM Tris/HCl (pH 7.6), 0.2 mM EDTA, 10 mM KCl, 1 mM MgCl2, 1 mM DTT and 250 mM sucrose [20]} to a concentration of 10 A260 units/ml.

Analysis of the 80S initiation complexes on sucrose-density gradient

The eluted ribosomes were separated on a 7–47% linear sucrose gradient in ice-cold buffer 50. The reactions were loaded on the gradients and spun (37000 rev./min for 2.5 h at 4°C) in a SW41 rotor. Gradients were fractionated and analysed by Cerenkov counting.

In vitro translation in RLL

Translation reactions in RRL were performed as described by the manufacturer (Promega). Reactions were incubated at 30°C for 90 min and included 100 mM KAc, 3.8 mCi [35S]Met and 250 nM of histone H4 mRNA transcript or mRNA–DNA chimaera (free or on the magnetic particles). Aliquots of translation reactions were analysed by SDS/PAGE (15% gel) and the [35S]-labelled histone H4 bands were quantified on a phosphorimager.

Toe-print analysis

Toe-print analyses were adapted from [21]. Initiation complexes were assembled in RRL in the presence of 1 mg/ml cycloheximide, or 10 mM edeine, 1 unit/ml RNaseOUT (Invitrogen) and 250 nM histone H4 mRNA (as a RNA–DNA chimaera immobilized on the biotin-coated paramagnetic particles or in the free RNA form). Ribosome assembly was obtained after 5 min of incubation at 30°C and the complexes were processed as follows. The complexes assembled on the paramagnetic beads were washed and eluted as described above. The complexes assembled on the mRNA in solution were processed without further purification. The two types of RNA–ribosome complexes were complemented with an equal volume of ice-cold buffer A [21] and placed on ice. In order to separate ribosomal complexes from the non-ribosomal, fraction samples were spun twice at 105000 g in a S45A rotor (Sorvall-Hitachi) at 4°C for 30 min. Ribosome toe-prints were analysed in the complete mixture (T) before centrifugation, in the supernatant fraction (S) and in the ribosome pellet (P). Ribosome pellets were dissolved in 40 μl of buffer A complemented with the same translation inhibitor. Deoxy oligonucleotide primer (4 pM) complementary to the sequence of bases 140–160 were added and incubated at 30°C for 3 min. Then, 1 μl of a 320 mM Mg(Ac)2 solution, 4 μl of a dNTP mixture (containing 5 mM of dATP, dGTP, dTTP and 1 mM dCTP), 1 μl of [α-32P]dCTP (~6000 Ci/mmol; Amersham Biosciences), 10 units of RNaseOUT and 1 unit/μl RNase H-minus MMLV (Moloney murine leukaemia virus) reverse transcriptase (Promega) were added and incubated for 45 min at 30°C. Copy DNA was purified and separated by denaturing PAGE, analysed by phosphorimaging and compared with appropriate dideoxynucleotide sequencing ladders.

Cryo-EM

Grids coated with home-made lacey carbon covered with a thin (~3 nm) continuous carbon film were used. The assembled complexes were applied to glow-discharged grids to increase the number of particles per field of view. Excess solution was blotted and the grids were flash-frozen in liquid ethane cooled by liquid nitrogen as described previously [22]. Grids were mounted for observation in a Tecnai F30 Polara cryo-EM at liquid nitrogen temperature (−190°C) (FEI). The images were collected under low-dose conditions (20 electrons per Å2; 1 Å=0.1 nm), at 100 kV, on film at ×59000 magnification and on a CCD (charge-coupled device) camera (4Kx4K Eagle, FEI) at ×76700 magnification. The best micrographs were digitized on a drum scanner (Heidelberger Druckmaschinen) with a step size of 4.6 μm. The digitized images were further selected according to their calculated power spectra and were coarsened by a factor of three, resulting in a pixel size corresponding to 2.4 Å per pixel at the specimen level. For the figures, the sampling of the CCD frames, originally at 1.96 Å per pixel, was scaled and adjusted to be the same as for the film data.

RESULTS

Strategy

We designed a strategy in which the ribosome initiation complex is assembled on the mRNA while being immobilized on a matrix. First, a chimaera mRNA–DNA biotinylated was assembled and immobilized on a matrix of streptavidin. To favour ligation of the chimaera, a splint DNA–oligonucleotide was used. Then, the ribosomal initiation complex was captured on to the chimaeric RNA bait immobilized on magnetic particles and eluted after specific DNA cleavage of the mRNA–DNA chimaera (Figure 1). The process was developed and refined for histone H4 mRNA.

Strategy of purification of translation initiation complex on histone H4 mRNA

Figure 1
Strategy of purification of translation initiation complex on histone H4 mRNA

Histone H4 mRNA is ligated to a biotinylated DNA oligonucleotide (in italic bold). The resulting chimaeric mRNA–biotinylated DNA is immobilized on streptavidin-coated ParaMagnetic Particles (S-PMP) and extensively washed to remove the splint DNA oligonucleotides (in italic bold). Assembly of translation initiation complex on the mRNA is performed in RRL. Final elution of the complex is obtained by cleavage of the DNA part of the chimaera by DNase I.

