Subcellular localization of ribosomal P0-like protein MRT4 is determined by its N-terminal domain

The International Journal of Biochemistry & Cell Biology 42 (2010) 736–748

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Subcellular localization of ribosomal P0-like protein MRT4 is determined by its N-terminal domain Barbara Michalec 1 , Dawid Krokowski 1 , Przemysław Grela, Leszek Wawiórka, Justyna Sawa-Makarska, Nikodem Grankowski, Marek Tchórzewski ∗ Department of Molecular Biology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland

a r t i c l e

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Article history: Received 18 September 2009 Received in revised form 21 December 2009 Accepted 10 January 2010 Available online 18 January 2010 Keywords: Ribosome Protein synthesis Ribosomal stalk P-protein

a b s t r a c t The Mrt4 protein, showing extensive sequence similarity to the ribosomal P0 protein, is classified as a ribosomal P0-like protein and acts as a trans-acting factor which modulates the assembly of the pre60S particle. In this report we investigated the biological nature of the human Mrt4 protein. First, we constructed a series of hybrid hMrt4-P0 proteins by replacing various domains of the P0 protein with corresponding protein fragments from hMrt4. We found that hMrt4 binds to the same site on the large ribosomal subunit as does P0, but despite the sequence homology it is not able to functionally complement the lack of P0. Using fluorescence microscopy and biochemical approaches we also show that hMrt4 occupies predominantly the nucleolar compartment, in contrast to P0 and P1/P2, which are located in the cytoplasm. The nucleolar accumulation of hMrt4 does not depend on a specific nucleolus localization signal, but rather occurs via interaction with established nucleolar components such as rRNA; however, nuclear import of hMrt4 is dependent on a short sequence in the N-terminal part of the protein. Functional analysis with specific inhibitors, actinomycin D and leptomycin B, showed that hMrt4 is a trans-acting factor involved in ribosome maturation, with nucleus-cytoplasm shuttling capacity. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Ribosome maturation in eukaryotic cells is dependent on a large number of trans-acting factors. So far, more than 150 factors have been implicated in the ribosome assembly, which associate with and dissociate from pre-ribosomal particles along the maturation pathway. The first well described precursor particle in ribosome biogenesis is the 90S ribonucleoprotein complex, which is cleaved to the pre-40S and pre-60S intermediate ribosomal particles with independent maturation pathways (Fatica et al., 2002; Fromont-Racine et al., 2003; Tschochner and Hurt, 2003; Johnson et al., 2002). The maturation pathway of the pre-60S particle has been characterized extensively, largely through tandem affinity purifications method of tagged trans-acting factors (Nissan et al., 2002; Harnpicharnchai et al., 2001; Fatica et al., 2002; Saveanu et al., 2003). The pre-60S particle goes through successive maturation steps including rRNA processing/modification, conformational changes, and transport through the nucleolus, the nucleoplasm,

∗ Corresponding author at: Department of Molecular Biology, Institute of Microbiology and Biotechnology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland. Tel.: +48 81 537 59 56; fax: +48 81 537 59 07. E-mail address: [email protected] (M. Tchórzewski). 1 These authors contributed equally. 1357-2725/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2010.01.011

and the nucleopore complex. Since most of the maturation takes place within the nucleus, also most of the trans-acting factors are nuclear and only a few are nuclear/cytoplasmic or strictly cytoplasmic (Zemp and Kutay, 2007). When the pre-60S particles arrive in the cytoplasm, they are not yet competent for translation and their fine tuning is required. Many pre-ribosomal factors have already left the cytoplasmic pre-60S particle, and only a few shuttling factors are present such as Nmd3 (Ho et al., 2000), Tif6 (Basu et al., 2001), Arx1 (Hung and Johnson, 2006), Alb1 (Lebreton et al., 2006), Rlp24 (Saveanu et al., 2003), and Nog1 (Kallstrom et al., 2003). Upon the fine tuning in the cytoplasm these factors are finally released by several strictly cytoplasmic ones, Lsg1 (Hedges et al., 2005), Efl1 (Senger et al., 2001), Drg1 (Pertschy et al., 2007), Rei1 (Hung and Johnson, 2006), Sdo1 (Menne et al., 2007) and Jjj1 (Meyer et al., 2007, Demoinet et al., 2007), a group of GTP/ATP-ases modulating the pre-ribosome to complete the maturation. Among all these early/late maturation factors, i.e. rRNA processing factors, RNA helicases, GTPases, methlytransferases, AAA-type ATPases, and factors required for the intranuclear movement of pre-ribosomes, one group deserves special attention, namely ribosomal-like proteins. This group comprises three well characterized proteins, Imp3 (Lee and Baserga, 1999), Rlp7 (Dunbar et al., 2000), and Rlp24 (Saveanu et al., 2003), which are similar to the ribosomal proteins Rps9, Rpl7 and Rpl24, respectively. It has been postulated that these ribosomal-like factors bind to the sites that later are occu-

