Exploring human 40S ribosomal proteins binding to the 18S rRNA fragment containing major 3′-terminal domain

Biochimica et Biophysica Acta 1854 (2015) 101–109

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Exploring human 40S ribosomal proteins binding to the 18S rRNA fragment containing major 3′-terminal domain Alexander V. Gopanenko, Alexey A. Malygin, Galina G. Karpova ⁎ Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 630090, Russia Novosibirsk State University, Novosibirsk 630090, Russia

a r t i c l e

i n f o

Article history: Received 3 July 2014 Received in revised form 10 October 2014 Accepted 4 November 2014 Available online 8 November 2014 Keywords: Human ribosomal protein Major 3′ domain of 18S rRNA Ribonucleoprotein assembly Chemical footprinting 40S ribosomal subunit

a b s t r a c t Association of ribosomal proteins with rRNA during assembly of ribosomal subunits is an intricate process, which is strictly regulated in vivo. As for the assembly in vitro, it was reported so far only for prokaryotic subunits. Bacterial ribosomal proteins are capable of selective binding to 16S rRNA as well as to its separate morphological domains. In this work, we explored binding of total protein of human 40S ribosomal subunit to the RNA transcript corresponding to the major 3′-domain of 18S rRNA. We showed that the resulting ribonucleoprotein particles contained almost all of the expected ribosomal proteins, whose binding sites are located in this 18S rRNA domain in the 40S subunit, together with several nonspecific proteins. The binding in solution was accompanied with aggregation of the RNA–protein complexes. Ribosomal proteins bound to the RNA transcript protected from chemical modification mostly those 18S rRNA nucleotides that are known to be involved in binding with the proteins in the 40S subunit and thereby demonstrated their ability to selectively bind to the rRNA in vitro. The possible implication of unstructured extensions of eukaryotic ribosomal proteins in their nonspecific binding with rRNA and in subsequent aggregation of the resulting complexes is discussed. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The central player of translation, ribosome, is one of the largest and most complicated cellular ribonucleoproteins. It consists of two subunits; each contains long ribosomal RNAs (rRNAs) and several dozen of ribosomal proteins (r-proteins). In the cell, the process of assembly of new ribosomal subunits from their constituents is governed and controlled by numerous assembly factors that provide folding of rRNAs and promote correct binding of r-proteins to them. In bacteria, the number of these factors is about two dozens (see for review [1]), whereas in eukaryotes, whose ribosomal subunits are significantly larger and more complex, the number of the assembly factors exceeds two hundreds (see for review [2]). However, the presence of the assembly factors is not prerequisite to assembly active prokaryotic ribosomal subunits in vitro. Experiments on searching for ways to reconstitute ribosomal subunits were successfully performed about 40 years ago with Escherichia coli ribosomes [3–5], and it has been demonstrated that active ribosomal subunits can be assembled from individual rRNAs and sets of total r-protein by their co-incubation under certain salt and temperature conditions. Thus, it was proved that information on the structures of ribosomal subunits is embedded in the structures of their RNA and proteins and that the subunits are capable of self-assembling. In the ⁎ Corresponding author at: Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 630090, Russia. Tel.: +7 383 363 5140; fax: +7 383 363 5153. E-mail address: [email protected] (G.G. Karpova).

http://dx.doi.org/10.1016/j.bbapap.2014.11.001 1570-9639/© 2014 Elsevier B.V. All rights reserved.

later studies, it was shown that ribosomal subunits of other prokaryotic species can be reconstituted under similar conditions [6,7], and that RNA transcripts obtained with the use of phage RNA polymerases and sets of recombinant r-proteins can substitute natural rRNAs and r-proteins in ribosomal subunit reconstitution protocols [8–10]. Moreover, separate morphological parts of the 30S ribosomal subunit can be also assembled in vitro with the use of the respective RNA transcripts and r-proteins [11–13]. It should be noted that methods of in vitro reconstitution of the prokaryotic ribosome subunits, which were extremely beneficial in biochemical studies on the ribosome structure in pre-crystal structure era (see e.g. [14,15]), are exploited nowadays, when atomic structure of prokaryotic ribosome became well understood due to X-ray analysis. In particular, these methods are successfully applied to studies on kinetics of r-proteins binding to rRNA during subunit assembly and on rRNA folding pathways accompanying this process (see for a review [16]). However, this sort of studies has not been performed so far with constituents of eukaryotic ribosome, whose atomic structure now is studied as well (see e.g. [17,18]), because of the lack of in vitro reconstitution methods. Therefore, development of approaches to the assembly of eukaryotic ribosomes similar to those working with prokaryotic ones is the actual task. Orderliness of in vivo assembly of eukaryotic small ribosomal subunit [19] resembles that of its bacterial counterpart [20], which suggests existence of in vitro assembly pathways for eukaryotic ribosomal subunits similar to those of prokaryotic ones. However, the approaches that worked well with prokaryotic ribosomal subunits turned out to

