The functional role of the C-terminal tail of the human ribosomal protein uS19

The functional role of the C-terminal tail of the human ribosomal protein uS19

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BBA - Gene Regulatory Mechanisms xxx (xxxx) xxxx

Contents lists available at ScienceDirect

BBA - Gene Regulatory Mechanisms journal homepage: www.elsevier.com/locate/bbagrm

The functional role of the C-terminal tail of the human ribosomal protein uS19 Konstantin Bulygina, Alexey Malygina,b, Alexander Gopanenkoa, Dmitri Graifera,b, ⁎ Galina Karpovaa,b, a b

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia Novosibirsk State University, Novosibirsk 630090, Russia

A R T I C LE I N FO

A B S T R A C T

Keywords: Human ribosomal protein uS19 Deletion of uS19 C-terminal amino acid residues Small ribosomal subunit Decoding site Translation elongation Aminoacyl-tRNA accommodation

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 (uS19FLAG) 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 uS19FLAG 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.

1. Introduction Protein synthesis is a key process in the cells of all kingdoms, because, being the last step in the implementation of genetic information, it provides cells with regulatory molecules that mainly predetermine their function and survival, thus affecting all aspects of cell life. During this process, information encoded as trinucleotide sequences (codons) in messenger RNAs (mRNAs) is translated into protein polypeptide sequences. Translation takes place on ribosomes, very complex ribonucleoprotein machines, whose small subunits are mainly responsible for decoding the genetic information, and large ones for the formation of peptide bonds. Amino acid residues are delivered to the ribosome for peptide synthesis, being attached to the 3′-terminal ribose residue of specialized adapter small RNA molecules (transfer RNAs, tRNAs), whose trinucleotide anticodons can recognize mRNA codons, which makes the basis for the decoding of genetic information. Ribosomes have three sites for the binding of tRNA molecules: an aminoacyl (A)

site for the incoming aminoacyl-tRNA (aa-tRNA), a peptidyl (P) site for retaining the tRNA with the nascent peptide, and an exit (E) site for binding the deacylated tRNA before it leaves the ribosome. Currently, the structural organization of both bacterial and eukaryotic ribosomes, as well as their functional sites, is well known mainly due to significant progress in cryo-electron microscopy (cryoEM) studies (for example, see [1–5]). These studies have provided the rationale for a number of earlier data obtained by biochemical approaches (e.g., see for review [6–8]). It is now generally accepted that the small subunit rRNA fragments responsible for decoding and the large subunit rRNA ones ensuring the activity of the ribosomal peptidyl transferase are strongly conserved from eubacteria to higher eukaryotes. At the same time, there are many gaps in knowledge on the contribution of ribosomal proteins and their particular fragments to the functions of ribosomes; especially this concerns those fragments of eukaryotic proteins that have no homology in the bacterial ones. The eukaryotic small (40S) subunit ribosomal protein uS19

⁎ Corresponding author at: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia. E-mail address: [email protected] (G. Karpova).

https://doi.org/10.1016/j.bbagrm.2020.194490 Received 4 October 2019; Received in revised form 15 January 2020; Accepted 15 January 2020 1874-9399/ © 2020 Elsevier B.V. All rights reserved.

Please cite this article as: Konstantin Bulygin, et al., BBA - Gene Regulatory Mechanisms, https://doi.org/10.1016/j.bbagrm.2020.194490

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uS19FLAG were incompetent in polysome formation. This implied that the deletion of the C-terminal pentadecapeptide fragment of uS19 made the 80S ribosomes defective in elongation. When discriminating the most probable stage of elongation, where the above fragment of uS19 could play a crucial role, we showed that 80S ribosomes with mutuS19FLAG were competent in the binding of aa-tRNAs at the A site, which allowed us to conclude that they were defective in the next stage, associated with transpeptidation. Ultimately, our findings suggest that the C-terminal tail of uS19 plays a key role in the accommodation of aatRNA at the A site, which insures the peptide bond formation by wildtype ribosomes.