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pied by the equivalent ribosomal proteins, but no specific direct interaction between a ribosomal-like protein and pre-rRNA has been documented to date. Imp3 is an important element of U3 snoRNP with critical roles in pre-RNA cleavage events which are important in small subunit (SSU) maturation (Lee and Baserga, 1999). The Rlp7 and Rlp24 maturation factors are involved in large subunit (LSU) maturation. Rlp7 localizes to the granular component of the nucleolus (Gadal et al., 2002) and is required for an early step in the processing of the pre-rRNA to the 5.8S and 25S rRNAs. When the pre-60S leaves the granular component and enters the nucleoplasm, Rlp7 dissociates from it (Nissan et al., 2002; Dez and Tollervey, 2004). In contrast, Rlp24 is regarded as a “core” pre-ribosomal protein associated with most, if not all, of the intermediates that are generated during 60S ribosomal subunit biogenesis and follows the pre-60S precursors from their formation in the nucleolus to the cytoplasm. A fourth ribosomal-like protein has been identified in Saccharomyces cerevisiae using the TAP method, the Mrt4 protein (Harnpicharnchai et al., 2001), which is similar to the ribosomal protein P0. Initially Mrt4 was found as a factor involved in mRNA turnover (Zuk et al., 1999), but a thorough analysis of all TAP data (Fromont-Racine et al., 2003) together with very recent experimental reports (Lo et al., 2009, Kemmler et al., 2009; Rodriguez-Mateos et al., 2009b) have indicated that the Mrt4 protein acts as a pre-60S trans-acting factor. The P0 protein, with which Mrt4 shows similarity, together with P1/P2 and L12 proteins, forms the ribosomal stalk, a structure recognized as the central part of the GTPase-associated-center (GAC), responsible for ribosome-mediated stimulation of translation factor-dependent GTP hydrolysis (Gonzalo and Reboud, 2003). The mechanism of Mrt4 involvement in ribosome maturation has been very recently investigated in S. cerevisiae but there is no any information about its human counterpart. Thus we have undertaken a characterization of the hMrt4 protein in the context of ribosome biogenesis and the ribosomal stalk assembly. The analyses showed that human Mrt4 is a trans-acting factor involved in ribosome maturation with a shuttling capacity between the nucleus and the cytoplasm, which binds to the same site in the ribosomal precursor as its equivalent ribosomal protein, P0.

2. Materials and methods 2.1. Genetic constructs Human DNA was PCR-amplified from a cDNA library from HeLa cells, whereas genes for the yeast Mrt4 and P0 proteins were amplified from genomic DNA of BY4741 strain. PCR reagents were from Fermentas. To obtain fluorescently tagged proteins the primers listed in Table S1 (Supplementary material) were used. The PCR products encoding human and yeast Mrt4 were introduced into the pDs-Red2-C1 or pDs-Red2-N1 vectors (Clontech Laboratories), and the PCR products encoding human ribosomal L12 and Rlp24 proteins were introduced into the pEGFP-C1/pEGFP-N1 vector (Clontech Laboratories). Truncated forms of hMrt4 gene were obtained by the same method as described for the whole gene, but the pEGFP-C1 vector was used. PCR product encoding human fibrillarin protein was introduced into pDs-Red2-C1 vector, SV40-NLS-GFP and hMrt4 94-97-GFP were amplified from pEGFP-C1 vector with an NLS sequence encoded in the primers and DNA fragments were introduced into pcDNA3.1B vector (Invitrogen). All genetic constructs were verified by DNA sequencing. The L9-GFP construct was described previously (Andersen et al., 2005). pEYFP-C1-Fib was a kind gift from Prof. A. Lamond (University of Dundee, Dundee, UK) (Trotta et al., 2003). pEGFP-C1-NMD3wt was kindly provided by Prof. James Dahlberg (Department of Biomolecular Chemistry, University of Wisconsin Madison, WI, USA) (Squatrito et al., 2006).