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be inapplicable with eukaryotic ones. In vitro reconstitution of active 40S and 60S subunits has been carried out so far only with proteins, which were split off from the subunits and then used for association with the “core particles” obtained as a result of the splitting [21,22]. Thus, in spite of success in the assembly of prokaryotic ribosomal subunits, methods for complete in vitro reconstitution of active subunits of the eukaryotic ribosome still remain unavailable. One might assume that the obstacles in the assembly of eukaryotic ribosomal subunits are caused by their large sizes. Actually, eukaryotic rRNAs are much longer than their prokaryotic counterparts (approximately 5000 nt with human 28S rRNA vs. 2500 nt with bacterial 23S rRNA and 1870 nt with human 18S rRNA vs. 1500 nt for bacterial 16S rRNA) and number of the eukaryotic r-proteins is greater than that of the bacterial proteins (approximately 80 vs. 47). However, the prokaryotic 50S ribosomal subunit, whose 23S rRNA is longer than 18S rRNA and number of proteins is greater than that in the eukaryotic 40S subunit, is able to self-assemble from rRNA and total protein, whereas the 40S ribosomal subunit cannot be assembled under the same conditions. Therefore, failure in in vitro assembly of active eukaryotic ribosomal subunits concerns other reasons, for instance, specific structural properties of eukaryotic ribosomal proteins. It was established that r-proteins of eukaryotes contain large unstructured regions, extensions [17,23,24], which are rigid when occupy certain positions in the subunits being associated with rRNA or neighbor r-proteins, but are likely highly flexible in free proteins. Apparently, difficulties in folding of these regions in the course of protein binding to rRNA prevent ribosome subunit selfassembly in vitro, and a possible role of factors is somehow to facilitate the folding. In this study, we have explored the possibility of self-assembly of the large morphological fragment, the head of the human 40S ribosomal subunit, from total 40S protein and an RNA transcript corresponding to the major 3′-terminal domain of the 18S rRNA, and revealed characteristic peculiarities of ribosomal proteins binding to this domain. We have shown that the resulting ribonucleprotein particles contain almost all of the expected r-proteins, whose binding sites are located in this domain in the 40S subunit. We have found that the bound r-proteins protected from chemical modification mainly those 18S rRNA nucleotides that turned out to be implicated in binding to r-proteins specific to the abovementioned 18S rRNA domain in the 40S subunits, and that the binding was accompanied with vigorous aggregation of the RNA–protein complexes. 2. Materials and methods 2.1. Ribosomes and ribosomal proteins 40S ribosomal subunits were isolated from full-term human placenta according to [25] and dissolved in water to concentration of about 8 pmol/μl. Total protein of the 40S subunits (TP40) was obtained by their overnight incubation in the presence of 2 M LiCl at 4 °C with following clearing from precipitated 18S rRNA by centrifugation [3]. 2.2. Construction of the 18S rRNA fragment The DNA template for synthesis of the RNA transcript 18S3DM RNA corresponding to the major 3′-terminal domain of the 18S rRNA (region 1203–1698) was obtained by PCR using the plasmid pAM18T7-2 [26] containing the full-length human 18S rRNA gene as a template and primers 1 (5′-TAATACGACTCACTATAGGGACGGAAGGGCACCACC-3′) and 2 (5′-GTGTACAAAGGGCAGGGA-3′). The resulting PCR-product contained T7 promoter due to the sequence inserted into the primer 1 (underlined) and was used for the synthesis of non-labeled and low 32 P-labeled (specific activity below 5000 cpm/pmol) 18S3DM RNA utilizing the T7 RNA polymerase as described [27]. Biotin was attached to the 3′ end of 18S3DM RNA by means of a hydrazide linkage. The RNA (1 nmol in 100 μl of water) was incubated

with 40 mM NaIO4 for 1 h at room temperature in the dark. After the reaction was stopped by addition of 100 μ1 of 50% ethylene glycol, the oxidized RNA was precipitated with ethanol, dissolved in 100 μl of 10 mM biotinamidocaproyl hydrazide (Sigma), and incubated at 37 °C for 2 h. The resulting hydrazone was reduced to the hydrazide by addition of 100 μl of 0.2 M NaBH4 and 200 μl of 1 M Tris–HCl (pH 8.0), followed by incubation for 30 min in the dark on ice. Biotin-derivatized 18S3DM RNA was purified from reactants using Sephadex G-25 spin column (GE Healthcare) according to manufacturer's protocol. 2.3. Binding of TP40 to the immobilized 18S3DM RNA Biotinylated 18S3DM RNA was refolded in water by heating at 90 °C for 2 min with the following cooling down to room temperature. Streptavidin sepharose beads (Sigma) (20 μl) were washed two times with 500 μl of water and incubated with 25 pmol of biotinylated 18S3DM RNA in 100 μl of buffer A (20 mM HEPES-KOH, pH 8.0, 400 mM NH4Cl, 16 mM MgCl2) on rotating platform for 4–16 h at 4 °C. After the incubation, the beads were washed four times with 100 μl of the same buffer and 25 pmol of LiCl-extracted TP40 in 100 μl of buffer B (20 mM HEPES-KOH, pH 8.0, 400 mM NH4Cl, 16 mM MgCl2, 3 mM spermidine-HCl, 0.1% Tween-20, 500 mM trimethylamine N-oxide (TMAO), 15% dimethylsulfoxide DMSO) was added to the beads. Binding of proteins to 18S3DM RNA in the mixture was carried out on rotating platform for 1 h at 25 °C. The beads were then washed four times with 100 μl of buffer B. Bound proteins were recovered with 15 μl of SDS sample loading buffer (0.125 mM Tris–HCl, pH 6.8, 2% SDS, 20% glycerol, 0.1 M DTT, and 0.02% bromophenol blue) and analyzed by SDS-PAGE in a 15% gel. 2.4. Binding of TP40 to 18S3DM RNA in solution One hundred pmol of TP40 extracted from 40S subunits with LiCl was diluted with 200 μl of buffer B lacking Tween-20 and mixed with 100 pmol of 18S3DM RNA refolded in 3 μl of water by incubation at 90 °C for 2 min and subsequent cooling to ambient temperature. Immediately after the mixing the solution was clarified by centrifugation at 14,000 g for 2 min and the supernatant was transferred to a new tube and incubated at 37 °C for 1 h. The resulting mixture was loaded onto a linear sucrose gradient (10–30% w/v sucrose in 20 mM HEPES-KOH, pH 8.0 containing 400 mM NH4Cl and 10 mM MgCl2) and centrifuged for 4.5 h at 52,000 rpm using Beckmann TS-60 rotor. The gradient fractions corresponding to ribonucleoproteins were collected and proteins were recovered from the fractions by shaking with StrataClean beads (Stratagen) according to the manufacturer protocol. Proteins bound to the beads were recovered and analyzed as described in Section 2.3. 2.5. Footprinting assay Binding of 18S3DM RNA with r-proteins was carried out in buffer A as described above with final concentrations of 0.25 μM 18S3DM RNA and 0.25 μM TP40. Hydroxyl radical and dimethyl sulfate (DMS) probing of the RNA in reaction mixture was performed as described [27]. Probing of 18S3DM RNA with CMCT was carried out by addition of 33 μl of freshly made 200 mM CMCT in 50 mM HEPES-KOH to 100 μl of the reaction mixture containing 18S3DM RNA and TP40. After 15 min incubation at 37 °C, the reaction was stopped by adding 1 μl of β-mercaptoethanol and the RNA was isolated by phenol deproteination. Purified RNA was used in reverse transcription reactions. Reaction mixtures of 10 μl volume containing 1 pmol of 18S3DM RNA fragment, 5 pmol of 5′-32P labeled primer complementary to one of 18S rRNA regions (1404–1417, 1544–1563, or 1681–1698), 0.5 mM dNTPs and 1 unit of AMV reverse transcriptase (NEB) in a buffer containing 50 mM Tris–HCl pH 8.3, 75 mM KOAc, 8 mM Mg(OAc)2 and 10 mM DTT were incubated at 37 °C for 30 min. The products of reaction were ethanol precipitated, dissolved in formamide (containing 0.1% bromophenol blue and 0.1%