(formerly known as S15e) is one of those whose role in the translation process has remained poorly defined. The conserved part of uS19 is located in the inter-subunit area on the head of the 30S subunit or the 40S one far from the decoding site, which lies at the groove between the subunit head and body (e.g., see [9,10]). However, studies on site-directed cross-linking of different mRNA analogues to mammalian ribosomes, carried out independently in three groups, have shown that they could cross-link to uS19 when their photoactivatable nucleotide residues were placed at the decoding site [11–16]. It should be noted that in early reports [11–13], this protein has been featured as a 19 kDa one. Later, the cross-linking site has been mapped to the C-terminal fragment (amino acid residues 131–145) of the protein [17]. Multiple alignment of amino acid sequences of ribosomal proteins from the uS19 family in Eukarya, Archaea and Bacteria has shown that the 15 amino acids long C-terminal sequence corresponding to this fragment is mainly conserved in the eukaryotic and archaeal proteins, but it has no homology with the C-terminal sequences of the bacterial ones [17]. Fifteen C-terminal amino acid residues of eukaryotic uS19 belong to a specific element of its spatial structure in the 40S subunit, an extended and mainly unstructured tail protruding away from the globular conserved part of the protein [17]. Large portion of this tail has long remained unresolved on the structures of 40S subunits and it has only recently been visualized at the decoding area [5]. The presence of the uS19 C-terminal tail in the decoding area is a feature that has no similarity in bacterial ribosomes, and this fact somewhat contradicts the widespread view on the universally conserved nature of the structural organization of the ribosomal decoding site in all kingdoms of life. Several hypotheses have been put forward regarding the role of the uS19 tail in the translation process. During elongation, the tail could improve the fixation the mRNA codon at the decoding site, increasing the accuracy of translation and reducing the probability of frameshift, and at termination, the tail could interact with eRF1 [17]. In the aforementioned report [5], it has been suggested that the interaction of the C-terminal tail of uS19 with aa-tRNA stabilizes its binding at the A site during decoding, which reduces the probability of undesired dissociation of cognate аa-tRNAs when proofreading. In the recent reports on the structure of yeast cytoplasmic pre-40S subunits [18,19] corresponding to the final step of maturation of the 40S subunits, uS19 is positioned close to small subunit assembly factors Tsr1 and Rio2. Analysis of the respective deposited pre-40S subunit models shows that these factors are at an interaction distance from the C-terminal part of uS19. Thus, one could suggest that the uS19 tail interacts with Tsr1 and/or Rio2, which, in turn, would imply that the tail is involved in the final maturation stage of the 40S subunit. However, all of the above hypotheses have not yet been experimentally examined, and the role of the C-terminal tail of uS19 in eukaryotic translation remains unknown. Here, we used HEK293T cells transfected with specially constructed plasmids enabling the production of wild type human uS19 carrying FLAG-tag (wt-uS19FLAG) and its mutant deprived of the 15 C-terminal aa residues (mut-uS19FLAG) to gain information on the importance of the C-terminal tail of uS19 for the ribosome assembly and translation in vivo. We focused on the deletion of exactly 15 C-terminal amino acid residues because (i) in the human uS19, the site of cross-linking of mRNA analogues with photoactivatable nucleotides in the A site codon has been mapped to the C-terminal pentadecapeptide fragment, and (ii) it is this one that is present in the 40S ribosomal decoding site (see above). On the first issue, we examined the levels of incorporation of wt-uS19FLAG and mut-uS19FLAG into the nuclear and cytosolic extracts of cells producing these proteins. On the second one, we explored the incorporation of wt-uS19FLAG and mut-uS19FLAG into 40S subunits, 80S ribosomes, and polysomes derived from the corresponding cytoplasmic extracts, and studied the effect of the deletion of the aforementioned amino acid residues of uS19 on the ability of 80S ribosomes to bind aatRNAs at the A site. It was found that mut-uS19FLAG could participate in the assembly of 40S subunits that were active in translation initiation, although 80S ribosome complexes consisting of those containing mut-

2. Materials and methods 2.1. Materials Oligodeoxyribonucleotides, including the random hexamer primer, were synthesized in the Laboratory of Biomedical Chemistry, the Institute of Chemical Biology and Fundamental Medicine (ICBFM), Siberian Branch of the Russian Academy of Sciences (SB RAS), (Novosibirsk, Russia). M-MuLV reverse transcriptase was produced in the Laboratory of Bioorganic Chemistry of Enzymes, ICBFM of SB RAS. DNA sequencing was performed in the SB RAS Genomics Core Facility based on ICBFM of SB RAS. Anti-mouse antibodies conjugated with horseradish peroxidase and mouse monoclonal antibodies specific for FLAG-peptide (M2, #F1804 and M5, #F4042) were purchased from Sigma; rabbit polyclonal antibodies specific for human uS19 (ab90902) and lamin B1 (ab16048) were purchased from Abcam; monoclonal antibodies specific for GAPDH (#60004-1-Ig) were from Proteintech; polyclonal rabbit antiserum against rat eS4 was kindly gifted by Dr. J. Stahl. 2.2. Preparation of plasmid constructions, transfection of cells and analysis of the production of FLAG-labeled target proteins DNA constructions with coding sequences for the proteins wtuS19FLAG and mut-uS19FLAG labeled with epitope FLAG at the N-terminus were prepared based on the original plasmid pAG-1 described earlier [20]. To synthesize minigenes for wt-uS19FLAG and mutuS19FLAG, total human cDNA was amplified with the use of forward primer 5′-acgtgaattcatggtggactacaaagacgatgacgacaaggcagaagtagagcagaag-3′ (containing a sequence coding for the epitope FLAG) and reverse primers 5′-acgtgaattcttacttgagagggatgaagc-3′ and 5′-acgtgaattcttaccggccatgctttac-3′, respectively. The resulting products were integrated into the vector pAG-1 linearized at the EcoRI site. The exact correspondence of the sequences inserted into the above constructions to the amino acid sequences of the target proteins was verified by sequencing. Transfecton of HEK293T cells (ATCC CRL-3216) with the obtained DNA constructions, preparation of lysates from the transfected cells, isolation of the total protein from the lysates and subsequent identification of the FLAG-labeled proteins by western blotting were carried out as described [21]. 2.3. Preparation of cytoplasmic and nuclear extracts from the cells producing FLAG-labeled target proteins Cytoplasmic extracts were prepared from the transfected cells according to [21]. Nuclear extracts were prepared from the pellets of nuclei obtained in the course of the cytoplasmic extracts preparation. The pellets were washed twice by resuspending them in 400 μl of 10 mM Tris-HCl buffer (pH 7.5) containing 2 mM MgCl2, 10 mM KCl, 0,05% Triton-X100, 1 mM EGTA, 40 mg/ml of phenylmethylsulfonyl fluoride and 10 μg/ml of Protease Inhibitor Cocktail (Sigma) with subsequent pelleting by centrifugation at 2000g for 5 min at 4 °C. Further manipulations with the pelleted nuclei were performed, as 2

