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Post-translational hydroxylation occurs in three mammalian ribosomal proteins, uS12, uL2, and uL15, which are located in the small (S) and large (L) subunits of the ribosome near the most important decoding and peptidyltransferase functional centers. We have used cell cultures, which produce protein uL15 labeled with the 3xFLAG epitope at the C-terminus (uL153xFLAG) or mutant forms of uL153xFLAG that contain His39Ala, His39Thr, or His40Ala substitutions, to examine the role of hydroxylated His39 of uL15 in maintaining the translational activity of ribosomes. It has been found that exogenous uL153xFLAG is able to functionally replace endogenous uL15 in HEK293 cells transfected with an appropriate DNA construct. However, the translational activity of ribosomes decreases by about 35% in cells that produce the above mutant forms of uL153xFLAG compared with that in cells that produce nonmutated uL153xFLAG. Analysis of the structural model of the human ribosome has allowed us to assume that the hydroxyl group in His39 is involved in the local stabilization of the ribosome structure through the formation of a hydrogen bond between this group and the nitrogen atom of the His40 imidazole ring. Given that His39 is located near the E site of the ribosome, we believe that this stabilization of the ribosome structure ensures the maintenance of its translational activity. [ABSTRACT FROM AUTHOR]

The ribosomal protein uS15 is one of the key proteins forming the small (40S) ribosomal subunit structure around the central domain of 18S rRNA. According to a number of proteomic studies based on mass-spectrometry analysis, there are many phosphorylation sites in this protein. However, when the protein uS15 is contained in the 40S subunit, it does not carry such posttranslational modification. In this study, it was found that the cytoplasmic protein kinases of HEK293T cells are able to phosphorylate the human recombinant ribosomal protein uS15 with significantly greater efficiency than that of the nucleus protein kinases. The effect of the amino acid substitutions Y38A and S48A preventing the phosphorylation of the ribosomal protein uS15 at the corresponding sites, on the transport of the recombinant protein uS15 fused to the green fluorescent protein in the nucleolus was studied. It was shown that single mutations at the above sites have little effect on the transport of this protein, whereas double mutation reduces the efficiency of this process by more than a quarter. The findings suggest the importance of phosphorylation of the ribosomal protein uS15 by cytoplasmic protein kinases at several sites, including Y38A and S48A, for its efficient transfer into the nucleolus, where pre-ribosomal subunits are assembled. [ABSTRACT FROM AUTHOR]

The analysis of data on genomic DNA sequences in the binding sites of human ribosomal protein uS3 in vivo, obtained earlier by ChIP-Seq method, was carried out; the presence of significant content of TGGAA repeats, belonging to the type III of human satellites, was found. The dependence of the affinity of the corresponding recombinant protein uS3 to single-stranded DNA and its AP lyase activity on the DNA sequence in vitro, in particular on the presence of TGGAA pentamer repeats, was investigated. It was shown that the presence of these repeats in the model DNAs did not provide an increased affinity to the isolated ribosomal protein uS3. The presence of TCC and TTC motifs in DNA complementary to the corresponding pentamer triplets only slightly increased the affinity of DNA to this protein. It was established that the AP lyase activity of the uS3 protein was also not increased when DNA containing abasic (AP) site contained TGGAA repeats and was independent on the position of the AP site in it. It was shown that the efficiency of single-stranded DNA cleavage at the AP site as a whole correlated with the affinity of uS3 protein to this DNA. It was concluded that the high content of the above repeats in the ribosomal protein uS3 binding sites on chromatin relate to high content of (TGGAA)n repeats in single-stranded DNA regions physically accessible for the protein binding but not to its higher affinity to the pentamer or DNA motifs complementary to it. [ABSTRACT FROM AUTHOR]