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xylene cyanol) and separated by denaturing PAGE on 8% gel. The gel was exposed to a Kodak phosphorimager screen.

the presence of all 40S subunit proteins in these preparations (data not shown).

2.6. Protein identification by MALDI TOF mass spectrometry analysis

3.2. Binding of TP40 with immobilized 18S3DM RNA

Protein samples were prepared as described [28] with modifications. Protein bands were excised from the CBB R250 stained gel, cut into pieces of approximately 1 mm width and destained by washing with 50% CH3CN containing 25 mM NH4HCO3. Proteins were in gel reduced by incubation in 25 mM NH4HCO3 containing 10 mM DTT at 56 °C for 1 h, then alkylated by 55 mM iodoacetamide for 45 min at room temperature in the dark, and digested with 30 μg/ml solution of trypsin (Promega, sequence grade) in 25 mM NH4HCO3 at 37 °C overnight. After the digestion, the solution was evaporated using a vacuum concentrator, and peptides were dissolved in 5 μl of 0.1% trifluoroacetic acid (TFA) in 50% CH3CN. The sample (1 μl) was mixed with 1 μl of the HCCA matrix (saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid in a mixture of acetonitrile and 0.1% aqueous solution of TFA, 1:2 v/v). Mass spectra were obtained by the staff of Mass Spectrometry Shared Facility at ICBFM SB RAS. Mass spectra were recorded using an Autoflex speed series MALDI TOF mass spectrometer (Bruker Daltonics, Germany) equipped with a pulsed N2 laser (337 nm) in a positive reflectron mode. Ions formed by a laser beam were accelerated to 23 keV. The final spectra were obtained by accumulating about 1500 single laser shot spectrum. External calibration in positive mode was done using Peptide Calibration Standard II (Part no. 222570, Bruker Daltonics, Germany). Mass accuracy ~ 15 ppm was usually achieved. Mass spectra were processed using flexAnalysis 2.4 software (Bruker Daltonics GmbH, Germany). The spectra were analyzed by means of site-licensed Mascot database searching program (200 ppm tolerance; Matrix Science, London, UK) using UniProtKB and human r-protein databases. Protein scores N 39 were considered as significant, that is, these indicate identity or extensive sequence homology (p b 0.05) when MS data were queried against the mentioned databases.

To find out which r-proteins are able to bind to 18S3DM RNA, we performed binding of TP40 with this RNA immobilized on streptavidin agarose resin through a biotin residue introduced at its 3′-end. The binding buffer contained high concentration of monovalent cations (400 mM NH+ 4 ), which is usually applied with prokaryotic ribosome subunits assembly in vitro to increase the specificity of r-proteins binding to rRNA (e.g. [4,31]). Furthermore, the buffer was supplied with 0.5 M TMAO as osmolyte, which has been revealed to stimulate the reconstitution of 50S ribosomes [32], and with 15% DMSO to prevent protein aggregation. Addition of these agents increased stability of TP40 solutions (data not shown). Immobilized 18S3DM RNA was incubated with a molar equivalent of TP40 and the proteins unable to bind to the RNA were rinsed off with the same buffer. Proteins bound to the immobilized RNA were eluted with SDS-containing solution and separated by SDS-PAGE (Fig. 2A). Comparing the electrophoretic patterns of the proteins selected by 18S3DM RNA and TP40 showed that a rather large set of r-proteins was capable, as expected, to bind to the RNA. To identify r-proteins selected by 18S3DM RNA, the protein bands were excised and treated with trypsin, and the resulting peptides were subjected to MALDI TOF mass spectrometry (spectra not shown). Peptide spectra were analyzed with the use of UniProtKB databases and human r-proteins database. As the result, we have found 21 of 33 proteins of the 40S ribosomal subunit in the pattern of the r-proteins bound to 18S3DM RNA. This set included 15 r-proteins (namely S2, S3, S5, S10, S12, S15, S16, S17, S18, S19, S20, S25, S26, S27a and S29) contacting the major 3′-terminal domain of the 18S rRNA and 6 rproteins (S3a, S7, S8, S13, S14 and S15a), which have no contacts with this domain in the 40S subunit (Table 1). Besides, three proteins known to contact the major 3′-terminal domain (RACK1, SA and S28) in 40S subunits were not detected in this set. These findings suggest that majority of r-proteins specific to the major 3′-terminal domain of the 18S rRNA were selectively bound to the model 18S3DM RNA, however with several r-proteins nonspecific binding to this RNA was observed.

2.7. Ribosome structure analysis The PyMol software [29] and PDB files noted in the text were used for the ribosome structure analysis.