Figure 1
Strategy of purification of translation initiation complex on histone H4 mRNA

Histone H4 mRNA is ligated to a biotinylated DNA oligonucleotide (in italic bold). The resulting chimaeric mRNA–biotinylated DNA is immobilized on streptavidin-coated ParaMagnetic Particles (S-PMP) and extensively washed to remove the splint DNA oligonucleotides (in italic bold). Assembly of translation initiation complex on the mRNA is performed in RRL. Final elution of the complex is obtained by cleavage of the DNA part of the chimaera by DNase I.

Design, assembly, binding and elution of the RNA–DNA chimaera

To design the splint oligo-deoxynucleotide complementary to both mRNA and DNA oligonucleotide sequences, we had to take into account that histone mRNAs are not polyadenylated, but end with a tightly folded 3′-terminal hairpin [23]. Thus, a direct hybridization of the 3′-UTR to the splint DNA during ligation could not be performed. Furthermore, the 3′-UTR is short (54 nt), therefore in order to prevent a sterical clash between the matrix and the ribosome assembly process, the histone H4 mRNA was extended by an RNA linker of 30 nt containing the sequence (CAA)9CAC. This extended histone H4 mRNA performs equally well in vitro translation assays when compared with the wild-type histone H4 mRNA (see below). The histone H4–(CAA)9CAC mRNA was used in a splint-ligation reaction to create the histone H4 mRNA–DNA–biotin chimaera. The chimaeric mRNA–DNA molecule was assembled by ligation of a biotinylated DNA oligonucleotide to the 3′-end of histone H4 mRNA in the presence of the DNA splint oligonucleotide.

Many runoff T7 transcripts have significant heterogeneity at their 3′-ends owing to the production of so-called N+1 products. To include these N+1 transcripts, a second splint oligonucleotide was added with an extra nucleotide (any four nucleotides at this position) at the junction site (Figure 1). Ligation was catalysed by T4 DNA ligase, which is able to ligate hybrids of ribo- and deoxyribo-nucleotide homopolymers in double-stranded regions [15,16,24]. However, efficient ligation of RNA substrates requires stoichiometric or greater concentrations of T4 DNA ligase because this enzyme cannot turnover effectively on RNA-containing duplexes [25]. With histone H4 mRNA or U7 snRNA (small nuclear RNA) as substrates, a nearly 100% ligation efficiency was obtained in stoichiometric conditions with ligase for an RNA concentration of 30 nM (results not shown). This is consistent with the low Km value of T4 DNA ligase for its nucleic acid substrates (from 10−8 to 10−7 M) [26] allowing efficient ligation at sub-micromolar concentrations of RNA. However, up-scaling the ligation reaction induced decreases of the ligation efficiency to about 40–50% (Figure 2) because the equimolar concentration of T4 DNA ligase could not be kept constant. We propose that this point of the protocol could be improved using more concentrated preparations of T4 DNA ligase. Using a His6-tagged version of the enzyme to produce highly concentrated enzyme solutions could solve the problem.

Ligation efficiency during chimaera production

Figure 2
Ligation efficiency during chimaera production

Denaturing PAGE showing the ligation efficiency of mRNA to the biotinylated oligonucleotide–DNA. Two RNAs were tested: histone H4 mRNA (A) and U7 snRNA (B). Ligation of the biotinylated oligonucleotide–DNA shifts migration of the 5′-end [α32P] cap-labelled RNAs. The efficiency of ligation quantified by a phosphorimager was 51 and 42% respectively. After digestion by RQ1 DNase of the chimaera, the original sizes of the RNAs were recovered.

Figure 2
Ligation efficiency during chimaera production

Denaturing PAGE showing the ligation efficiency of mRNA to the biotinylated oligonucleotide–DNA. Two RNAs were tested: histone H4 mRNA (A) and U7 snRNA (B). Ligation of the biotinylated oligonucleotide–DNA shifts migration of the 5′-end [α32P] cap-labelled RNAs. The efficiency of ligation quantified by a phosphorimager was 51 and 42% respectively. After digestion by RQ1 DNase of the chimaera, the original sizes of the RNAs were recovered.

To immobilize the ribonucleoprotein complex on the solid matrix, we used streptavidin-coated magnetic beads. Biotin was positioned on the 3′-end of the mRNA–DNA chimaera to enable ribosome binding to occur freely on the 5′-end and to ensure that only intact mRNAs (full-length) were bound to the affinity column. The high affinity of biotin for streptavidin (Kd=10−15M) ensures almost irreversible binding and prevents dissociation of the immobilized molecules and unspecific release of the complexes during the different purification steps. Streptavidin-coated magnetic beads also facilitated the rapid separation of complexes from the RRL and rapid washing steps.

Extensive washing of the immobilized mRNA is essential for removal of remaining DNA splint oligonucleotide in order to avoid RNA cleavage by endogenous RNase H activity of RRL [27,28]. After several washing steps, the immobilized mRNA–DNA chimaera is ready for ribosomal complex capture. It can also be stored at −20°C for several months.