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described [22], using 0.5% Nonidet P40 instead of 0.1% Igepal-CA630. 2.4. Analysis of the presence of FLAG-labeled target proteins and specific RNAs in the fractions of polysome profiles from cytoplasmic extracts of transfected cells Obtaining polysome profiles from cytoplasmic extracts of cells producing FLAG-labeled target proteins and analysis of the incorporation of these proteins into fractions of the profiles with the use of antiFLAG antibodies were carried out as described [21]. Immunoprecipitation of 40S subunits containing FLAG-labeled protein from the respective fractions of the above polysome profiles, isolation of total RNA from these subunits, synthesis of cDNA on this RNA and subsequent analysis of the presence of mature 18S rRNA, 18S-E prerRNA and particular mRNAs by real-time PCR, were performed according to [21]. Immunoprecipitation of 80S ribosomes containing FLAG-labeled protein and isolation of total RNA from these 80S ribosomes were carried out in a similar manner. Analysis of tRNA molecules bound to 80S ribosomes containing FLAG-labeled protein was performed with the Cu2+-treated and 3′-end-[32P]-labeled total RNA as described [23]. 3. Results 3.1. Characterization of transfected cells producing FLAG-tagged proteins wt-uS19FLAG or mut-uS19FLAG Cell cultures used for the inducible production of exogenous FLAGtagged proteins wt-uS19FLAG or mut-uS19FLAG were prepared by analogy with those utilized in our previous study [21] by transiently transfecting HEK293T cells with plasmids containing DNA sequences encoding the respective proteins, which were derived from the mammalian expression vector pAG-1 [20]. The production of the target proteins by the transfected cells was examined by western blotting using anti-FLAG monoclonal antibodies applied to the total protein of the whole cell lysates separated by SDS PAGE. It was shown that the transfection of cells with the above plasmids results in the appearance of FLAG-tagged polypeptides, whose molecular weights correspond to those expected for the target proteins wt-uS19FLAG and mut-uS19FLAG (Fig. 1, A and B). However, the level of mut-uS19FLAG in the cells was considerably (approximately 8 times) less than that of wt-uS19FLAG, which points to the importance of the uS19 C-terminal tail for the protein production, although the levels of mRNAs encoding these proteins were only slightly different (see Fig. S2). Indeed, it is known that proteins with C-terminal sequences similar to that of mut-uS19FLAG are prone to enhanced degradation (see [24,25]). Therefore, the reason for the significantly decreased cellular level of mut-uS19FLAG may be associated with a higher rate of its degradation compared to that of wtuS19FLAG. The application of anti-uS19 antibodies to the same lanes that were examined with anti-FLAG antibodies made it possible to compare the production of endogenous and exogenous proteins, which could be easily distinguished from each other due to differences in their molecular weights (see Supplementary material). It was found that the cellular level of wt-uS19FLAG was somewhat lower (about 1.5 times) than that of the endogenous one, while with mut-uS19FLAG this difference increased approximately up to 8-fold (Fig. S1), which was expected due to less effective production of this protein by transfected cells compared to that of wt-uS19FLAG (Fig. 1B). With all this, the transfection of cells with plasmids encoding wt-uS19FLAG or mutuS19FLAG did not significantly affect the overall level of endogenous uS19 there (see Supplementary material). Western blot analysis applied to total protein samples from cytoplasmic and nuclear extracts obtained from cells producing wtuS19FLAG or mut-uS19FLAG showed the presence of each of the target proteins in these cell fractions (Fig. 1C). With both cell types, the level of the target protein in the cytoplasmic extract was higher than that in

Fig. 1. Production of target proteins in HEK293T cells transfected with DNA constructions with coding sequences for the proteins wt-uS19FLAG and mutuS19FLAG and their distribution between different cellular compartments. Western blot analysis of total protein samples from lysates of cells producing wt-uS19FLAG or mut-uS19FLAG (A and B) and from the respective cytoplasmic (cyt) and nuclear (nuc) extracts (C) for the content of the target proteins after transferring their patterns separated by SDS-PAGE from the gel onto a nitrocellulose membrane using anti-FLAG peptide antibodies. Panel A is the stained nitrocellulose membrane, where the positions of the protein molecular weight markers are shown on the left. Before western blotting, it was cut into several sheets (the cut lines are shown as dotted lines). In panel B, the bands of eS4, which was used as a reference ribosomal protein, were developed using anti-eS4 antiserum. Designations “wt” and “Δ” correspond to the experiments with the cells producing wt-uS19FLAG or mut-uS19FLAG, respectively, and designation “-” refers to the experiments with non-transfected cells. Arrows indicate the positions of wt-uS19FLAG and mut-uS19FLAG, as well as eS4. The relative intensities of the bands corresponding to the FLAG-tagged proteins are shown on the bottom of panel B. In panel C, lanes 1 and 3 correspond to a total protein obtained from 105 cells, whereas lanes 2 and 4 were loaded with a total protein from 2 × 105 cells. Panel D shows the quality of the subcellular fractions prepared from the above transfected cells, examined on the example of cells producing wt-uS19FLAG by western blot detection of protein markers lamin B1 and GAPDH specific to nuclear and cytoplasmic extracts, respectively. The data presented imply that cross-contamination between the extracts is negligible.

the nuclear one, and the difference between the levels of the target protein in these extracts, as estimated by a quantitative analysis of the data in Fig. 1C, was approximately 5-fold. This meant that the wtuS19FLAG and mut-uS19FLAG proteins could be transported from the cytosol, where they were synthesized, to the nucleus, which implied that the deletion of the C-terminal tail of uS19 did not interfere with this transport. For all that, the level of mut-uS19FLAG in the each kind of extract was about 8 times lower than the level of wt-uS19FLAG, which was consistent with the difference in the levels of these proteins in extracts from the respective whole cell lysates (Fig. 1B).