3.3. Binding of TP40 with 18S3DM RNA in solution 3. Results 3.1. RNA and r-proteins used for reconstitution of the human 40S subunit head The head constitutes a large morphological part of the small ribosomal subunit comprising specific ribosomal proteins (r-proteins) and the major 3′-terminal domain of the 18S rRNA. Eighteen from 33 small subunit r-proteins form polar contacts with this domain according to the cryo-EM model of the human 40S subunit ([30], PDB ID: 3J3A and 3J3D) (Table S1). To elucidate a possibility of reconstitution of a ribonucleoprotein corresponding to the head of the 40S subunit, we studied the interactions of the human r-proteins with the major 3′-terminal domain using an RNA modeling this domain and determined those proteins, which can recognize the domain selectively. This RNA (18S3DM RNA) was 498 nt long and contained a region corresponding to the fragment 1203–1698 of human 18S rRNA as well as two additional G residues at the 5′ end derived from the T7 promoter sequence in the corresponding template (Fig. 1). Total protein of human 40S ribosomal subunits (TP40) was obtained by deproteination of the subunits with 2 M LiCl. Under the conditions used, r-proteins dissociated from the 18S rRNA completely and remained in supernatant, whereas 18S rRNA was precipitated. Thus, preparations of TP40 isolated by this method did not contain RNA impurities and their electrophoretic patterns in SDS-PAGE did not differ from patterns of the proteins isolated from 40S subunits by a standard procedure indicating

To examine whether the use of immobilized 18S3DM RNA caused the observed nonspecific protein binding, we have carried out binding of TP40 with this RNA in solution. 32P-labeled 18S3DM RNA was mixed with equivalent amount of TP40 in the same binding buffer that was used in the experiments with immobilized RNA (see above). Notably, while both TP40 and 18S3DM RNA were stable in this buffer, on completion of incubation of their mix, up to 50% of radioactive material was found as an insoluble material, indicating partial aggregation of the resulting RNA–protein complexes, which occurred despite the use of the binding buffer containing the abovementioned additions. We have attempted to isolate remaining soluble 18S3DM RNA complexed with r-proteins by ultracentrifugation of the supernatant in a 10–30% sucrose gradient (Fig. 3). One can see that shapes of 18S3DM RNA peaks in the profiles of sedimentation of free RNA and the RNA incubated with r-proteins differ, indicating r-proteins binding, but the peak corresponding to complexed 18S3DM RNA was reduced approximately by two fold, which could be caused by precipitation of 18S3DM RNA in composition of higher molecular weight RNA–protein aggregates formed in the course of centrifugation. Actually, the 32P-labeled material was found at the bottom of the centrifuge tube and its quantity was nearly the same as that of 32P-labeled 18S3DM RNA detected in the peak of complexed RNA. Proteins collected from the gradient fractions containing 18S3DM RNA peak were separated in SDS-PAGE and the obtained protein pattern was compared with those of TP40 and r-proteins selected by the

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A A U U GG U U U A 980 G C AC U C C GA G G C AAUUC A GC G C G G C G C A AG A C G U A A C G 1380 1400 1420 G AU A GA A GAG G GC C GC GU U A U GC A CG A A U GGA GA U C C A UG AC C C U U U CUC U G G C C U A AC UA G U U A CGA A C G G A C C CCC G A G C C C U 1000 G 940 U C G 840 960 G A U G C GA GU G G A GA UU U C A A CC C C C U G C G G C U G 1360 U G UGG UUG C G G C CU UU G A C U G U G G C A U G G 1440 U A 1480 A 1300 G A U A G C U A A GC U G G U A G C A A G A U A U U A G G C A U GC A G A 1060 A U C 1340 U A G U C UG U C A U UG A G C G U A C U A C U GA U A GA C C A C G G C C U A 820 C U G C U A G A U A U U A U G C U G U C 900 U A G U G C G C G U C G A A A C G A C C G C U U A G UC C A G C U A C G C 1460 G U A A U U A G C G G A G C G C G UG G U A A U U G A CUU A AG U 880 G G U 1020 1290 U A AU A 860 G C U C G U G CA U U C A G G G A A U U GU GA A U G 1500 C G G A UU U A G A G G C C C C G U A A C 1330 G G C 920 G A CA UC G C G A U G A A U U G C U U G C G G UC U G A C U A UU A G GUG U G C GA U A UG A U U A A G A AU 800 U C C G C G U A G A U G C A U GG C CG GU C G C G 1080 A A GG C G A C AAG A U C U 1120 U A G U A CG C G C C C C CCAU U C A AG C 780 G G U GG G G C C A AA U CC UU CC G C G G G G U G G CU C A C G 1260 A C G C G A U 1100 C G GU C GC C 680 A C C CA C G C A C GG A GU UGC U CCC A 740 C G CUG AU UC C C U G C C GC C A CG C G G G G G 1560 G G GU A G A C C A G A C 760 A CUC G AC A G C G G UU U G 620 G U G U C A C GA G GCC G A CC G U A C CC A G 1520 C A C G UC C G U GC UU A GC C C C A C U A AU G A C C A G C A C C 1140 U G UGG UG G G U G C C G U G C U A A U C G U G 1240 C G G CG G G A U A UUU C G G A G A G C G C A G U CUC C C A A G C C U G C AG C G U G G C C C A C C A C C C G 660 G C A G 700 G G 1170 A A U U U C G C G A U A 1580 C A G UA C G A C 1540 C G C G U U U 1180 C C 580 G U G A G A GGG GGUU G U G C CA G CU G G C U C A U U G A 720 G C U CU A UU A C A UU U C AG C G C C A U U A C UUC U CGA A A G G C G A AG UC C G A G A 1220 A A U C A A A G C A UU A 1210 C G C C A AG C C GA G CU G A GA A G G G GG G A C A U U G A C G A A G G G C AC CA CC G G G U A A 20 U G 600 A C U U A 1640 A U U G A C C G G G 1600 AU CCG CCU G C C U G UGA C 640 G C A U C U A C U GU U U C C C GU U G A A A A G A G U C U C A CCC A GU U GA U 560 A AU G A UG C A G G C A 1690 G A C A G C U C G C G A GG C C U G UA A U U G G C U AU 1700 C U AG U A G U A A C G U U A A U 1 A A C A U C U CA G GA C A U A A C A A U AA A G C G U G U G A G 520 A A U C U C 540 U U A U A G A C G G G 1840 U U G ACG C C G C G C G UC U C G G G C U U C C G U A G GU 1670 G C G A GU C G C G G G G G U A G G U C A U U G C A U A A U C A C G U A A GGC GU C C A C U A G C AG CCC C C A UA G U U G A 40 A G 1860 U A C U C A G 500 CA AA U U AA A U C A UAA A U U C C G G C U A 1620 GA C A A A U U C G 480 G U C G A U C GA G A A 1869 G C A G G CA A 1720 U U A U A G 3’ G U A C G G G C A C C A U A GC A G C 450 G A U A 60 U G U A C U A C CG G C A G 460 CUA A A C G G CC A U A A GCU A GU GU AC CC U G G G 420 A U U G G U CC G A G G CG U CA UG A U G AAG A C AU G GG G C A U U GAU G 80 A U A 1800 C G U A CU G A A CC AA A G G A G C G G G U G C U G G C G U GC A 100 C G U A G C G U U 1740 C G C 400 A G U A UA A C A CG CC C G C C G C G A U A A U G A G A G C A G U U U U G U A U G C G A A C 350 CG A C U G U U UG A A U G C C A U C G C UG A U G C C A G U G C G U G C C A C U C G C GC A U 120 C G 1780 G U U U G C U U G U A C G C GC CU CC C G C C G U G CG C G U C 380 340 C U U G C U 150 C C G C C A G C 1760 G C AUAA C G U G A CU U G G C UGU GGU A GU G G C G G C G A C C G C A G C C G A GA G A UC U U C C C C G C C G C UA G U A CA A CCC C G UA A U C 160 320 C C C GG G C U G G C C U A GG C G C 180 A 300 G C A A C G U U A CU U G C G U A GA U U U C A U U A G 220 GG C G A A G C G G C A G C U G C C G C G U A U C U A U G C A 230 U A 200 C C C C CAA G C G G C G 280 G U