Working with RNA–DNA chimaeras conferred an obvious advantage for specific elution of the assembled complexes on the solid matrix by DNase treatment, keeping the mRNA within the initiation complex intact. Another advantage of the DNase I elution is that it is very specific compared with other elution processes based on pH or salt variations which often result in complex dissociation and/or unspecific elution of the other biomolecules bound aspecifically to the beads.

The protocol of binding/washing/elution of the mRNA–DNA chimaera on the beads was first tested and validated without the ribosome assembly step (Figure 3A). Chimaeric histone H4 (6.6 μg) mRNA (55 pmol) was incubated with 0.6 ml of prewashed streptavidin-coated beads (about 0.45 mg of beads). Although the amount of RNA did not exceed the beads’ capacity (450 pmoles), a certain amount of RNA (about 30–40% of the starting RNA) did not bind to the particles and was found in the flow through fraction (FT lane). This unbound RNA might correspond to the unligated mRNA that still contaminated the mRNA–DNA chimaeras after ligation (see Figure 2). It is also possible that some steric occlusion might have prevented the streptavidin–biotin interaction. After capture on the magnetic stand and washing of the beads, the captured chimaeric mRNA could be eluted with RQ1 DNase (Figure 3, lanes E, about 20% of the initial amount of RNA), whereas a fraction resistant to DNase digestion was found in the urea-extracted fraction (Figure 3, lane U, about 20% of the initial amount of RNA). This observation suggests that RQ1 DNase is less efficient on the chimaeras bound to the beads when compared with the digestion of same chimaera in solution (Figure 2, right-hand lane). Nevertheless, this experiment showed that chimaeric mRNAs could be successfully captured on the beads and eluted by the RQ1 DNase digestion, with an estimated yield of 20%.

Binding and elution of the mRNA bait and affinity purification of translation-initiation complexes

Figure 3
Binding and elution of the mRNA bait and affinity purification of translation-initiation complexes

(A) Affinity purification of H4 mRNA on streptavidin-coated beads. A representative denaturating urea PAGE gel is shown. The chimaeric mRNA–biotinylated DNA (lane C) was loaded on the beads (0.2 μg is shown). The excess of chimaeric molecules is shown in the flow-through fraction (lane FT). After three wash steps (lanes W1–W3) to remove unspecific RNA binding, the bound molecules were eluted with DNase I (lane E). A fraction of the bound chimaeras was resistant to DNase I digestion as shown by the urea final extraction step (lane U). (B) Translation-initiation complexes were assembled in RRL. Lane C contains the histone H4 mRNA/biotinylated DNA chimaera (2 μg is shown), which was used for the purification of ribosomal complexes. Unbound rRNAs and tRNAs are shown in the flow-through fraction (lane FT). After four wash steps [buffer 250 (lanes W250), buffer 500 (lane W500) and buffer 50 (lane W50)] to remove unspecific RNA binding, the bound molecules were eluted with DNase I (lane E). Fractions were separated on a 1% agarose gel and visualized by ethidium-bromide staining. Several bands of the DNA ladder (1 Kb Plus DNA Ladder, Invitrogen) are highlighted and the positions of the ribosomal RNAs, tRNAs and histone H4 mRNA are indicated.

Figure 3
Binding and elution of the mRNA bait and affinity purification of translation-initiation complexes

(A) Affinity purification of H4 mRNA on streptavidin-coated beads. A representative denaturating urea PAGE gel is shown. The chimaeric mRNA–biotinylated DNA (lane C) was loaded on the beads (0.2 μg is shown). The excess of chimaeric molecules is shown in the flow-through fraction (lane FT). After three wash steps (lanes W1–W3) to remove unspecific RNA binding, the bound molecules were eluted with DNase I (lane E). A fraction of the bound chimaeras was resistant to DNase I digestion as shown by the urea final extraction step (lane U). (B) Translation-initiation complexes were assembled in RRL. Lane C contains the histone H4 mRNA/biotinylated DNA chimaera (2 μg is shown), which was used for the purification of ribosomal complexes. Unbound rRNAs and tRNAs are shown in the flow-through fraction (lane FT). After four wash steps [buffer 250 (lanes W250), buffer 500 (lane W500) and buffer 50 (lane W50)] to remove unspecific RNA binding, the bound molecules were eluted with DNase I (lane E). Fractions were separated on a 1% agarose gel and visualized by ethidium-bromide staining. Several bands of the DNA ladder (1 Kb Plus DNA Ladder, Invitrogen) are highlighted and the positions of the ribosomal RNAs, tRNAs and histone H4 mRNA are indicated.