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3.2. Effect of deletion of 15 C-terminal amino acid residues of uS19 on maturation of 40S subunits

amino acid residues of uS19 does not considerably increase the share of immature rRNA in cytosolic 40S subunits.

Given the interaction distances between the C-terminal part of uS19 and the small subunit assembly factors Tsr1 and Rio2 [18,19], one could assume that the deletion of the 15 C-terminal amino acid residues of uS19 impaired the assembly and/or maturation of 40S subunits. To clarify this issue, we examined the effect of this deletion on the final stage of maturation of the 40S subunits occurring in the cytosol, where specific pre-rRNA cleavage takes place to form normal length 18S rRNA, in the same way as in our previous study with FLAG-labeled ribosomal protein uS3 and its mutant form [21]. In particular, we compared the content of mature 18S rRNA and immature one (the socalled 18S-E pre-rRNA) in the fractions corresponding to peaks of 40S subunits from polysome profiles obtained by the centrifugation of cytoplasmic extracts of cells producing uS19FLAG or mut-uS19FLAG in sucrose gradients (these profiles are presented in Section 3.3, where they are more appropriate). To this end, 40S subunits were immunoprecipitated from the respective fractions with anti-FLAG antibodies, followed by isolation of the total RNA from the resulting samples, which was utilized to synthesize total cDNA using either a random hexamer primer or a primer complementary to the 3′-end of 18S-E prerRNA. Conventional PCR performed with a pair of primers specific to 18S rRNA on the total cDNA obtained with the random primer showed the presence of a product of the expected length with both wt-uS19FLAG and mut-uS19FLAG, as in the previous study [21]. On the contrary, with a pair of primers specific to the 18S-E pre-rRNA 3′-terminus portion, no specific PCR product was observed even at 40 cycles (Fig. S4). This meant that both types of 40S subunits immunoprecipitated from the respective polysome profile fractions contained almost only mature 18S rRNA. The PCR product, which could be attributed to 18S-E pre-rRNA, was detected, as earlier [21], only with cDNA obtained with the primer complementary to the 3′-end of 18S-E pre-rRNA and using the pair of primers specific to the 18S-E pre-rRNA 3′-terminus portion (Fig. S4). Quantitative PCR analysis performed on this cDNA with the above pair primers showed that the content of immature 18S-E pre-rRNA in 40S subunits with mut-uS19FLAG was 3 times higher than with wt-uS19FLAG (Fig. 2). However, since the overall amount of this kind of RNA in 40S subunits was incomparably lower than that of the mature 18S rRNA (see above), one can conclude that the deletion of the 15 C-terminal

3.3. Effect of deletion of 15 C-terminal amino acid residues of uS19 on the protein incorporation into 40S subunits, 80S ribosomes and polysomes To find out the effect of the deletion of 15 C-terminal amino acid residues of uS19 on the formation of 40S subunit and 80S ribosome complexes and polysomes, we examined the content of FLAG-labeled polypeptides in the respective fractions from the aforementioned polysome profiles by Western blotting using anti-FLAG antibodies. The results presented in Fig. 3A show that wt-uS19FLAG is distinctly detected in all fractions containing 40S subunits (free subunits, 80S ribosomes and polysomes), which implies that uS19 with the deleted C-terminal pentadecapeptide fragment can participate in all translation stages, by analogy with the endogenous protein. With the cells producing mutuS19FLAG, the target protein is well seen in the fractions of 40S subunits and 80S ribosomes, and the ratio of the intensities of the signals corresponding to these fractions (Fig. 3B) is similar to that for the cells producing wt-uS19FLAG (Fig. 3A). This meant that the deletion did not affect significantly the share of 40S subunits passing through initiation steps up to the assembly of 80S ribosomal complexes. This suggestion was confirmed by end-point and quantitative PCR analyses of the content of mRNAs of abundant proteins in fractions of 40S subunits immunoprecipitated by anti-FLAG antibodies carried out by analogy with that made for 18S rRNA and 18S-E pre-rRNA (see above) using the total cDNA obtained with the random primer and pairs of appropriate primers, respectively. These analyses showed the presence of mRNAs in the tested samples of 40S subunits containing wt-uS19FLAG or mutuS19FLAG and did not reveal considerable differences between these samples (Figs. S5 and S6), indicating the ability of both types of 40S subunits to bind mRNAs, i.e. to form 48S pre-initiation complexes. A striking difference between the results obtained in the experiments described above concerned the polysome fractions where no mut-uS19FLAG was detected at all, in contrast to wt-uS19FLAG (Fig. 3B). This indicated that the deletion of the 15 C-terminal amino acid residues of uS19 did not prevent the incorporation of the protein into the 40S subunits and that mut-uS19FLAG-containing 40S subunits could join with 60S subunits to form 80S ribosomes, but the latter were incompetent in polysome formation. Therefore, 80S ribosomes containing mut-uS19FLAG were crucially defective in one of steps of translation elongation. 3.4. Comparative analysis of the competence of 80S ribosomes containing wt-S19FLAG or mut-S19FLAG in tRNA binding at the A site The elongation cycle includes several stages, starting with the EF1Aand GTP-dependent binding of incoming aa-tRNA at the ribosomal A site and subsequent accommodation of aa-tRNA at the A site, which shifts its 3′-end to the position favorable for the peptide bond formation. After transpeptidation, peptidyl-tRNA elongated by a new amino acid residue translocates from the A to the P site in an EF2- and GTPdependent manner, and deacylated tRNA moves from the P to the E site. To determine the elongation stage affected by the deletion of the 15 Cterminal amino acid residues of uS19, we applied an approach based on the analysis of tRNA molecules bound to 80S ribosomes containing wtuS19FLAG or mut-uS19FLAG, which has been used in our previous works [23,26]. This analysis involves hydrolysis of the complex ester bond between tRNA and the aminoacyl residue by Cu2+ ions, which does not affect the same bond in peptidyl-tRNA, and the labeling of the liberated tRNA 3′-OH termini with [32P]pCp and T4 RNA ligase, followed by separation of the labeled tRNAs by denaturing PAGE. The band corresponding to the initiator tRNAiMet can be uniquely identified by the control lane, onto which the tRNA sample from the 40S subunit fraction is loaded, because it can contain only such tRNA, while any other tRNA band appearing on the lane corresponding to the 80S ribosome fraction