H23

H35

H37

H22

H39

H38

H36

H24

H20

H40

H34

H33

H26

H32

H25

H31

H18

H30

H19

H27

H41

H29

H2

H17 H16

H3

H1

H28

H45

H42

H4

H15

H43

H5

H14

H13

H44

H6

H12

H11

H7

H10

H9

C G C A C G C G G C G C G C C G C G U G C G U G C G C C G C G G C G C C C

250

A A U U U U G C G C U C G 1380 1400 1420 AU A GA G A U A UG AC C CUC U G G C C U A AC UA G U U G G A C C CCC G A G A CGA A C C G G G G A GA UU U C A A CC C C C U G C G G C U G GA GU 1360 U G UGG UUG CU A C U G U G 1440 U A 1480 A 1300 A U A U A C A G AG U A G C U GC A G A 1340 U A C C A G U A C U C G G C C U A U A U U A C G U G C G U C G A C G C U G UC C C G C 1460 A U G G A G G UG G A CUU 1290 U U A A U U U C A U GU G 1500 G G G C C A C 1330 G C CA G U G U G C G GG C G UU GU A A C G A A GG C GU C C U G C G C C G 1260 U C C CA C G GG A C C G C C A G 1560 G A A G C AC G G U U C G A G U C CC G 1520 G UC C U GC C C A C U A AU G A G C U G G G C G A U 1240 C G U A A G G C U CUC A U G AG C G C C A C C C G U U C C G 1580 1540 C G C U G A U G G A G A CU A C A C G C CU A C C G A 1220 U G UU 1210 A C A GG G C C C G A GGG A C G A A G G G C A A C U U U A 1640 C G 1600 C G C A A C U GU UU C C C GU U G A A U G C CCC A G A G 1690 G A G C G U U A G A U A A A A G U G G U U C U U A G G G C C G C C G G G G C 1670 U G G C U A U U C U C U U G A A G AA U U C UAA

H37

H35

H39

H38

H36

H40

H34

H33

H32

H31

H30

H41

H29

H28

H42

H43

1620

260

18S rRNA

18S3DMRNA

Fig. 1. The 18S3DM RNA used for reconstitution of the human 40S subunit head. Left, the secondary structure of the 18S rRNA of human 40S ribosome [30] where major 3′-domain is outlined. Right, the secondary structure of the 18S3DM RNA with enumerated nucleotides and helices.

Fig. 2. Analysis of proteins of the human 40S ribosomal subunits bound to 18S3DM RNA in 15% SDS-PAGE. A, the r-proteins associated with immobilized 18S3DM RNA. Lane −RNA, r-proteins bound to uncharged streptavidin sepharose resin; lane +RNA, r-proteins bound to the resin laden with biotinylated 18S3DM RNA. B, the r-proteins complexed with 18S3DM RNA in solution. Lane RNP, r-proteins isolated from the complex purified by centrifugation in the sucrose gradient. Lanes TP40, total protein of 40S subunits. R-proteins identified in the gels and their positions (marked with bars) are noted on the right. C, the protein content of sucrose gradient fractions containing RNP. Lane M — protein molecular weight marker.

A.V. Gopanenko et al. / Biochimica et Biophysica Acta 1854 (2015) 101–109 Table 1 Ribosomal proteins bound to 18S3DM RNA immobilized on streptavidin agarose and identified by MALDI-TOF mass-spectrometry.

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Table 2 Ribosomal proteins bound to 18S3DM RNA in solution and identified by MALDI-TOF massspectrometry.