Isolation of ribosomal initiating complexes assembled on histone H4 mRNA

Ribosome–mRNA complexes were isolated using nuclease-treated RRL. Ribosomal 80S complexes were assembled on the beads on the chimaeric mRNA in RRL in the presence of cycloheximide and hygromycin (blocking the translocation step; see also [18]). After incubation, the beads were thoroughly washed with buffers containing different salt stringencies, always in presence of cycloheximide. Finally, the bound complexes were released from the beads by RQ1 DNase digestion. The final product contains equimolar amounts of 18S, 28S and 5S/tRNA. A small portion of histone H4 mRNA bait molecules did not trap ribosomes during the process as indicated by the small molar excess of histone H4 mRNA (Figure 3B). These molecules were also released by RQ1 DNase digestion and required an additional step of centrifugation to be eliminated. The digestion mix was centrifuged in a micro-ultracentrifuge at 680000 g for 1 h. Under these conditions, only the ribosomal complexes were recovered in the pellet, whereas free mRNA stayed in the supernatant. This step provided an easy way to separate both species and to concentrate initiating ribosomal complexes. It has the advantage, compared with the conventional sucrose-gradient ultracentrifugation, to be less labour consuming and does not need further concentration or sucrose elimination. However, it is only efficient on high molecular mass complexes such as ribosomal complexes that can be pelleted on centrifugation.

Purification yield

To quantify ribosome assembly on histone H4 mRNA, the whole process was executed in the presence of [32P]-labelled chimaeric RNA in order to monitor its distribution in the different fractions (Figure 4). The process was also performed on U7 snRNA, a non-coding RNA that should not assemble ribosomes. [α32P]m7G-capped histone H4 mRNA and U7 snRNA were first ligated to the biotinylated oligonucleotide DNA. The radioactive chimaeras (about 2 pmol) were gel-purified to remove unligated RNAs and were added to unlabelled histone H4 mRNA and U7 snRNA chimaeras (final concentration of 4.7 μM) before being immobilized on streptavidin-coated beads. The radioactivity of the fractions was quantified in all the subsequent steps until the final elution step of the initiation complex or snRNA. A small part of the radioactivity was not retained on the beads and was eluted in the flow-through fraction and wash steps as seen in Figure 4. After washing and the addition of the RRL to the beads a first significant peak of radioactivity was observed. This loss might be explained by the endogenous RNase H action, which cleaves specifically the remaining mRNA/DNA duplexes that still contained the splint oligonucleotide. More extensive washing of the immobilized hybrid RNA could improve the removal of the splint molecules and the global yield. Alternatively, we could add an antisense splint oligonucleotide to favour the release of the splint oligonucleotide. The addition of DNase I during the elution step led to a recovery of 17% and 20% of the initial counts for the histone H4 and U7 chimaeras respectively. This might be considered as the amount of mRNA successfully binding the beads and being released by the DNase digestion (Figure 4). A high peak of radioactivity resistant to DNase digestion was remaining on the beads at the end of the process. This resistance might be partly owing to the presence of ribosomes on the RNA, which might create some steric hindrance; however, U7 snRNA that did not assemble ribosomes showed the same level of DNase resistance. Another possibility might be that the chimaera interacted aspecifically with the beads, acquiring by that way resistance to DNase.

Elution profile of the whole purification process and purification yield

Figure 4
Elution profile of the whole purification process and purification yield

(A) mRNA elution profile during purification of 80S particles on histone H4 mRNA. To track the mRNA, a radioactive [α32P]G-cap was introduced at the 5′-end of the mRNA. The radiolabelled chimaera was gel-purified to eliminate free unincorporated [α32P]GTP and unligated radiolabelled mRNA that would perturb the yield calculation. A fraction of [α32P]-labelled mRNA was lost during the mRNA loading step. Then, a new release of mRNA was observed during the RRL addition step (endogenous RNase H activity of RRL). Release of initiation complexes by RQ1 DNase digestion (DNase I elution step) yielded elution of 17% of the initial c.p.m. The residual c.p.m. were resistant to elution and remained irreversibly bound to the column. Radiolabelled mRNAs were counted using the Cerenkov method. (B) mRNA elution profile during purification of U7 snRNA. The same process was repeated on U7 snRNA–DNA chimaera. Although no ribosomes were assembled, the whole process of snRNA purification could be quantified. The elution profile is very similar to the histone H4 mRNA profile, with a purification yield of 20% of U7 snRNA. (C) The mRNA concentration in the RRL-assembly mix was gradually increased during the purification process and initiation complexes were quantified at 260 nm. The linear curve suggests that the beads were not saturated by the maximal mRNA concentration used.

Figure 4
Elution profile of the whole purification process and purification yield

(A) mRNA elution profile during purification of 80S particles on histone H4 mRNA. To track the mRNA, a radioactive [α32P]G-cap was introduced at the 5′-end of the mRNA. The radiolabelled chimaera was gel-purified to eliminate free unincorporated [α32P]GTP and unligated radiolabelled mRNA that would perturb the yield calculation. A fraction of [α32P]-labelled mRNA was lost during the mRNA loading step. Then, a new release of mRNA was observed during the RRL addition step (endogenous RNase H activity of RRL). Release of initiation complexes by RQ1 DNase digestion (DNase I elution step) yielded elution of 17% of the initial c.p.m. The residual c.p.m. were resistant to elution and remained irreversibly bound to the column. Radiolabelled mRNAs were counted using the Cerenkov method. (B) mRNA elution profile during purification of U7 snRNA. The same process was repeated on U7 snRNA–DNA chimaera. Although no ribosomes were assembled, the whole process of snRNA purification could be quantified. The elution profile is very similar to the histone H4 mRNA profile, with a purification yield of 20% of U7 snRNA. (C) The mRNA concentration in the RRL-assembly mix was gradually increased during the purification process and initiation complexes were quantified at 260 nm. The linear curve suggests that the beads were not saturated by the maximal mRNA concentration used.