The elastic oscillations of air bubbles separated from an air cavity compressed by the ponderomotive forces of a magnetic field in a magnetic fluid are accompanied by the appearance of an alternating magnetic field component. The frequency of the alternating component corresponds to the frequency of radial bubble oscillations, and this fact is used to determine the bubble size. A great body of experimental data has been obtained from six magnetic fluid samples with different viscosities. Based on these data, histograms illustrating the bubble radius distribution are plotted. The appearance of the alternating magnetic field component caused by bubble oscillations in a magnetized magnetic fluid can be used to develop a fundamentally new method for supplying small metered gas shots to a reactor, as well as to study the boiling process in a magnetic fluid. [ABSTRACT FROM AUTHOR]

Human ribosomal protein (rp) S26e is known to bind to the first intron of its pre-mRNA and, thus, inhibit mRNA splicing. In this work, the hydroxyl radical footprinting was applied for the detailed mapping of the rpS26e binding site on the RNA transcript that corresponds to the rpS26e pre-mRNA fragment containing the first intron flanked by the sequences of the first exon and a part of the second exon. Nucleotides protected from the hydroxyl radical attack in the presence of rpS26e were identified. Most of them were found in the region of the 3′ splice site of the first intron within the purine-rich sequence, which forms a loop between two helices in the predicted secondary structure of the rpS26e pre-mRNA fragment, and the remaining nucleotides are located near the 5′ splice site. A comparison of the arrangements of the rpS26e binding sites on the pre-mRNA and 18S rRNA secondary structures revealed similar elements in the organization of these sites. It was found that both sites contained a uniform structural motif, namely an extended purine-rich loop between two helices, which could be recognized by rpS26e upon binding to these RNAs. The results shed light on the structural aspects of the RNA-protein interactions, which underlie the autoregulation of the human RPS26e gene expression at the splicing step. [ABSTRACT FROM AUTHOR]

Initiation of translation of genomic RNA (gRNA) of hepatitis C virus (HCV) is provided by a highly structured fragment in its 5′-untranslated region, the so-called Internal Ribosome Entry Site (IRES). In this work, the exposed NH-groups of proteins in the 40S subunit of the human ribosome and in its binary complexes with RNA transcripts corresponding to the full-size HCV IRES or its fragments were probed using the N-hydroxysuccinimide derivative of the fluorescent dye Cy3. Comparison of efficiencies of modification of ribosomal proteins in free subunits and in their binary complexes with the RNA transcripts revealed ribosomal proteins involved in the HCV IRES binding. It was found that binding of the 40S subunits with the RNA transcript corresponding to full-size HCV IRES results in a decrease in modification levels of ribosomal protein (rp) S27 and, to a lesser extent of rpS10; also, a noticeable decrease in the efficiency of labeling of proteins RACK1/S2/S3a was observed. When a fragment of HCV IRES containing the initial part of the open reading frame (ORF) of the viral gRNA was deleted, the level of rpS10 modification became the same as in free subunits, whereas the levels of modification of rpS27 and the RACK1/S2/S3a group remained virtually unchanged compared to those observed in the complex of 40S subunit with the full-size HCV IRES. Binding of 40S subunits to a fragment of the HCV IRES lacking an ORF and domain II increased the modification level of the RACK1/S2/S3a proteins, while the efficiencies of labeling of rpS10 and rpS27 remained the same as upon the deletion of the ORF fragment. Comparison of these results with known structural and biochemical data on the organization of 40S subunit and the location of the HCV IRES on it revealed structural elements of the IRES contacting exposed lysine residues of the above-mentioned ribosomal proteins. Thus, it was found that the majority of exposed lysine residues of rpS27 are involved in the binding of the HCV IRES region formed by the junction of subdomains IIIa, IIIb, and IIIc with the central stalk of domain III, and that several lysine residues of rpS10 participate in the binding of the HCV IRES region corresponding to the initial part of the ORF of the viral gRNA. In addition, we concluded that lysine residues of rpS3a are involved in the binding of domains II and III of HCV IRES. [ABSTRACT FROM AUTHOR]