#

Protein namea

Number of peptide matches

Sequence coverage (%)

Score

#

Protein namea

Number of peptide matches

Sequence coverage (%)

Score

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S2 (uS5) S3a (eS1) S3 (uS3) S5 (uS7) S7 (eS7) S8 (eS8) S10 (eS10) S12 (eS12) S13 (uS15) S14 (uS11) S15 (uS19) S15a (uS8) S16 (uS9) S17 (eS17) S18 (uS13) S19 (eS19) S20 (uS10) S25 (eS25) S26 (eS26) S27a (eS31) S29 (uS14)

6 10 10 5 11 10 6 3 5 4 11 4 7 6 6 9 7 5 13 8 3

22 44 45 21 26 47 41 10 32 34 44 39 55 25 29 53 27 29 66 37 30

40 51 80 43 163 82 59 52 59 52 149 56 41 86 47 152 104 64 193 141 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S2 (uS5) S3a (eS1) S3 (uS3) S5 (uS7) S7 (eS7) S10 (eS10) S12 (eS12) S13 (uS15) S15 (uS19) S15a (uS8) S16 (uS9) S17 (eS17) S18 (uS13) S19 (eS19) S20 (uS10) S25 (eS25) S26 (eS26) S27a (eS31) S28 (eS28) S29 (uS14) SAb (uS2)

9 10 18 7 22 8 4 7 8 7 9 7 12 11 11 8 4 3 3 4

26 40 69 21 61 52 18 43 32 52 56 27 57 60 55 42 40 11 30 28

143 120 84 109 68 91 71 60 130 70 89 98 182 93 184 119 67 58 59 66

a Proteins contacting the major 3′-domain of 18S rRNA in 40S subunits according to [30] are typed in bold. Names of r-proteins according to new nomenclature [48] are given in parenthesis.

a

See footnote to Table 1. Protein SA was not subjected to mass-spectrometry analysis because the protein migrates in the SDS-PAGE as a distinctive band allowing it to be unambiguously identified.

immobilized 18S3DM RNA (Fig. 2B). In general, pattern of r-proteins bound to 18S3DM RNA in solution was similar to that of the proteins bound to the immobilized RNA except that it did not contain a band corresponding to nonspecific protein S8 and included a band corresponding to protein SA, which contacts the major 3′-terminal domain of the 18S rRNA in 40S subunit [33]. We also determined other proteins in bands of this pattern using mass spectrometry (spectra not shown). It turned out that the pattern of r-proteins bound to 18S3DM RNA in

solution (Table 2) contained, in addition to SA, r-protein S28 specific to the 3′ domain of 18S rRNA [34] and did not include nonspecific r-protein S14. Thus, the specificity of r-protein binding to 18S3DM RNA in solution (Table 2) was distinctly higher than that to the immobilized RNA (Table 1), while total yield of the complex was lower because of its substantial aggregation. As for the relatively large width of the peak of 18S3DM RNA bound to r-proteins, it could suggest that the complex formed is heterogeneous, however, examination of the r-protein content in fractions of the peak (Fig. 2C) revealed only slight differences in intensities of low molecular weight protein bands in lanes corresponding to fractions 10–11, and to fractions 6–7 and 8–9, indicating that the composition of the RNA–protein complexes in the peak was practically uniform. Notably, binding of r-proteins to 18S3DM RNA did not result in shift of the respective peak towards the bottom of the sucrose gradient as it was earlier observed with ribonucleoprotein (RNP) assembled with 3′ terminal domain of 16S rRNA [11]. Apparently, increase in the mass of 18S3DM RNA and its compaction due to r-protein binding equally contributed to the migration capacity of the resulted RNP, that could be a reason why the peak of 18S3DM RNA complexed with r-proteins was not shifted relatively to the peak of isolated 18S3DM RNA.

b

3.4. Chemical probing of 18S3DM RNA bound with r-proteins

Fig. 3. Isolation of the complex of r-proteins with 18S3DM RNA by centrifugation in the sucrose gradient. A, sedimentation profile of free 18S3DM RNA. B, sedimentation profile of 18S3DM RNA bound with r-proteins. Fractions taken for analysis of r-protein content are marked with arrows.

To clarify the question concerning the specificity of r-protein interaction with 18S3DM RNA in solution, it was tempting to determine whether the major 3′-terminal domain of 18S rRNA nucleotides contacting r-proteins in the 40S subunit was implicated in the interaction. To this end, we performed chemical probing of the 18S3DM RNA structure in complex with r-proteins using DMS, CMCT and hydroxyl radical probes to reveal nucleotides protected from the probe attack and to compare the results with known information on contacts of r-proteins with the 3′-terminal domain of 18S rRNA in 40S subunits. DMS methylates positions N1 of adenines and N3 of cytosines and CMCT attacks positions N1 of guanines and N3 of uracils that are not involved in base-pairing [35], whereas hydroxyl radicals attack the C4′ atom of exposed ribose moieties and thus cause strand scissions in the RNA phosphodiester bonds [36,37]. All these modifications and breaks can be detected by 32P labeled primer extension assay, because reverse transcriptase makes stops at nucleotides 3′ to the modified base or

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break that is displayed as enhancement of radioactive signal at this nucleotide. We inspected practically all 18S3DM RNA sequence with exception for the ultimate 3′ terminal region containing a sequence for primer hybridization; results of this footprinting are shown in Fig. S1 and displayed in Table S2. We have found that in the presence of r-proteins a significant part of 18S3DM RNA nucleotides became protected from hydroxyl radical attack and a lesser part of nucleotides were protected from attack by DMS and CMCT. It should be noted that these protections may be caused not only by contacts of nucleotides with r-proteins but also by shielding of some 18S3DM RNA regions due to RNA folding as a result of r-proteins binding. Therefore, to verify what nucleotide protections of 18S3DM RNAs could correspond to protein contacts, we compared data on footprinting of the complexed RNA with information on contacts of r-proteins with the major 3′-terminal domain of the 18S rRNA gained from spatial structure model of the human 40S subunit (Fig. 4, Table S1).

In addition, we performed a comparison of the footprinting results with the data on the exposed regions of 18S rRNA in the 40S subunit head obtained by hydroxyl radical probing in [33] to learn whether protein binding has occurred properly. It turned out that the large part of nucleotides involved in the interactions with r-proteins in 40S subunit appeared protected from modification or cleavage in 18S3DM RNA bound with r-proteins, and these protections corresponded to binding of almost all of the expected r-proteins contacting the major 3′-terminal domain in the 40S subunit. But at the same time, many regions exposed in 40S subunit according to [33] were protected from hydroxyl radical attack in 18S3DM RNA complexed with r-proteins, although a similar number of regions uncovered in 40S subunits were accessible to the attack. Hence, we conclude that the main part of r-proteins bound to 18S3DM RNA is positioned in sites corresponding to those in the 40S subunit, i.e. their binding is specific. As for 18S3DM RNA protections unrelated to interactions with specific r-proteins, they could arise because

Fig. 4. Chemical footprinting of 18S3DM RNA bound to r-proteins. Nucleotides protected from base reactive probes (DMS or CMCT), hydroxyl radical or both base reactive probes and hydroxyl radicals are shown in red, blue or green circles, respectively. Non-analyzed sequences are shown in magenta. Positions of r-protein contacts with nucleotides of 18S rRNA in the human 40S ribosomal subunit structure according to the 40S model (PDB ID: 3J3A and 3J3D, [26]) are designed by the names of the respective proteins (see also Table S1). Regions of 18S rRNA exposed in the 40S subunit head according to [33] are marked by dark-pink lines.