Scaling up the purification process

The mRNA concentration in the RRL-assembly mix was gradually increased and purified initiation complexes were quantified at 260 nm (Figure 4C). We observed a linear increase in the purified ribosomal complex formation compared with the increase in chimaeric mRNA, suggesting that the beads were not saturated. Therefore, for routine purifications, we used 35 μg (335 pmol) of the histone mRNA–DNA chimaera and 200 μl of RRL. The procedure yielded routinely up to 6 μg (about 6 pmol) of pure tag-less histone H4 mRNA/80S particles.

Homogeneity and purity of the affinity-purified ribosome complexes

The composition and purity of the eluted ribosomal particles was verified by several analytical techniques. Sucrose-gradient centrifugation was used to fractionate and analyse complexes assembled on [32P]-labelled histone H4 mRNA. The radioactivity profile of a typical sucrose-gradient isolation procedure performed on ribosome-histone H4 mRNA complexes, formed in the presence of both cycloheximide and hygromycin, is depicted in Figure 5(A). The radiolabelled histone H4 mRNA formed a main peak that sediments as a typical 80S particle corresponding to the ribosome assembled on histone H4 mRNA. A peak of 48S particles was also detected, suggesting that a fraction of the initiating complexes did not achieve the complete process of assembly. However, the amount of 48S particles remained low as shown by equimolar concentrations of 18S and 28S rRNA in the agarose gel (Figure 5B). This is supported by the cryo-EM analysis, which revealed a remarkable homogeneity of the sample consisting of only 80S particles and no 48S particles (Figure 5C, also see below).

Characterization of the histone H4 mRNA–80S complexes

Figure 5
Characterization of the histone H4 mRNA–80S complexes

Ribosomal complexes were assembled in RRL on histone H4 [32P]-end labelled mRNA, affinity-purified and analysed. (A) Mobility of translation initiation complexes during centrifugation through a 7–47% linear sucrose density gradient. Initiation complex was tracked using [32P]-end labelled H4 mRNA. Sedimentation was from right- (7%) to left-hand side (47%). The 80S and 48S complexes are indicated. The percentage of RNA bound represents the mean of two independent experiments. (B) After ultracentrifugation, the initiation complexes assembled on the different RNAs were analysed on an agarose gel stained by ethidium bromide. The rRNAs, U7 snRNA and histone H4 mRNA are indicated. The far left-hand lane is a molecular mass ladder (1 Kb Plus DNA Ladder, Invitrogen). (C) Cryo-EM micrograph of the preparation. Scale bar, 500 Å.

Figure 5
Characterization of the histone H4 mRNA–80S complexes

Ribosomal complexes were assembled in RRL on histone H4 [32P]-end labelled mRNA, affinity-purified and analysed. (A) Mobility of translation initiation complexes during centrifugation through a 7–47% linear sucrose density gradient. Initiation complex was tracked using [32P]-end labelled H4 mRNA. Sedimentation was from right- (7%) to left-hand side (47%). The 80S and 48S complexes are indicated. The percentage of RNA bound represents the mean of two independent experiments. (B) After ultracentrifugation, the initiation complexes assembled on the different RNAs were analysed on an agarose gel stained by ethidium bromide. The rRNAs, U7 snRNA and histone H4 mRNA are indicated. The far left-hand lane is a molecular mass ladder (1 Kb Plus DNA Ladder, Invitrogen). (C) Cryo-EM micrograph of the preparation. Scale bar, 500 Å.