At high concentrations, Mg ions are known to stabilize the structure of the 16S rRNA in a conformation favorable for binding to the ribosomal proteins in the course of the eubacterial 30S ribosomal subunits assembly in vitro. The effect of Mg on the formation of the 18S rRNA structure during 40S subunit assembly remains poorly explored. In this work, we show that a sequential increase in the Mg concentration from 0.5 to 20 mM leads to a significant decrease in the affinity of recombinant human ribosomal protein S13 (rpS13e) to the RNA transcript that corresponds to the central domain fragment of 18S rRNA (18SCD). The regions near the rpS13e binding site in 18SCD, including the nucleotides of helices H20 and H22, the availability of which to hydroxyl radicals depended on the Mg concentration, were determined. The increase in the Mg concentration was shown to result in the enhanced accessibility of nucleotides G933-C937 and C1006-A1009 in helix H22 and reduced accessibility of nucleotides A1023, A1024, and C1026-A1028 in the H20 helix. A comparison of the obtained results with crystallographic data of the central domain of 18S rRNA in the 40S ribosomal subunit of Saccharomyces cerevisiae has led to the conclusion that an increase in the Mg concentration resulted in the reorientation of helices H20 and H24 relative to to helices H22 and H23 and the formation of a structure in which these helices were positioned the same way as in 40S subunit. Hence, the saturation of the central domain of 18S rRNA with coordinated Mg ions causes the same changes in its structure as rpS13e binding, and results in the decreased affinity of this domain to the protein. [ABSTRACT FROM AUTHOR]

Human ribosomal proteins S5 and S16 (hrpS5 and hrpS16) are homologous, respectively, to prokaryotic ribosomal proteins S7p and S9p, which, according to X-ray crystallography data on the Thermus thermophilus 30S ribosomal subunit, contact the 3′-terminal 16S rRNA region formed by helices H28–H30 and H38–H43. Footprinting was used to study the mutual effect of hrpS5 and hrpS16 on their binding with an RNA transcript corresponding to 18S rRNA region 1203–1236/1521–1698 (helices H28–30 and H41–43), which is homologous to the16S rRNA region known to contain the binding site for S7p and part of the binding site for S9p. A simultaneous binding of hrpS5 and hrpS16 with the RNA transcript was shown to cause RNA conformational changes that stabilized the complex by involving new RNA parts, which did not interact with hrpS5 or hrpS16 in the respective binary complexes. [ABSTRACT FROM AUTHOR]

Human S3 (hS3) is a structural component of the ribosome and, in addition to its role in translation, possesses apurinic/apyrimidinic (AP) lyase activity, characteristic of DNA repair enzymes. Recombinant hS3 was isolated from inclusion bodies, refolded under different conditions, and tested for the ability to bind and cleave oligodeoxyribonucleotide substrates with various lesions abundant in genomic DNA: AP sites, uracil, 8-oxoguanine, 8-oxoadenine, 5,6-dihydrouracil, and hypoxanthine. It was found that hS3 is capable of cleaving AP sites via the β-elimination mechanism, producing a Schiff base covalent intermediate, but cannot cleave substrates with the other lesions. Refolding in the presence of Fe2+ and S2− did not increase hS3 activity, suggesting the absence of an iron-sulfur cluster. The binding of hS3 with DNA ligands containing oxidized or deaminated bases was less efficient than with intact DNA. It was assumed that the catalytic activity of hS3 towards AP sites is most likely unimportant for global DNA repair in vivo, but is possibly involved in repairing DNA sites in certain genome regions. [ABSTRACT FROM AUTHOR]