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pEGFP-C3-Ebp1 was a kind gift from Prof. Massimo Squatrito (European Institute of Oncology, Milan, Italy) (Squatrito et al., 2006). 2.2. Genetic manipulation in yeast cells Hybrid genes were constructed in pCM184 plasmid (ARS1/CEN4, TRP1) (Gari et al., 1997) by ligation of two PCR-amplified coding sequences: hMrt41–118 with P0103–312 ; hMrt41–238 with P0200–312 ; and human Mrt41–221 with P0200–312 , additionally, full hMrt4 gene and hMrt4 lacking the last 17 amino acids were subcloned in the same vector. Briefly, hMrt4 DNA fragments were PCR-amplified with primers listed in Table S1 (Supplementary material), introducing BglII and NotI restriction sites at the 5 and 3 ends, respectively. The DNA fragments were introduced into the pCM184 BamHI and NotI sites. Then, DNA fragments encoding truncated P0103–312 or P0200–312 were amplified with specific primers, with NotI and PstI restriction sites and introduced into the same vector bearing sequence respective hMRT4 constructs. As a control full P0 gene was reconstructed from two parts of P0, 1–102 and 103–312, using the same approach. All genetic constructs were verified by DNA sequencing. Yeast manipulations were performed on the C12 yeast strain having the pUG36-EGFP-P0 construct, complementing the lack of the genomic gene for the P0 protein (MAT˛, his31; leu20; lys20; ura30; rpp0::KanMX4, (CEN6/ARSH4, URA3, PMET25 -EGFPRPP0)). The strain was transformed with pCM184 plasmids carrying the gene of interest under the tetO-PCYC1 inducible promoter. The cells were grown for 12 h on YPD in the presence of methionine (100 mM) in order to repress expression of the wild-type P0, and subsequently spoted on SD medium containing 5-fluoroorotic acid (5-FOA) to get rid of pUG36-EGFP-P0 vector. The expression of hybrid proteins was verified with the aid of Western blotting using antibodies against yeast P0 or hMrt4. In cases when the genetic constructs were unable to complement the P0 null mutation, expression of the hybrid protein was tested in the W303-1b strain after selection on SD medium, and the expression level of hybrid proteins was probed with specific antibodies. 2.3. Cell cultures and transient transfection NIH3T3 cells were cultured in a humid atmosphere of 5% CO2 at 37 ◦ C in Dulbecco’s minimum essential medium (DMEM) supplemented with GlutaMax, non-essential amino acids, 10% bovine serum, 100 IU/ml penicillin and 100 ␮g/ml streptomycin (Gibco, Invitrogen). DNA constructs were transiently transfected into NIH3T3 cells using FuGENE (Roche). In brief, 5 × 104 cells were plated on a glass coverslip placed in a 35 mm tissue culture dishes. After 24 h, cells were transfected with 1.2 ␮g of DNA according to the manufacturer’s instruction. Before microscopic observations cells were fixed with 4% formaldehyde (Sigma), permeabilized with ice-cold acetone and washed several times with PBS. Nucleus was stained with 2 ␮g/ml Hoechst 33342 (Serva). When indicated, 1 day after transfection, cells were treated with 2.5 nM leptomycin B (LMB) (Sigma) or 50 ng/ml actinomycin D (ActD) (Sigma), and incubated for a further 16 h. For recovery experiments after treatment with ActD, the cells were washed with fresh medium without ActD and cell visualization was performed after 8 h. 2.4. Preparation of specific antibodies Coding sequences for human Mrt4 protein (hMrt4) or truncated form of human P0 (hP0C(1–293) ) were amplified from HeLa cDNA library using primers listed in Table S1, and introduced into pGEX4T-1 vector (Amersham Biosciences). The constructs were verified by DNA sequencing. Expression of the GST-hMrt4 and GSThP0C proteins was performed in E. coli BL21(DE3)RIL (Clontech). The recombinant fusion proteins were purified on a GST-Trap col-