To analyse further the protein content of the assembled purified complexes, we performed tandem MS investigations. Ribosome complexes were assembled in usual conditions and the complexes bound to the particles were subjected to trypsic digestion. The buffer was exchanged with the help of a magnetic stand to a buffer compatible with trypsin digestion: 25 mM ammonium bicarbonate. The proteins were reduced with 5 mM DTT at 95°C, alkylated with 10 mM iodoacetamide and digested with trypsin (1:10 trypsin/protein) for 16 h. The digest was analysed by nano-LC-MS/MS (liquid chromatography tandem MS) on a Nano LC 2D Plus with cHIPLC nanoflex module (Eksigent) coupled with a TripleTOF 5600 (Sciex). The data were analysed against the UniProt human database with a false detection rate<0.5%. The proteins showing the highest scores (for MS) were ribosomal proteins. All 32 ribosomal proteins of the small subunit [in addition to RACK1 (receptor for activated C kinase 1)] could be detected. For the large subunit, 44 out of the 46 ribosomal proteins could be detected. The two missing proteins RPL39 (ribosomal protein L39) and RPL41 (ribosomal protein L41) are very small (51 and 25 amino acids respectively) and are both arginine- and lysine-rich proteins. Therefore trypsin digestion of these proteins cleaves them into peptides that are clearly too small to be detected by MS. Altogether, this analysis confirmed the integrity of the translation initiation complex purified by our approach. With lower scores we could also detect eight non-ribosomal proteins, most being considered as non-specific RNA- or DNA-binding proteins. For instance, poly(rC)-binding protein 1 and 2 are single-stranded nucleic acid-binding proteins that bind preferentially to oligo(dC) [29]. YB-1 (nuclease-sensitive element-binding protein 1) is a general RNA-binding protein involved in many mRNA processes [30]. Sterile α motif domain-containing protein 9 is a member of the group carrying the sterile α motif present in a wide variety of proteins involved in many biological processes [31]. The heterogeneous nuclear ribonucleoprotein K is a multifunctional protein known to be involved in the regulation of transcription, translation, nuclear transport and signal transduction [32]. PTB1 (polypyrimidine tract-binding protein 1) is a member of the poly(rC)-binding protein family. PTB1 is a regulator of alternative pre-mRNA splicing, and also stimulates the initiation of translation dependent on many viral IRESs and is considered as an ITAF (IRES-trans-acting factor) [33]. The replication protein A 70 kDa is known to be a single-stranded DNA-binding protein essential for multiple processes in cellular DNA metabolism. It binds and stabilizes single-stranded DNA intermediates [34]. By binding the DNA moiety of the chimaera, this protein may hide it from digestion by the DNase during elution of the complexes. This could explain the partial recovery of the assembled complexes on to the particles.

Except for unspecific RNA/DNA-binding proteins at moderate levels, the MS analysis showed that the initiation complex preparation was remarkably pure and did not contain any translation initiation factor, confirming that the assembly of 80S was completed.

We also repeated the ribosome assembly and purification procedure on a non-coding RNA (U7 snRNA) in order to check if spontaneous and aspecific binding of ribosomes could occur on the chimaera or on the beads. For this purpose, the U7 snRNA was extended by the adaptor sequence (CAA)9CAC and ligated to the biotinylated oligo DNA (Figure 2). Then the whole purification process was repeated and the DNase I elution fraction was analysed on an agarose gel (Figure 5B). No ribosomal RNAs could be eluted from the U7 snRNA bait, confirming the absolute requirement of the AUG codon to assemble the 80S particles. Similarly, no proteins could be eluted from the beads (results not shown).

Functional assays on the 80S assembled particles

To test the functional ability of the 80S particles assembled on histone H4 mRNA, two assays were performed. The translation ability of histone H4 mRNA bound to the magnetic particles was compared with the unbound biotinylated chimaeric histone H4 mRNA, and to the free native histone H4 mRNA. Translation was performed in RRL in the presence of equal concentrations of mRNAs (250 nM). In the case of the biotinylated chimaeric mRNA bound to the magnetic particles, the excess of free mRNA was carefully eliminated and translation was directly performed on the beads. The three mRNAs showed the same ability to synthesize histone H4 (Figure 6A). This demonstrates that the 80S ribosomal particles assembled and purified by our method are fully functional.

Translation assays and ribosome toe-print analysis on complexes assembled on histone H4 mRNA

Figure 6
Translation assays and ribosome toe-print analysis on complexes assembled on histone H4 mRNA

(A) Translation products were formed in RRL in the presence of [35S]methionine and were separated by 15% SDS/PAGE. Histone H4 mRNA concentration was 250 nM in order to avoid exhaustion of the compounds from the translation extracts. The biotinylated chimaeric mRNA was bound to the streptavidin-coated ParaMagnetic Particles (S-PMP) and thoroughly washed to eliminate the free mRNA chimaera. The translation product, histone H4, is shown by the arrow. (B) Initiation complexes were assembled on histone H4 mRNA immobilized on the magnetic particles starting from RRL extracts. Cycloheximide (1 mg/ml) or edeine (10 μM) were used to stall the initiation complexes at the translocation step or 48S binding respectively. Initiation complexes affinity-purified (lane T, for total) were fractionated in pellet (P) and supernatant (S) fractions. Primer extensions were performed with MMLV reverse transcriptase. Reaction samples were treated as described in the Experimental section and separated by 8% denaturing PAGE together with the appropriate dideoxynucleotide sequencing ladder (shown on the left-hand side). The position of the AUG triplet is indicated; arrows on the right-hand side indicate toe-print positions. (C) Control of initiation complexes assembled on histone H4 mRNA in solution in the presence of cycloheximide. No purification step on the streptavidin-coated beads was performed in this experiment.