Fig. 2. Comparative analysis of the content of 18S-E pre-rRNA in samples of 40S subunits immunoprecipitated from fractions corresponding to 40S subunit peaks from polysome profiles obtained from cytoplasmic extracts of cells producing wt-uS19FLAG or mut-uS19FLAG. The content of 18S-E pre-rRNA normalized to that of 18S rRNA in 40S subunits containing wt-uS19FLAG or mutuS19FLAG determined using quantitative RT-PCR, is presented by columns as mean of arbitrary units (A.U.) ± SD (**p < 0.005; p values are calculated using Student's t-test). The content of 18S-E pre-rRNA normalized to that of 18S rRNA for wt-uS19FLAG-containing 40S subunits was taken as one A.U. 4

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Fig. 3. Western blot analysis of fractions of polysome profiles from cytoplasmic extracts of transfected cells for the content of FLAG-labeled target proteins using anti-FLAG peptide antibodies. Polysome profiles obtained by fractionation of cytoplasmic extracts of cells producing wt-uS19FLAG (A) or mut-uS19FLAG (designated as uS19Δ) (B) in sucrose density gradients with marked peaks of 40S subunits, 80S ribosomes and polysomes. The results for one of three biological replicates are presented as an example. The numbers of the gradient fractions shown on the X axes of the profiles correspond to the numbers of the lanes on the western blots shown below the panels with the profiles. The content of the ribosomal protein eS4 in the same fractions was determined as a control using anti-eS4 antiserum. Separate lanes “in” (input) correspond to western blot analysis of proteins obtained from aliquots equal to 1/30 of the amounts of extracts applied to sucrose gradients. Note that the western blot with fractions corresponding to mut-uS19FLAG-producing cells (B) was developed using longer exposure time than blot with those corresponding to wt-uS19FLAG-producing ones (A) because of the lower cellular level of mut-uS19FLAG (see above).

should be assigned to elongator tRNAs. Although this analysis does not detect peptidyl-tRNA, two labeled tRNA species should be found in the 80S ribosome fraction if it contains one of two types of complexes differing in the states of tRNAs bound at the A and P sites and the E site occupancy. One is a complex containing two aa-tRNAs (namely, MettRNAiMet at the P site and an elongator aa-tRNA at the A site) before the transpeptidation. Another is a complex with deacylated tRNA (namely, tRNAiMet) at the E site, peptidyl-tRNA at the P site and aa-tRNA at the A site resulting from translocation in the first elongation cycle and subsequent binding of the newly arrived aa-tRNA at the A site. The existence of complexes of the latter type has been shown for translating mammalian ribosomes in vivo [27]. If the analysis reveals only one kind of labeled tRNA, it indicates two possible types of 80S complexes, one of which contains peptidyl-tRNA at the A site and deacylated tRNA at the P site (pretranslocation complex), and another has peptidyl-tRNA at the P site, deacylated tRNA at the E site and the empty A site (posttranslocation complex). To apply the approach described above to answer the question about the effect of deletion of the 15 C-terminal amino acid residues of uS19 on translation elongation, we isolated FLAG-containing 80S ribosomes and 40S subunits (control) from the respective fractions of the polysome profiles (Fig. 3) by immunoprecipitation using anti-FLAG antibodies. As could be seen from Fig. S3, the immunoprecipitation yield was almost quantitative (about 97%) and similar for samples of 80S ribosomes and 40S subunits, which allowed a direct comparison of the tRNA content in the above fractions. The results presented in Fig. 4 show that in all lanes corresponding to 40S subunit fractions, there is a single main tRNA band (obviously corresponding to tRNAiMet), while in the lanes of 80S ribosome fractions, two tRNA bands with comparable intensities are observed. One of these bands represents tRNAiMet (a product with the same electrophoretic mobility as in the 40S subunit lanes), and another corresponds to elongator tRNAs, which is consistent with complexes of 80S ribosomes with aa-tRNA at the A site described above. One can see that the intensities of RNA bands in all lanes corresponding to samples immunoprecipitated from fractions of polysome profiles from cytoplasmic extracts of mut-uS19FLAG-producing cells are significantly weaker compared to those of wt-u19FLAG-producing ones, which is consistent with reduced amounts of 40S subunits and 80S ribosomes containing mut-uS19FLAG in the respective cells (see above). Such a difference was not observed in lanes corresponding to control fractions 40S subunits and 80S ribosomes treated without immunoprecipitation, which was expectable due to the similar total content of ribosomes (endogenous + exogenous) in these two kinds of