Human ribosomal protein S16 is homologous to prokaryotic S9, which contacts the 16S rRNA region formed by helices H29, H30, H38–H41, and H43 by X-ray data. A study was made of the interaction of recombinant human S16 with an RNA transcript corresponding to human 18S rRNA region 1203–1236/1521–1698 (helices H28–30 and H41–43), homologous to the 16S rRNA region harboring part of the S9-binding site. S16 specifically bound to the transcript with an apparent dissociation constant of (1.3 ± 0.1) · 10−8 M at 20°C. Enzymatic and chemical footprinting was used to identify the nucleotides whose accessibility for RNases and modifying chemical agents changes upon S16 binding. In the presence of S16, a substantially higher accessibility was observed for C1544 (internal loop of hairpin H41), C1618-U1622, C1629-A1634 (hairpin H42), C1521-C1523, U1530, C1532 (helix H30), C1645, C1646, and G1648 (base of hairpin H43) and an appreciably lower accessibility was observed for C1670-A1675 (hairpin H43). Many of the 16S rRNA nucleotides corresponding to the above 18S rRNA nucleotides tightly contact S9 residues in the bacterial 30S ribosomal subunit. [ABSTRACT FROM AUTHOR]

Complex measurements of the following elastic-magnetic parameters of a magnetic fluid suspended by magnetic levitation within a horizontal tube in a strong magnetic field were performed: the oscillation frequency and decay coefficient; the static, ponderomotive, and dynamic elasticity coefficients; the fluid displacement under hydrostatic pressure; magnetization curve; and the magnetic field strength and gradient. Calculations based on a model of ponderomotive elasticity with correction for the resistance of a viscous fluid in motion and on the fluid column displacement for two magnetic fluid samples agree well with the experimental magnetization curve. The discussed technique holds promise for research into magnetophoresis and nanoparticle aggregation in magnetic colloids. [ABSTRACT FROM AUTHOR]

Based on the concept of mapping the free surface geometry of a weakly magnetic medium by the topography of isolines of the magnetic field strength magnitude, the shape of the free surface of a magnetic fluid is studied in the static state at two stages: the initial stage, where an annular magnet is moving toward the surface of the magnetic fluid column in a tube, and at the stage where the air cavity is pressed to the bottom. It is shown that the separation of bubbles from the air cavity occurs at the magnet axis in the immediate vicinity of the magnet symmetry plane. A method and an experimental setup are proposed to investigate possible electromagnetic indication of the size of air bubbles formed in the magnetic fluid. The results of an experimental study of bubble separation from an air cavity held in the magnetic fluid and compressed by the pondermotive forces of the magnetic field are discussed. The results may be of importance for designing a new technique for metering small gas shots to a reactor. [ABSTRACT FROM AUTHOR]

The paper describes the process of an air cavity rising in a magnetic fluid filling a tube with a bottom, transport, and retention of the cavity by magnetic levitation forces. The elastic and dissipative properties of a vibratory system with an inertial element that is a column of a magnetic fluid over an air cavity are considered. The possibility of using a transported air cavity as a movable reflector for a sound wave is evaluated. [ABSTRACT FROM AUTHOR]

The possible involvement of 18S rRNA fragment 1638-1650, including basements of the helices h44 and h28, as well as nucleotides of the ribosomal decoding site in the cap-independent mode of the initiation of the translation of plant ribosomes is studied. This rRNA fragment is shown to be accessible for complementary interactions in the 40S ribosomal subunit. It is found that the sequence that is complementary to the 18S rRNA fragment 1638-1650 is able to enhance the efficiency of the reporter mRNA translation when placed just after the initiation codon. The obtained results indicate that, in the course of the cap-independent mode of the initiation of translation, complementary interactions can occur between the mRNA coding sequence and 18S rRNA fragment in the region of the ribosomal decoding site. [ABSTRACT FROM AUTHOR]

putative implication 3′-terminal 18S rRNA segment in the cap-independent initiation of translation on plant ribosomes was studied. It was shown that 3′-terminal segment (nucleotides 1777-1811) of 18S rRNA including the last hairpin 45 was accessible for complementary interactions within 40S ribosomal subunits. Oligonucleotides complementary to this segment of rRNA, when added to wheat germ cell-free protein synthesizing system, specifically inhibited translation of uncapped reporter mRNA encoding β-glucuronidase. In the 5′-untranslated region (UTR), the reporter mRNA contained a leader sequence of potato virus Y (PVY) genomic RNA with fragments complementary to the region 1777-1811. A sequence corresponding to nucleotides 291-316 of PVY, which was complementary to most of the 3′-terminal 18S rRNA segment 1777-1808, was shown to enhance translational efficiency of the reporter mRNAs when placed into 5′-UTR. The obtained results suggest that complementary interactions between 5′-UTR of mRNA and 3′-terminal segment of 18S rRNA can take place during cap-independent translation initiation. [ABSTRACT FROM AUTHOR]