Figure 6
Translation assays and ribosome toe-print analysis on complexes assembled on histone H4 mRNA

(A) Translation products were formed in RRL in the presence of [35S]methionine and were separated by 15% SDS/PAGE. Histone H4 mRNA concentration was 250 nM in order to avoid exhaustion of the compounds from the translation extracts. The biotinylated chimaeric mRNA was bound to the streptavidin-coated ParaMagnetic Particles (S-PMP) and thoroughly washed to eliminate the free mRNA chimaera. The translation product, histone H4, is shown by the arrow. (B) Initiation complexes were assembled on histone H4 mRNA immobilized on the magnetic particles starting from RRL extracts. Cycloheximide (1 mg/ml) or edeine (10 μM) were used to stall the initiation complexes at the translocation step or 48S binding respectively. Initiation complexes affinity-purified (lane T, for total) were fractionated in pellet (P) and supernatant (S) fractions. Primer extensions were performed with MMLV reverse transcriptase. Reaction samples were treated as described in the Experimental section and separated by 8% denaturing PAGE together with the appropriate dideoxynucleotide sequencing ladder (shown on the left-hand side). The position of the AUG triplet is indicated; arrows on the right-hand side indicate toe-print positions. (C) Control of initiation complexes assembled on histone H4 mRNA in solution in the presence of cycloheximide. No purification step on the streptavidin-coated beads was performed in this experiment.

In addition, proper assembly of the 80S complexes on the initiation codon was verified by the ribosome toe-printing assay, which directly identifies the position of the ribosome on the mRNA. In the toe-printing assay on histone H4 mRNA, reverse transcription stops 19–20 nt downstream of the adenine of the AUG start codon [18]. Although this position 19–20 differs from the standard position 16–18, we have demonstrated that the 19–20 toe-print position detected on histone H4 mRNA with MMLV reverse transcriptase is equivalent to a 16–18 toe-print detected with AMV (avian myeloblastosis virus) reverse transcriptase [18]. In the present study the toe-printing reaction was carried out on the affinity-purified histone H4 mRNA particles and on the 80S particles assembled on free histone H4 mRNA. Ribosome assembly was performed in the presence of cycloheximide, which blocks the translocation step and thus stalls the ribosome on the AUG codon. Identical ribosome toe-prints were detected at position 19–20 in both experiments (Figures 6B and 6C). This shows that the affinity-purified 80S ribosomes are assembled at the proper position with the AUG codon within the ribosomal P-site base-paired with Met-tRNA(Met)i. We could also notice that the toe-print positioning was shifted from position +20 to +19 after the ultracentrifugation step (Figures 6B and 6C, lane P) as the result of the loss of one aminoacyl-tRNA molecule from the ribosomal A site [35]. Toe-printing experiments were also performed after incubation with edeine instead of cycloheximide. Edeine is an inhibitor that interferes with AUG recognition by 40S-eIF2-GTP/Met-tRNA(Met)i and prevents formation of 48S particles on the AUG codon during canonical initiation [36,37]. Indeed, no initiation complexes could be assembled and no ribosome toe-print could be detected in this condition (Figure 6B). Altogether, the toe-printing data and the translation results clearly show the correct assembly of 80S particles on histone H4 mRNA immobilized on the beads.

Cryo-EM characterization of the sample

Flash-freezing and cryo-EM visualization of the eluted 80S complexes revealed that our purification protocol was efficient and of appropriate quality for biophysical characterization and structural analysis. Such techniques often require high purity and homogeneity of rather concentrated samples. Single-particle images obtained from the ribosome stalled in the initiation state confirmed that the complexes prepared with this purification method were sufficiently concentrated and that the majority of the particles were monosomes. The image contrast showed compatibility of the elution buffer with the chosen biophysical technique and the absence of macromolecular size impurities (Figure 5C and Ménétret, J.-F. and Klaholz, B.P., unpublished work). Statistical analysis and classification of the isolated particles demonstrated sufficient homogeneity in the sample which should allow determination of the three-dimensional structure of the complex. The fact that only full 80S molecules rather than 40S and 60S subunits are seen in the cryo-EM images illustrates that the affinity purification is a mild procedure avoiding dissociation of the ribosomal subunits.

DISCUSSION

The goal of the present study was to develop a simple, rapid and efficient method of purification which generates pure, homogenous eukaryotic ribosomal complexes suitable for cryo-EM studies. The protocol that we describe is an affinity purification procedure using an in vitro-transcribed mRNA linked to biotin through a DNA oligonucleotide. The protocol for isolation of eukaryotic initiation complexes was developed and tested using the mRNA encoding murine histone H4 and RRL as a source of translation initiation factors and ribosomes.

The method combines two innovations compared with previous reported protocols of purification of ribosomal particles. First, it uses an RNA–DNA chimaera instead of an RNA/DNA hybrid to link the mRNA to the biotin molecule. This provides intrinsic resistance to the RNase H activity present in RRL extracts and does not require synthesis of expensive modified oligonucleotides resistant to this activity [14]. Another advantage is that the chimaera does not require denaturating/annealing steps that may modify the mRNA structure and alter initiation complex formation or even induce dissociation. In addition, the biotinylated chimera can readily be eluted from the beads by DNase I digestion under native conditions, keeping the initiation complexes intact. Secondly, the method allows assembly of ribosomal particles on mRNAs that do not bear exogenous structural tags (aptamers) used for the affinity purification. These aptamers have demonstrated their efficiency in diverse affinity-based purification procedures; however, these procedures suffer from recurrent problems stemming from the nonspecific binding of either proteins or RNAs found in crude translation extracts and native RNA folding disturbance.