Fig. 4. Analysis of tRNAs contained in fractions of 80S ribosomes (lanes 1) and 40S subunits (lanes 2) of polysome profiles obtained from cytoplasmic extracts of cells producing wt-uS19FLAG or mut-uS19FLAG (designated “wt” and “Δ”, respectively). Total RNA was isolated from the above fractions, labeled at the 3′-termini with [32P]pCp and T4 RNA ligase and resolved by denaturing PAGE followed by autoradiography of the gel. Designations “+IP” and “−IP” correspond to fractions subjected to immunoprecipitation using anti-FLAG antibodies or not, respectively. The bands of initiator and elongator tRNAs are designated as “tRNAi” and “tRNAe”, respectively; besides, the positions of the bands of labeled 5S and 5.8S rRNAs in the fractions of 80S ribosomes are shown.

transfected cells. Since immunoprecipitation did not practically change the tRNA patterns of 80S ribosome fractions, one could conclude that the presence of the FLAG-tag in uS19 did not significantly affect the ability of 80S ribosomes to bind aa-tRNA at the A site. The qualitative similarity of the results obtained with the samples of 80S ribosomes containing wt-uS19FLAG or mut-uS19FLAG allowed the conclusion that the deletion of the C-terminal pentadecapeptide fragment of uS19 had no substantial effect on the binding of aa-tRNA at the 80S ribosomal A site in the first elongation cycle. Thus, the deletion of the 15 C-terminal amino acid residues in uS19 only slightly affects the final step of maturation of the 40S subunit in the cytosol, does not impair translation initiation up to the formation of the 80S post-initiation complex, and does not prevent the binding of the first aa-tRNA at the 80S ribosomal A site. However, the deletion completely deprives the 80S ribosome of the ability to form polysomes, which is most likely associated with the elongation stage following the binding of aa-tRNA at the A site.

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4. Discussion

rearrangements underlying the elongation process [28]. In particular, mutations in amino acid residues in positions 104–105 and 112–114 of the yeast uS19 implicated in the formation of the universal intersubunit bridge B1a affect the ribosomal rotational equilibrium and inhibit the binding of elongation factor eEF2 responsible for ribosomal translocation [28]. However, the eukaryote/archaea-specific C-terminal tail is not a component of an intersubunit bridge and does not belong to the conserved globular part of the protein [17] in contrast to the abovementioned uS19 fragments. Therefore, implication of the C-terminal tail of uS19 in the ribosomal rotational equilibrium or eEF2 binding seems highly unlikely. On the other hand, there are several lanes of data mentioned in the Introduction, which clearly indicate that the uS19 tail is involved in the formation of the decoding site. Site-directed crosslinking of the protein to the modified nucleotides in the A site mRNA codon or in the codon immediately downstream of it has been reported with ribosomal complexes where the P site was occupied with a tRNA molecule and the A site was vacant [11–17,29–31]. Occupation of the A site with a tRNA molecule [30] or with translation termination factor eRF1 [31] shields uS19 from cross-linking to mRNA, indicating that the tail of the protein, which is implicated in the cross-link [17], interacts with mRNA mainly only if the A site is vacant. This suggests that when the A site becomes occupied, the uS19 tail switches to interact with the A site ligand fragment located at the decoding area (Fig. 5). Such kind possibility has been discussed in the cryo-EM study, where the major part of the C-terminal tail of uS19 (except the very five C-terminal amino acid residues) had been visualized in various kinds of 80S ribosomal complexes [5]. In the mentioned study, the authors have suggested that the C-terminal tail of uS19 interacts with both the P site and the A site tRNAs, and a hypothesis has been proposed about the stabilizing effect of this tail on the binding of cognate aa-tRNA at the A site at the steps of initial selection and accommodation. Our results do not provide any indication of the significance of the uS19 tail for the Psite tRNA molecule, but suggest a significant role of the tail in the binding of aa-tRNA in the A site in vivo. It is known that aa-tRNA initially binds at the A site in a labile manner before the codon-anticodon interaction is established, which corresponds to the so-called hybrid A/T state. The accommodation of aa-tRNA at the A site, i.e., its transition to the A/A state, where codonanticodon interaction occurs, is critically required for the peptide transfer since the 3′-terminal fragment of aa-tRNA arrives to the ribosomal peptidyl transferase center only when aa-tRNA is the A/A state (for a review, see [32]). Our results show that 80S ribosomes with mutuS19FLAG can form stable complexes containing aa-tRNA at the A site, i.e., those in which there is a codon-anticodon interaction at the decoding site. The incompetence of these complexes in the elongation stages following recognition of the A site codon by a cognate aa-tRNA is most likely due to the inability of this aa-tRNA to accommodate to the A site when uS19 lacks the C-terminal amino acid residues. Accordingly, the crucial importance of the C-terminal tail of uS19 for translation elongation can be related to its direct participation in maintaining the conformation of the codon-anticodon duplex at the A site, favorable for the placement of the 3′-terminal part of aa-tRNA at the peptidyl transferase center. Our statement is in accordance with the above assumption regarding the participation of the C-terminal tail of uS19 in the stabilization of aa-tRNA at the A site, previously made based on cryo-EM data [5]. Recently obtained data on the cross-linking of mRNA analogues carrying 4-thiouridine residues at definite locations to human ribosomes in vitro [33] have shown that indeed uS19 interacts with the A site mRNA codon only if this site is not occupied by the tRNA molecule and that it does not contact the P site codon. The intracellular 4-thiouridine-dependent cross-linking of mRNAs to ribosomes has also not revealed any uS19 contacts with mRNAs in complexes, where the translocation of peptidyl-tRNA from the A site to the P site was blocked by cycloheximide [33]. It seems likely that switching the uS19 tail interaction from the A-site mRNA codon to the anticodon of cognate aatRNA upon accommodation of the latter to the A site makes a