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of either the RNA folding induced by proteins binding as it was already mentioned above or binding of nonspecific r-proteins; besides, the possibility of wrong folding of some parts of 18S3DM RNA in the conditions used cannot be ruled out. 4. Discussion In the present study, attempting to reconstruct the head of the 40S ribosomal subunit, we learned which r-proteins from the total protein of human 40S ribosomal subunit are capable of binding in vitro to an RNA transcript corresponding to the major 3′ domain of 18S rRNA. This domain of human 18S rRNA comprises region 1202–1697, whose folded structure includes helices 28–43. Strands at the 5′ and 3′ termini of this 18S rRNA domain are paired in the long helix 28, so that the overall domain is structurally isolated from remaining regions of the 18S rRNA and does not substantially contact them. In the composition of 40S ribosomal subunit, the major 3′ terminal domain of 18S rRNA forms a scaffold, around which a large morphological part of the 40S subunit, the head, is organized. We showed that incubation of RNA modeling this rRNA domain with TP40 results in formation of a complex containing practically all r-proteins specific to the subunit head, but at the same time vigorous aggregation of the RNA–protein complexes occurred. Applying chemical footprinting with the use of probes specific to ribose or nucleotide bases, we revealed that among nucleotides protected in this complex from the attack by the probes, mostly those nucleotides appear, which are involved in binding to the respective r-proteins in the 40S ribosomal subunit, whereas nonspecific protections are observed to a lesser extent. A principal possibility of autonomous in vitro reconstitution of the head of the small ribosomal subunit has been demonstrated earlier with the ribosomes from bacteria E. coli [12] and Thermus thermophilus [11] in experiments similar to those performed in our work. In particular, r-proteins S2p, S3p, S4р, S7p, S9p, S10p, S13p, S14p and S19p (index “p” means prokaryotic), which are homologs of eukaryotic proteins SA, S3, S9, S5, S20, S18, S29 and S15, respectively, were shown to bind to an RNA transcript corresponding to the major 3′ domain of 16S rRNA E. coli when being in the 30S subunits total protein [12]. This set included practically all proteins contacting this 16S rRNA domain in the 30S ribosome with exception for S5p (homologous to S2e) and, in addition, it contained r-protein S4p, which is specific to the 5′ domain of 16S rRNA; authors explained the presence of S4p in this set by its interaction with the other proteins bound to the RNA transcript specifically [12]. In another work, a set of ribosomal proteins of T. thermophilus bound to an RNA corresponding to the major 3′-domain of 16S rRNA was almost the same as in the work mentioned above [11], but it did not include S2p, and the amount of S4p in the resulting complex was greatly reduced as compared to those of other r-proteins [12], apparently, because of more rigid conditions of the head assembly used. As for S2p, its contacts with 16S rRNA are known to be rather poor [38] and it is prone to dissociation from the 30S subunit [39], therefore its binding to the major 3′ domain of 16S rRNA could be too weak to be detected with confidence. Interestingly, protein S5p that contacts both the 3′ and 5′-terminal domains of 16S rRNA in the 30S subunit, was not found in the composition of the assembled particles in both works. Binding of S5p to the 16S rRNA was shown to depend on preliminary binding of the r-proteins participating in formation of the body of the 30S subunits [5], therefore the reasons for S5p loss could relate to lack of the binding sites of r-proteins specific for the ribosome body; apparently, its own contacts with the 3′ domain of the 16S rRNA are too weak to provide association of the protein with this domain. The set of r-proteins contacting the major 3′ domain of 18S rRNA in the 40S subunit of mammals is much larger than that of the respective proteins in the 30S subunit despite similar lengths of these domains in 18S and 16S rRNAs (496 nt for human 18S rRNA vs. 476 nt for 16S rRNA of E. coli). This set includes 10 eukaryote-specific proteins in addition to 8 ones that, although they have bacterial homologs (Table S1),