The strategy of construction of the mRNA–biotinylated DNA chimaera is a widely applicable and inexpensive procedure for the purification of other RNPs depending on the RNA-binding motif used or for mRNA-bound ribosomes. The RNA-binding motif can be indistinctly tagged by the biotinylated oligonucleotide DNA part on the 3′- or 5′-side. We choose to tag the 3′-end to assemble translation initiation complexes on the 5′-end, but 5′-end tagging could also be used to isolate complexes on the mRNA 3′-end such as processing or polyadenylation complexes. The method is also fast and, starting from the mRNA–biotinylated DNA chimaera kept at −20°C, the purified RNPs can be isolated within 3 h. Considering the efficiency, we frequently observed that, starting from a rather low amount such as 35 μg of crude mRNA transcript, 6 μg of 80S ribosomal initiation complex could be isolated with high quality enabling cryo-EM investigations. Potentially, purifying the chimaera and depleting more efficiently the splint could increase the yield of purification by improving binding to the beads and limiting the RNase H degradation. The DNase I digestion step could also be improved by extending the length of the oligonucleotide DNA for a better access to the enzyme. For some analytical applications, it would also be preferable to replace the DNase I elution step by a self-cleavage step catalysed by a ribozyme inserted upstream of the oligonucleotide DNA. The method could easily be adapted for that and this would prevent the addition of exogenous proteins that could affect mass spectrometry analysis.

The mRNA concentration in the RRL-assembly mix was gradually increased and purified initiation complexes were quantified at 260 nm (Figure 4C). We observed a linear increase in the purified ribosomal complex formation compared with the increase in chimaeric mRNA, suggesting that the beads were not saturated. Therefore, for routine purifications, we used 35 μg (335 pmol) of the histone mRNA–DNA chimaera and 200 μl of RRL. The procedure yielded routinely up to 6 μg (about 6 pmol) of pure tag-less histone H4 mRNA/80S particles. Compared with other methods that yield milligram quantities of 48S particles [10], this amount remains modest; however, it is adequate for performing high homogeneity demanding techniques such as cryo-EM and MS studies, two approaches widely used to investigate high-molecular-mass complexes.

To our knowledge, this is the first time that an entire eukaryotic cellular mRNA is used in its native state to specifically assemble and purify ribosomal initiation complexes from a crude translation extract (RRL). The materials purified with this procedure contain 80S monosomes complexed to intact histone H4 mRNA. The homogeneity of ribosomal complexes purified by this method represents a significant technical improvement for the study of eukaryotic translation. Another advantage is that the purification procedure is simple and rapid, so that a large number of different samples can be processed at the same time. The amount of starting biological material needed to analyse associated proteins and mRNAs can be scaled down, thereby allowing qualitative experiments to be performed, in which multiple conditions of ribosome assembly can be assessed.

To conclude, we have shown in the present study a novel affinity-based chromatography procedure allowing specific purification of RNA-associated RNP complexes as illustrated by the preparation of eukaryotic 80S ribosomes stalled in the initiation state. This simple inexpensive method may be easily adapted to study the assembly of complexes on to binding motifs or complete cellular RNAs.

Abbreviations

     
  • CCD

    charge-coupled device

  •  
  • cryo-EM

    cryo-electron microscopy

  •  
  • DTT

    dithiothreitol

  •  
  • HCV

    hepatitis C virus

  •  
  • IRES

    internal ribosome entry site

  •  
  • KAc

    potassium acetate

  •  
  • Mg(Ac)2

    magnesium acetate

  •  
  • MMLV

    Moloney murine leukaemia virus

  •  
  • ORF

    open reading frame

  •  
  • PTB1

    polypyrimidine tract-binding protein 1

  •  
  • RRL

    rabbit reticulocyte lysate

  •  
  • snRNA

    small nuclear RNA

  •  
  • UTR

    untranslated region

AUTHOR CONTRIBUTION

Sharief Barends, Gilbert Eriani and Franck Martin designed the research. Lydia Prongidi-Fix, Laure Schaeffer, Angelita Simonetti, Sharief Barends and Franck Martin performed the research. Jean-François Ménétret and Bruno Klaholz performed the cryo-EM analysis. Gilbert Eriani and Franck Martin analysed the data and wrote the paper.

We thank Philippe Hammann, Laurianne Kuhn and Johana Chicher (Core Facility of IBMC-Esplanade, Université de Strasbourg, Strasbourg, France) for the MS analysis.

FUNDING

This work was supported by CNRS (Centre National de la Recherche Scientifique), the Association pour la Recherche sur le Cancer [grant number 3814] and Agence Nationale pour la Recherche [grant numbers ANR-06-BLAN-0206-01 and ANR-2011-SVSE8025-01].

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