The binding of the 18S rRNA of the 40S subunits of wheat germ ribosomes to an oligodeoxyribonucleotide complementary to the 1112–1123 region of the central domain of this RNA molecule has been studied. The selective binding of this oligomer to the complementary RNA fragment and the inhibition of the translation of uncapped chimeric RNA containing enhancer sequences in the 5′-untranslated region upstream of the reporter sequence coding for β-glucuronidase has been shown in a cell-free protein-synthesizing system. The use of a derivative of the aforementioned oligomer containing an alkylating group at the 5′ end allowed for the demonstration that the 1112–1123 region of 18S RNA can form a heteroduplex with the complementary sequence of the oligomer. The data obtained show that the 1112–1123 region in loop 27 of the central domain of 18S RNA of 40S ribosomal subunits is exposed on the subunit surface and probably participates in the cap-independent binding of the subunits to mRNA due to the complementary interaction with the enhancer sequences. [ABSTRACT FROM AUTHOR]

Recombinant human ribosomal protein S16 (rpS16) is shown to bind specifically to a fragment of its own pre-mRNA that includes exons 1 and 2, intron 1, and part of intron 2, and to inhibit the splicing of the fragment in vitro. The weaker binding of other recombinant human ribosomal proteins, S10 and S13, to this pre-mRNA fragment indicated that the binding of rpS16 was specific. Besides, the poly(AU) and rpS16 mRNA fragment insignificantly affected the binding of rpS16 to its pre-mRNA, providing another evidence that the interaction was specific. rpS16 specifically inhibited the splicing of the pre-mRNA fragment, whereas recombinant rpS10 and rpS13 did not affect intron excision from this pre-mRNA fragment in contrast to rpS16. Those positions in rpS16 pre-mRNA fragment that were protected by rpS16 from cleavage by RNases T1, T2, and V1 were found to be located closely to the branch point and 3’ splice site in the pre-mRNA. The obtained results suggest the possibility of the autoregulation of rpS13 pre-mRNA splicing through the feedback mechanism. [ABSTRACT FROM AUTHOR]

Polyclonal and monoclonal antibodies (MABs) to human laminin-binding protein (LBP) can efficiently block the penetration of some alphaand flaviviruses into the cell. A panel of 13 types of MABs to human recombinant LBP was used for more detailed study of the mechanism of this process. Competitive analysis has shown that MABs to LBP can be divided into six different competition groups. MABs 4F6 and 8E4 classified under competition groups 3 and 4 can inhibit the replication of Venezuelan equine encephalitis virus (VEEV), which is indicative of their interaction with the receptor domain of LBP providing for binding with virions. According to enzyme immunoassay and immunoblotting data, polyclonal anti-idiotypic antibodies to MABs 4F6 and 8E4 modeling paratopes of the LBP receptor domain can specifically interact with VEEV E2 protein and tick-borne encephalitis virus (TBEV) E protein. Mapping of binding sites of MABs 4F6 and 8E4 with LBP by constructing short deletion fragments of the human LBP molecule has shown that MAB 8E4 interacts with the fragment of amino acid residues 187–210, and MAB 4F6 interacts with the fragment of residues 263–278 of LBP protein, which is represented by two TEDWS peptides separated by four amino acid residues. This suggested that the LBP receptor domain interacting with VEEV E2 and TBEV E viral proteins is located at the C-terminal fragment of the LBP molecule. A model of the spatial structure of the LBP receptor domain distally limited by four linear loops (two of which are represented by experimentally mapped regions of amino acid residues 187–210 and 263–278) as well as the central β-folded region turning into the α-helical site including residues 200–216 of the LBP molecule and providing for the interaction with the laminin-1 molecule has been proposed. [ABSTRACT FROM AUTHOR]