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umn (Amersham Biosciences) according to the protocol provided by the supplier. The GST-tag was cleaved off with bovine thrombin (Sigma) (10 U thrombin/1 mg protein, 1 h, 37 ◦ C). The obtained recombinant proteins were resolved on SDS-PAGE and further purified by electroelution from the gel. The identity of the electroeluted proteins was confirmed by mass spectrometry. Heterodimer of the human P1/P2 proteins was purified as described previously (Grela et al., 2008). Antiserum directed against hMrt4, hP0C(1–293) or hP1/P2 was prepared as follows: 1 mg of the protein or protein complex was mixed with an equal volume of Freund’s complete adjuvant (Sigma–Aldrich) and the emulsion was injected into rabbits intradermally. After 4 weeks, the rabbits were boosted with 0.5 mg of emulsified recombinant proteins using incomplete Freund’s adjuvant. After another 2 weeks the procedure was repeated. Serum was collected 10 days after the third immunization. Specific antibodies were further purified using affinity chromatography, where proteins used for immunization were immobilized on nitrocellulose membrane (Whatman). 2.5. Immunological techniques Subcellular fractionation was performed as described previously (Tchorzewski et al., 2003), with the following modification: cells were lysed in dezintegration buffer (50 mM Tris pH 7.5, 80 mM KCl, 12.5 mM MgCl2 , 0.5 mM EDTA, 1% Triton X-100) and cell extract was centrifuged: 30 min at 16,000 × g to obtain the total cell extract, and then for 2.5 h at 100,000 × g in a 70.1 Ti rotor (Beckman) for ribosome isolation. Ribosomes were resuspended in buffer (50 mM Tris pH 7.5, 80 mM KCl, 10 mM MgCl2 , 0.5 mM EDTA, 5 mM DTT and 50% glycerol). Immunoprobing was performed with polyclonal rabbit anti-yeast-P0 sera (Krokowski et al., 2002), rabbit anti-human-P0C(1−293) , anti-human-P1/P2 or anti-human-hMrt4. Immunolocalization of the proteins was performed using a standard procedure; briefly, NIH3T3 cells were grown on coverslips and fixed as described above. Coverslips were incubated for 1 h at room temperature (r.t.) with appropriate antibodies diluted 1:1000 in 2% BSA (Sigma) in PBS. After washings with PBS, Alexa-Fluor 488-conjugated secondary antibody was added (1 ␮g/ml at r.t. for 1 h) (BD Bioscience). Stained cells were examined using an inverted microscope (Olympus CKX-41) equipped with epifluorescence filter sets and photographed using a digital camera (Olympus). Digital images were collected using QuickPHOTO PRO 2.0 program and processed with Corel Photo-Paint 12. Immunoelectron microscopy was performed using HeLa cells. The cells were scrapped off, fixed with 4% glutaraldehyde in 100 mM cacodylate buffer and postfixed in 1% osmium tetroxide (Sigma). Then, the cells were dehydrated in a series of ethanol and acetone and embedded in LR White resin (Sigma). Ultrathin sections were cut and collected on nickel grids. The specimens were washed with PBS and incubated for 1.5 h with rabbit anti-human hP0C(1–293) polyclonal antibodies diluted 1:50 or rabbit anti-hMrt4 polyclonal antibodies diluted 1:25 in PBS. Pre-immune serum was used as a negative control. After thorough washing with PBS, secondary antibody conjugated with 10 nm gold particles (Sigma) was used in 1:5 dilution. Grids were washed with pure water, dried and examined in a transmission electron microscope (Leo, Zeiss). 3. Results 3.1. The P0 and Mrt4 proteins have the same binding site on the ribosome The Mrt4 protein has been classified as a ribosomal P0-like protein because of the sequence similarity to the ribosomal P0 protein. To study the mode of human Mrt4 protein action, as a first approach