The results obtained with transiently transcfected HEK293T cells producing wt-uS19FLAG or mut-uS19FLAG for the first time made it possible to establish the functional role of the eukaryote/archaea-specific C-terminal tail of the ribosomal protein uS19. We showed that human uS19 with deleted 15 C-terminal amino acid residues was able to participate in the assembly and maturation of 40S subunits (although the share of mature 18S rRNA in them turned out to be somewhat reduced) and in the translation stages associated with the formation of 80S ribosomal complexes containing aa-tRNA at the A site. As mentioned in the Introduction, the C-terminal part of uS19 in pre-40S subunits is located close to the 40S subunit assembly factors Tsr1 and Rio2, which are arranged at the region of the A and P sites and thereby prevent premature binding of initiation factors and tRNA there [18,19]. Therefore, one could expect a strong effect of the above deletion on the ability of the protein to participate in the assembly and/or maturation of 40S subunits. However, the results of this study show that the deletion of the 15 C-terminal amino acid residues of uS19 does not significantly affect the above processes. In particular, the distribution of FLAG-labeled protein between the nucleus and the cytosol is similar for wt-uS19FLAG and mut-uS19FLAG, which implies that uS19 truncation does not prevent the transport of pre-40S subunits from the nucleus to the cytosol. Besides, the rRNA in the cytoplasmic 40S subunits with the truncated uS19 is represented mainly by the mature 18S rRNA, as in those with wt-uS19FLAG. Therefore, a 3-fold increase in the ratio of the content of immature 18S-E pre-rRNA to that of mature 18S-rRNA in 40S subunits containing mut-uS19FLAG, compared with the respective ratio in subunits containing wt-uS19FLAG, seems insignificant. Thus, one can assume that the C-terminal pentadecapeptide fragment of uS19 in the pre-40S subunits does not physically contact the aforementioned assembly factors Tsr1 and Rio2, or, if the corresponding contacts exist, they do not significantly affect the interaction of the factors with the pre-40S subunits. The dramatic effect of the deletion of the 15 C-terminal amino acid residues of uS19 is manifested in the complete inability of the 80S ribosome complexes containing the truncated protein to form polysomes, that is, to participate normally in translation elongation. With all types of 80S ribosomes, including those containing uS19 with the deleted Cterminus, we proved the formation of complexes with two tRNA molecules, initiator and elongator ones. The detection of these tRNAs can indicate the presence in the analyzed samples of two types of complexes corresponding to the states of 80S ribosomes, in which the A site is occupied by aa-tRNA (see above). One is the 80S post-initiation complex with Met-tRNAiMet at the P site, which contains an elongator aatRNA at the A site; another is a complex corresponding to the next elongation cycle, which leads to the appearance of tRNAiMet at the E site and dipeptidyl-tRNA at the P site. Considering that wt-uS19FLAG is able to be incorporated into polysomes, one can suggest that the corresponding 80S ribosome sample is a mixture of the above complexes. At the same time, the sample containing mut-uS19FLAG that is not found in polysome fractions from the respective cells, is most likely the complex with Met-tRNAiMet at the P site and an aa-tRNA at the A site. In general, our findings show that the C-terminus deletion of uS19 does not make the 80S ribosomes defective in binding of aa-tRNA at the A site. Obviously, the effect of this deletion is manifested in other stages of the elongation cycle, namely, peptide bond formation or translocation, although the obtained results do not allow an accurate determination of the elongation stage, at which the process stops after binding of aatRNA at the A site, when the ribosomes contain truncated uS19. Nevertheless, analysis of the available data presented below suggests that the deletion of the 15 C-terminal amino acid residues of uS19 affects the transfer of the Met residue to the A site aa-tRNA, rather than translocation. It is known that several fragments of the C-terminal part of the eukaryotic uS19 are important players in the ribosomal conformational 6

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Fig. 5. Positioning of uS19, and mRNA and tRNA molecules at the A, P and E sites on the 40S ribosomal subunit according to the near-atomic model of the ribosomal complex [5] (PDB ID: 5LZS), where the C-terminal tail of uS19 was resolved better than in other available models of mammalian ribosomes. General view from the interface side of the 40S subunit and zoomed structure of the fragment containing the C-terminal tail of uS19, mRNA codons at the A and P sites and anticodon stemloop fragments of the A site and the P site tRNAs (on the right). uS19 is depicted as magenta cartoon, and its ten C-terminal amino acid residues in positions 131–140 resolved on the model are shown in spheres (the very 5 of theC-terminal amino acid residues of uS19 are not resolved). mRNA is shown as yellow spheres, and tRNA molecules in the A, P and E sites are presented in dark blue, red and green cartoons, respectively. On the bottom, it is presented a zoomed view of the same model where C-terminal amino acid residues of uS19 after that in the position 130 were deleted. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the implication of the C-terminal tail of uS19 in the elongation revealed in this study is associated only with its participation in the rRNA folding since the rRNA arrangements at the 30S and 40S ribosomal decoding sites are very similar [7,34]. Therefore, we have grounds to suggest that the involvement of the C-tail of eukaryotic uS19 in the accommodation of aa-tRNA takes place due to the interaction of the tail with the A site ligand (aa-tRNA and/or eEF1A), which may be either direct or mediated by another ribosomal component. The next frontier is to understand why additional interactions appeared to accommodate aa-tRNA at the ribosomal A site in the course of evolution of the translational machinery from bacteria to eukaryotes.