Ribosomal protein p40 is a structural component of the eukaryotic 40S ribosomal subunit, is partly homologous to prokaryotic ribosomal protein S2, and has a long eukaryote-specific C-terminal region. The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA was tested for the binding to 40S ribosomal subunits deficient in p40, saturated with recombinant p40, or pretreated with monoclonal antibody (MAB) 4F6 against p40. The apparent association constant of HCV IRES binding to 40S subunits was shown to directly depend on the p40 content in the subunits. MAB 4F6 prevented HCV IRES binding to 40S subunits and blocked translation of IRES-containing RNA in a cell-free translation system. The results implicate p40 in the binding of the HCV IRES to the ribosome and, therefore, in translation initiation on HCV RNA. [ABSTRACT FROM AUTHOR]

Ribosomal protein SA (rpSA), or p40, is a structural element of the small subunit of the eukaryotic ribosome. The N-terminal and central parts of rpSA are homologous to prokaryotic S2, whereas its C-terminal part is specific to eukaryotes. Preparations of 40S ribosomal subunits isolated from full-term human placenta proved to be deficient in SA to a varying extent. To study the rpSA binding to human 40S subunits, recombinant rpSA and its mutant forms with N-and C-terminal deletions were synthesized. The full-size and N-truncated rpSA variants bound to 40S subunits, while deletion of the C-terminal domain completely abolished the binding. [ABSTRACT FROM AUTHOR]

Protein S15 is a characteristic component of the mammalian 80S ribosome that neighbors the mRNA codon at the decoding site and the downstream triplets. The S15 fragment juxtaposed in the human ribosome to mRNA nucleotides +4 to +12 relative to the first nucleotide of the P-site codon was determined. S15 was modified using a set of mRNA analogs containing the triplet UUU/UUC at the 5′ end and a perfluorophenyl azide-carrying uridine at various positions downstream of this triplet. The mRNA analogs were positioned on the ribosome with the use of tRNAPhe, cognate to the UUU/UUC triplet, targeted to the P site. Modified S15 was isolated from complexes of 80S ribosomes with tRNAPhe and the mRNA analogs after irradiation with mild UV light and hydrolyzed with cyanogen bromide, cleaving the polypeptide chain after Met residues. Analysis of the modified oligopeptides resulting from hydrolysis demonstrated that the crosslinking site was in C-terminal fragment 111–145 of S15 in all cases, suggesting the involvement of this fragment in the decoding site of the eukaryotic ribosome. [ABSTRACT FROM AUTHOR]

The arrangement of the stop codon and its 3′-flanking codon relative to the components of translation termination complexes of human 80S ribosomes was studied using mRNA analogs containing the stop signal UPuPuPu (Pu is A or G) and the photoreactive perfluoroarylazido group, which was linked to a stop-signal or 3′-flanking nucleotide (positions from +4 to +9 relative to the first nucleotide of the P-site codon). Upon mild UV irradiation, the analogs crosslinked to components of the model complexes, mimicking the state of the 80S ribosome at translation termination. Termination factors eRF1 and eRF3 did not change the relative arrangement of the stop signal and 18S rRNA. Crosslinking to eRF1 was observed for modified nucleotides in positions +5 to +9 (that for stop-codon nucleotide +4 was detected earlier). The eRF1 fragments crosslinked to the mRNA analogs were identified. Fragment 52–195, including the N domain and part of the M domain, crosslinked to the analogs carrying the reactive group at A or G in positions +5 to +9 or at the terminal phosphate of nucleotide +7. The site crosslinking to mRNA analogs containing modified G in positions +5 to +7 was assigned to eRF1 fragment 82–166 (beyond the NIKS motif). All but one analog (that with modified G in position +4) crosslinked to the C domain of eRF1 (fragment 330–422). The efficiency of crosslinking to the C domain was higher than to the N domain in most cases. It was assumed that the C domain of eRF1 bound in the A site is close to nucleotides +5 to +9, especially +7 and +8, and that eRF1 undergoes substantial conformational changes when binding to the ribosome. [ABSTRACT FROM AUTHOR]