we examined the ribosomal binding site for the human Mrt4 protein (hMrt4). A sequence alignment showed that the Mrt4 and P0 proteins are similar in their rRNA-binding domains, suggesting that they have the same binding site on the 60S subunit (Fig. 1A). To check if indeed the hMrt4 binds the same rRNA fragment as the P0 protein does, a hybrid protein was constructed in a way that the rRNA-binding domain of yeast P0 was replaced by a similar domain from hMrt4. The hybrid protein hMrt41–118 -P0103–312 (Fig. 1B) was expressed in yeast lacking wild-type P0, which is indispensable for cell survival (Fig. 2). The strain expressing the hybrid protein was viable, indicating that the analyzed protein construct complemented the lack of the wild-type P0. However, the growth rate of the hMrt41–118 -P0103–312 -expressing strain was significantly diminished, the doubling time being 220 min vs. 95 min of the BY4741 reference strain and 100 min of a strain with plasmidborne P0 expression. The slow growth was probably related to the fact that the hybrid protein bound to the rRNA was less stably, as it could be easily washed off from the ribosomes by salt treatment (data not shown). This result was verified by shifting yeast cells with the hybrid protein hMrt41–118 -P0103–312 onto YPD plate containing tetracycline as a repressor of the hybrid gene expression. In this case no growth was observed, which confirmed that the hybrid protein is necessary for survival of cells lacking native P0 (Fig. 2A). The presence of the chimeric protein in the whole ribosomal fraction and thus presumably bound to the ribosome was verified by Western blotting using antibodies against yeast P0, and also appropriate protein band was cut out from SDS-PAGE and analyzed by mass spectrometry (Fig. S1). 3.2. hMrt4 by itself cannot support protein biosynthesis in yeast cells lacking P0 protein Despite substantial homology between yeast and human P0 proteins, the Mrt4 counterparts seem less closely related. There is no extensive amino acid homology between these two proteins moreover, some significant differences are apparent, especially at the C-terminus (Fig. 1). To check if the human and yeast Mrt4 proteins have retained homology, the mrt4 deletion yeast strain, which exhibits slow growth phenotype, was complemented with hMrt4 protein. As shown in Fig. 3A, the human counterpart was able to rescue the growth of the mrt4 mutant strain, but not fully. The yeast and human Mrt4 proteins show sequence similarity to the P0 protein, not only within the rRNA-binding domain; additionally, a cluster of about 65 amino acids at the C-terminal part of the hMrt4 proteins is similar to P0 (Fig. 1A): regions 147–213 in yeast and 160–226 in human Mrt4 proteins are similar to regions 129–195 and 131–197 in the yeast and human P0 proteins, respectively. This P0 region is probably the interacting interface with translation factors (Santos et al., 2004). Moreover, in the amino acid sequence of hMrt4 an additional cluster of acidic amino acids is present, resembling the C-terminus of the P-proteins generally regarded as their functional part (Fig. 1A). Since yeast growth can be supported by a minimal stalk structure containing only the P0 protein (Santos and Ballesta, 1995), and the rRNA-binding domain of hMrt4 does bind to the yeast ribosome and is able to functionally replace the rRNA-binding domain of P0, it was interesting to see whether hMrt4 could support yeast growth in the absence of the P0 protein. Upon removal of the plasmid encoding yeast P0 protein, no cell growth was observed in a yeast strain, in which full hMrt4 was expressed. A truncated form of hMrt4 lacking the C-terminal part (17 amino acids) (hMrt4C, Fig. 1B), also failed to support the yeast growth (Fig. 3). Both hMrt4 variants were expressed in a control strain, as shown in Fig. 3B. These results indicate that the hMrt4 protein is unable to support ribosome function in yeast lacking the P0 protein, despite having a C-terminus resembling that of the P-proteins. To exclude the possibility that the lack of a P-domain with a bind-