significant contribution to the structure of the ribosome complex competent in peptide transfer. Thus, the results of this study indicate that in vivo eukaryote/archaea-specific C-terminal tail of uS19 does not participate in the aatRNA binding at the A site, but it is most probably critically implicated in its accommodation there, a feature that has no analogy in bacterial ribosomes. Structural and functional differences between eukaryotic and bacterial ribosomes have been discussed in detail as soon as nearatomic structures of the 40S and 60S subunits have been reported (e.g., see [34]). These differences have been attributed mainly to the solvent sides of the subunits and to several intersubunit bridges; a number of eukaryote-specific tails of ribosomal proteins have been discussed as well. It has been suggested that such kind tails contribute to proper rRNA folding [34]. Likely contacts of some of these tails (but not the tail of uS19) with components of translational machinery derived from the structural studies have been considered (e.g., see [34,35]), but the specific contribution of the tails in particular stages of the translation process has remained mainly unknown. Our results provide the first example of a ribosomal protein tail that plays specific and crucially important role at a particular step of translation. It seems unlikely that

5. Conclusions This study provides the first unambiguous evidence that the long Cterminal tail of uS19, whose sequence has no homology in the bacterial protein uS19, is an important player in elongation of translation in vivo. We show that the C-terminal pentadecapeptide fragment of uS19 is not involved in the assembly and maturation of the 40S subunit, as well as in the initiation of translation, but it is important for the ability of 80S 7

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ribosomes to form polysomes, which suggests its participation in the elongation process. Our findings demonstrate that the deletion of the above uS19 fragment does not prevent the binding of aa-tRNA at the 80S ribosomal A site, which indicates that the deleterious effect of this mutation on elongation is associated with peptide transfer or translocation. Analysis of the currently available data on uS19 in the eukaryotic ribosome allows us to conclude that the protein tail is most likely involved in the interaction with aa-tRNA at the A site to facilitate its accommodation there, which is a prerequisite for the participation of the amino acid residue of aa-tRNA in peptide bond formation. Thus, the results of this study explain the functional significance of the C-terminal tail of the eukaryotic uS19, whose occurrence in the ribosomal decoding area has no analogy in bacteria.

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Authors' contributions A.M. and G.K. planned experiments; K.B. and A.G. performed experiments; all authors were involved in the analysis and discussion of the results obtained in this study; D.G. and G.K wrote the paper. 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. Acknowledgements Russian Foundation for Basic Research [17-04-00609 to G.K.], Russian state funded budget of ICBFM SB RAS [project АААА-А17117020210022-4], Russian Ministry of Science and Higher Education under 5-100 Excellence Programme. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bbagrm.2020.194490. References [1] T.V. Budkevich, J. Giesebrecht, E. Behrmann, J. Loerke, D.J. Ramrath, T. Mielke, J. Ismer, P.W. Hildebrand, C.S. Tung, K.H. Nierhaus, K.Y. Sanbonmatsu, C.M. Spahn, Regulation of the mammalian elongation cycle by subunit rolling: a eukaryotic-specific ribosome rearrangement, Cell 158 (2014) 121–131. [2] S. Matheisl, O. Berninghausen, T. Becker, R. Beckmann, Structure of a human translation termination complex, Nucleic Acids Res. 43 (2015) 8615–8626. [3] J.L. Llacer, T. Hussain, L. Marler, C.E. Aitken, A. Thakur, J.R. Lorsch, A.G. Hinnebusch, V. Ramakrishnan, Conformational differences between open and closed states of the eukaryotic translation initiation complex, Mol. Cell 59 (2015) 399–412. [4] H. Khatter, A.G. Myasnikov, S.K. Natchiar, B.P. Klaholz, Structure of the human 80S ribosome, Nature 520 (2015) 640–645. [5] S. Shao, J. Murray, A. Brown, J. Taunton, V. Ramakrishnan, R.S. Hegde, Decoding mammalian ribosome-mRNA states by translational GTPase complexes, Cell 167 (2016) 1229–1240. [6] D. Graifer, G. Karpova, Roles of ribosomal proteins in the functioning of translational machinery of eukaryotes, Biochimie 109 (2015) 1–17. [7] D. Graifer, G. Karpova, Interaction of mRNA with ribosomes in the course of translation in higher eukaryotes, in: I. Fernandez, L. Jackson (Eds.), mRNA: Molecular Biology, Processing and Function, Nova Science Publishers Inc, New York, 978-1-53613-169-7, 2018, pp. 1–42 (eBook), ISBN: 978-1-53613-168-0 (hardcover). [8] A.G. Hinnebusch, Structural insights into the mechanism of scanning and start codon recognition in eukaryotic translation initiation, Trends Biochem. Sci. 42 (2017) 589–611. [9] D.E. Brodersen, W.M. Clemons Jr., A.P. Carter, B.T. Wimberly, V. Ramakrishnan, Crystal structure of the 30S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA, J. Mol. Biol. 316 (2002) 725–768.

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