Recombinant human ribosomal protein (rp) S13 was shown to specifically bind with its own pre-mRNA fragment containing the first exon, first intron, second exon, and a part of the second intron and to inhibit its splicing in vitro. The binding of rpS13 was specific: recombinant human rpS10 and rpS16 bound with the fragment to a lower extent. Moreover, rpS13 binding with the rpS13 pre-mRNA fragment was inhibited by non-labeled poly(AU) and an adenovirus pre-mRNA fragment to a lower extent than by the nonlabeled rpS13 pre-mRNA fragment. The specificity of splicing inhibition was inferred from the finding that, in contrast to rpS13, recombinant rpS10 and rpS16 did not affect the efficiency of first intron excision from the rpS13 pre-mRNA fragment. Enzymatic footprinting was used to determine the rpS13 pre-mRNA nucleotides whose accessibility to RNases T1, T2, and V1 changed in the presence of rpS13. Such nucleotides were detected close to the 5′ and 3′ splicing sites of the first intron. Analysis with the EMBOS-Align program showed that the nucleotide sequence of the first intron of the mammalian rpS13 pre-mRNA is conserved to a greater extent as compared with the other introns. It was assumed that the first intron plays an important role in regulating the expression of the rpS13 gene at the splicing level in all mammals. [ABSTRACT FROM AUTHOR]

The human protein uS3, a component of the small ribosomal subunit, has a long-known extra-ribosomal activity as an enzyme of base excision DNA repair displayed in its ability to cleave DNA at abasic (AP) sites. It has been found that the efficacy of DNA cleavage by uS3 in vitro depends on the DNA sequence. To clarify the issue on the sequence specificity of uS3 as an AP lyase in general, we applied a combinatorial approach based on the use of a model single-stranded circular DNA with an AP site flanked with random trinucleotides at both sides. The cleavage of this DNA by uS3 under conditions when only its minor portion undergoes the reaction resulted in the formation of the linear DNA with random triplets at the 5' and 3' termini. NGS sequencing of the DNA library derived from this DNA allowed identifying the contexts within which uS3 cleaves DNA the most and the least effectively. Given that the AP lyase reaction occurs via the formation of a covalent intermediate (Schiff base), we determined the region comprising the active center of the uS3 protein. By digesting of uS3 cross-linked to a radiolabeled AP site-containing model DNA with specific proteolytic agents followed by analysis of the resulting modified oligopeptides, the cross-link was mapped to the region 155-192 (likely, to R173/R178). Thus, our results clarified two previously unstudied features of the uS3 AP lyase activity, one related to the recognition of sequences in DNA surrounding the AP site, and the other to the protein region directly contacting this site., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Proteins belonging to the universal ribosomal protein (rp) uS19 family are constituents of small ribosomal subunits, and their conserved globular parts are involved in the formation of the head of these subunits. The eukaryotic rp uS19 (previously known as S15) comprises a C-terminal extension that has no homology in the bacterial counterparts. This extension is directly implicated in the formation of the ribosomal decoding site and thereby affects translational fidelity in a manner that has no analogy in bacterial ribosomes. Another eukaryote-specific feature of rp uS19 is its essential participance in the 40S subunit maturation due to the interactions with the subunit assembly factors required for the nuclear exit of pre-40S particles. Beyond properties related to the translation machinery, eukaryotic rp uS19 has an extra-ribosomal function concerned with its direct involvement in the regulation of the activity of an important tumor suppressor p53 in the Mdm2/Mdmx-p53 pathway. Mutations in the RPS15 gene encoding rp uS19 are linked to diseases (Diamond Blackfan anemia, chronic lymphocytic leukemia and Parkinson's disease) caused either by defects in the ribosome biogenesis or disturbances in the functioning of ribosomes containing mutant rp uS19, likely due to the changed translational fidelity. Here, we review currently available data on the involvement of rp uS19 in the operation of the translational machinery and in the maturation of 40S subunits, on its extra-ribosomal function, and on relationships between mutations in the RPS15 gene and certain human diseases., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The conserved ribosomal protein uS3 in eukaryotes has long been known as one of the essential components of the small (40S) ribosomal subunit, which is involved in the structure of the 40S mRNA entry pore, ensuring the functioning of the 40S subunit during translation initiation. Besides, uS3, being outside the ribosome, is engaged in various cellular processes related to DNA repair, NF-kB signaling pathway and regulation of apoptosis. This review is devoted to recent data opening new horizons in understanding the roles of uS3 in such processes as the assembly and maturation of 40S subunits, ensuring proper structure of 48S pre-initiation complexes, regulation of initiation and ribosome-based RNA quality control pathways. Besides, we summarize novel results on the participation of the protein in processes beyond translation and consider biomedical implications of previously known and recently found extra-ribosomal functions of uS3, primarily, in oncogenesis., (© 2020 Wiley Periodicals LLC.)