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contain long eukaryote-specific extensions [17,23]. All this implies that assembly of the 40S ribosome head is much more complicated process than that of the 30S subunit head. Remarkably, from 18 r-proteins contacting the major 3′ domain of 18S rRNA in the 40S subunit, 17 were capable of specific binding to the RNA transcript corresponding to this domain in solution. The remaining unbound protein RACK1 has very few contacts with the 18S rRNA in the 40S subunit (Table S1) and therefore it was not surprising that this protein was not found among those bound to the 18S3DM RNA. As for the r-proteins that are uncharacteristic for the 40S subunit head but appeared bound to 18S3DM RNA (namely, S3a, S7, S13 and S15a bound to the RNA in solution as well as all these proteins together with S8 and S14 bound to the immobilized RNA), their binding could occur through the interaction with some specific proteins, like it was observed with S4p in the work mentioned above [12]. Actually, S3a and S15a have numerous contacts with S26 and S2 in the 40S subunit, respectively, whereas S7 and S13 tightly contact each other and S15a. Besides, binding of the abovementioned r-proteins with 18S3DM RNA could arise because of the nonspecific interactions of their highly positively charged unstructured extensions (see, e.g. [30] PDB ID: 3J3A) with exposed RNA regions, and, possibly, those footprints that did not correspond to binding of specific r-proteins with 18S3DM RNA (Figs. 4 and S1) occurred due to these interactions. For instance, S3a has the 27 and 33 amino acids long disordered extensions at its N- and C-terminal regions respectively, S7 has the 20 amino acids long KR-rich part in the middle of the protein, whereas S13 has 28 amino acids long extension at the N-terminal part [30]. Despite rather satisfactory reconstitution of ribonucleoprotein corresponding to the 40S subunit head, the substantial problem remained unsolved in our work was the aggregation, which accompanied the formation of RNA–protein complexes. Notably, this aggregation was observed only when RNA transcript and r-proteins were mixed together and our attempts to preclude it using high salt concentration with addition TMAO and DMSO only partially solved the problem. In our opinion, the most probable reason for the aggregation also relates to the presence of long unstructured positively charged extensions in eukaryotic and, in particular, mammalian r-proteins. R-proteins are known to interact with rRNA both through globular parts and extensions (see, e.g. [40]). Kinetic experiments performed with r-protein S4p and minimal rRNA containing its binding site indicated that the globular part of the protein binds to the RNA much faster than its N-terminal extension, whose binding at the specific site is accompanied by structural rearrangements of the RNA [41]. Similar behavior is expected with many other r-proteins. If so, r-protein could bind simultaneously to two different RNA molecules; one of them could interact specifically with its globular part and another one could bind non-specifically to its extension, leading to formation of large associates and their subsequent aggregation. This sort of binding should be especially expressed with eukaryotic r-proteins since long positively charged extensions are typical for them, and a number of r-proteins associated with the 3′ domain of rRNA in 40S subunits are larger than that in 30S subunits. Consequently, it is not surprising that binding of TP40 with 18S3DM RNA resulted in formation of large insoluble associates. Remarkably, new method of the reconstitution of E. coli ribosome at physiological conditions resembling those in cytoplasm was recently described [42,43]; this method of utilizing ribosome-free cellular extract enables rRNA transcription, ribosome assembly and translation to occur in the same compartment like it takes place in vivo. Perhaps, the application of a similar approach to reconstitute eukaryotic ribosome could help to avoid or at least to decrease the aggregation; however, it is not evident at first sight and this topic requires special studies. The question is why similar unproductive associates of r-proteins with rRNA are not formed during assembly of ribosomal subunits in the living cell. One can suggest that numerous post-translational modifications of r-proteins, which were observed in many proteomic studies of mammalian cells (see PhosphoSitePlus database [44]), protect r-proteins from formation of these associates. These modifications

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(phosphorylation and acetylation, predominantly) being mostly located in the regions of extensions (see PhosphoSitePlus database [44]), reduce their high positive charges and thus facilitate specific binding of r-proteins to rRNA. It is worth to note that r-proteins isolated from the maturated ribosomal subunits as a rule do not contain this sort of modifications [45–47]. It suggests that when the assembly of ribosomal subunits is completing, the post-translational modifications are removed to allow r-protein extensions to be accurately located in the ribosomal subunits. But it so far remains unclear how these modifications are ultimately lost, and the cellular mechanism that governs this process is not understood as well. On the other hand, taking into account implication of more than 200 various factors in the ribosome biogenesis in eukaryotes [2], one cannot rule out that some of them directly participate in structural adjustment of r-proteins providing their proper binding to the rRNA during ribosomal subunits assembly. Thus, our results show a principal possibility of in vitro reconstitution of eukaryotic ribosomal subunits, in particular of their definite morphological parts. We have revealed that the majority of the r-proteins specific to the major 3′ domain of 18S rRNA were capable of binding to the RNA simulating it, though several nonspecific proteins were registered in the complex too. We have also identified nucleotides of the RNA protected from chemical modification by the bound proteins and found that a large part of these protections match with nucleotides contacting r-proteins in the 40S subunit head, and that unspecific protections take place as well. Certainly, at present we cannot determine in what extent reconstituted RNP corresponds to the 40S subunit head and perhaps, cryo-EM study could provide the answer. Additionally, our findings highlight the aggregation of the resulting complexes as a characteristic feature of the interaction of rRNA with eukaryotic r-proteins that seems to impede strongly the process of reconstruction of active 40S subunits without a set of assisting factors. We have proposed that both the nonspecific binding and aggregation are caused by nonspecific binding of unstructured extensions of r-proteins with RNA. Acknowledgements We gratefully thank D. Graifer and O. Kossinova for critical reading of the manuscript. This work was supported by a grant from the Russian Foundation for Basic Research (14-04-00740) to A.A.M. and by a Program of the Presidium of the Russian Academy of Sciences (Molecular and Cell Biology) to G.G.K. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbapap.2014.11.001. References [1] M. Kaczanowska, M. Ryden-Aulin, Ribosome biogenesis and the translation process in Escherichia coli, Microbiol. Mol. Biol. Rev. 71 (2007) 477–494. [2] K. Karbstein, Inside the 40S ribosome assembly machinery, Curr. Opin. Chem. Biol. 15 (2011) 657–663. [3] F. Dohme, K.H. Nierhaus, Total reconstitution and assembly of 50 S subunits from Escherichia coli ribosomes in vitro, J. Mol. Biol. 107 (1976) 585–599. [4] P. Traub, M. Nomura, Structure and function of E. coli ribosomes. V. Reconstitution of functionally active 30S ribosomal particles from RNA and proteins, Proc. Natl. Acad. Sci. U. S. A. 59 (1968) 777–784. [5] W.A. Held, S. Mizushima, M. Nomura, Reconstitution of Escherichia coli 30 S ribosomal subunits from purified molecular components, J. Biol. Chem. 248 (1973) 5720–5730. [6] M.E. Sanchez, P. Londei, R. Amils, Total reconstitution of active small ribosomal subunits of the extreme halophilic archaeon Haloferax mediterranei, Biochim. Biophys. Acta 1292 (1996) 140–144. [7] M.E. Sanchez, D. Urena, R. Amils, P. Londei, In vitro reassembly of active large ribosomal subunits of the halophilic archaebacterium Haloferax mediterranei, Biochemistry 29 (1990) 9256–9261. [8] G.M. Culver, H.F. Noller, Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins, RNA 5 (1999) 832–843.

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