The eukaryotic ribosomal protein uS19 has a C-terminal tail that is absent in its bacterial homologue. This tail has been shown to be involved in the formation of the decoding site of human ribosomes. We studied here the previously unexplored functional significance of the 15 C-terminal amino acid residues of human uS19 for the assembly of ribosomes and translation using HEK293-based cell cultures capable of producing FLAG-labeled uS19 (uS19 FLAG ) or its mutant form deprived of the mentioned amino acid ones. The examination of polysome profiles of cytoplasmic extracts from the respective cells revealed that the deletion of the above uS19 amino acid residues barely affected the assembly and maturation of 40S subunits and the initiation of translation, but completely prevented the formation of polysomes. This implied the crucial importance of the uS19 tail in the elongation process. Analysis of tRNAs associated with 40S subunits and 80S ribosomes containing wild type uS19 FLAG or its truncated form showed that the deletion of the C-terminal pentadecapeptide fragment of uS19 did not interfere with the binding of aminoacyl-tRNA (aa-tRNA) at the ribosomal A site. The results led to the conclusion that the transpeptidation, which occurs on the large ribosomal subunit after decoding the A site codon by the incoming aa-tRNA, is the most likely elongation stage, where this uS19 fragment can play a critical role. Our findings suggest that the uS19 tail is a keystone player in the accommodation of aa-tRNA at the A site, which is a pre-requisite for the peptide transfer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Conserved ribosomal protein uS3 contains a decapeptide fragment in positions 55-64 (human numbering), which has a very specific ability to cross-link to various RNA derivatives bearing aldehyde groups, likely provided by K62. It has been shown that during translation in the cell-free protein-synthesizing system, uS3 becomes accessible for such cross-linking only after eIF3j leaves the mRNA binding channel of the 40S ribosomal subunit. We studied the functional role of K62 and its nearest neighbors in the ribosomal assembly and translation with the use of HEK293T-derived cell cultures capable of producing FLAG-tagged uS3 (uS3 FLAG ) or its mutant form with amino acid residues at positions 60-63 replaced with alanines. Analysis of polysome profiles from the respective cells and cytosol lysates showed that the mutation significantly affected the uS3 ability to participate in the assembly of 40S subunits, but it was not essential for their maturation and did not prevent the binding of mRNAs to 40S subunits during translation initiation. The most striking effect of the replacement of amino acid residues in the above uS3 positions was that it almost completely deprived the 40S subunits of their ability to form 80S ribosomes, suggesting that the 48S pre-initiation complexes assembled on these subunits were defective in the binding of 60S subunits. Thus, our results revealed the previously unknown crucial role of the uS3 tetrapeptide 60 GEKG 63 in translation initiation related to maintaining the proper structure of the 48S complex, most likely via the prevention of premature mRNA loading into the ribosomal channel., (Copyright © 2019 Elsevier B.V. All rights reserved.)