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Fig. 1. Multiple sequence alignment of P0 and Mrt4 proteins from S. cerevisiae and H. sapiens. (A) Alignment was performed on amino acid sequences of yeast Mrt4 (yMrt4) and P0 (yP0) and their human counterparts hMrt4 and hP0 using ClustalW. rRNA-binding domain and translation factor (TF) binding domain are framed. Bold font marks putative NLS of Mrt4 proteins predicted by analysis with PSORT II. Tetrapeptide of basic amino acids likely to be involved in nuclear localization of truncated hMrt4 is underlined. Cluster of acidic amino acids at the C-terminus of hMrt4 is underlined with a wavy line. “*” below the alignment marks residues identical in all sequences. “:” marks conserved substitutions, “.” marks semi-conserved ones. (B) Schematic representation of hMrt4 and P0 hybrid constructs: rRNA-BD, rRNA-binding domain; TF-BD, translation factor-binding domain; P-D, P domain; C, conserved cluster of acidic amino acids. Numbers indicate position of amino acids.

ing site for P1/P2 proteins makes hMrt4 unable to support protein biosynthesis in yeast, additional genetic constructs were prepared; hMrt4 or hMrt4C were fused with region 200–312 of the P0 protein harboring the binding site for the P1/P2 proteins (Fig. 1B). The two hybrid proteins were expressed in the absence of P0 protein, and as it was the case for the wild-type hMrt4, the hybrid proteins did not support the growth of the yeast. It should be noted that the hybrid proteins were efficiently synthesized in control wild-type yeast (Fig. 3B). 3.3. Subcellular localization of the hMrt4 protein There are three types of trans-acting factors in respect to their localization in the cell, namely nuclear, nuclear/cytoplasmic (shut-

tle factors), and cytoplasmic, it was therefore of interest where hMrt4 is localized in the cell. For this analysis a fusion protein construct was used, where hMrt4 was attached to the fluorescent marker DsRed, yielding the DsRed-hMrt4 fusion protein. The appropriate genetic construct was transiently tranfected into NIH3T3 cells, and the cells were observed after 24, 48, and 72 h after transfection. Initially (24 h after transfection) the protein was present mainly in the nucleolus (Fig. 4A), but upon prolonged cell growth (48 or 72 h after transfection) the signal was also detected in the cytoplasm, with nucleoli still well stained. The results were the same for N- and C-tagged hMrt4 (data not shown). The nucleolar localization of hMrt4 was confirmed by co-transfection of NIH3T3 cells with two genetic constructs, YFP-fibrillarin and DsRed-hMrt4 (Fig. 4B). Expression of full-length DsRed-hMrt4 fusion protein was

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Fig. 2. Complementation of yeast P0 protein by hybrid protein hMrt4(1–118)-P0(103–312). (A) Growth of yeast strains: (1) BY4741 (MAT˛; his31 leu20 ura30 met150), (2) pCM184-P0 (MAT˛; his31; leu20; lys20; ura30; rpp0::KanMX4, (CEN4/ARS1, TRP1, PCYC1/tetO -P0), and (3) pCM184-hMrt4(1–118)-P0(103–312) (MAT˛, his31; leu20; lys20; ura30; rpp0::KanMX4, (CEN4/ARS1, TRP1, PCYC1/tetO -hMrt4(1–118)-P0(103–312))) on YPD plates and after tetracycline (TET) dependent promoter repression (YPD + TET). Plates were incubated for 4 days at 30 ◦ C. (B) Cells carrying the indicated plasmids were spotted in serial 10-fold dilutions onto YPD plates and incubated at 30 ◦ C for 3 days. pCM184-P0 and pCM184-P0(1–102)-P0(103–312) drive expression of wild-type P0 and P0(1–102)-P0(103–312) a reconstructed P0 with a short linker, present in hMrt4-P0 hybrid protein. (C) SDS-PAGE and immunoblotting of ribosomal fractions toward detection of wild-type P0 protein (pCM184-P0) and hybrid protein pCM184-hMrt4(1–118)-P0(103–312). Arrows indicate position of P0 and hybrid protein.

confirmed by Western blotting with specific antibodies recognizing the fluorescent protein, and the same type of fractionation was performed for non-transfected cells and probed with specific antibodies recognizing hMrt4. In both cases, hMrt4 or its fusion with DsRed was found in the cytoplasmic, ribosomal, and nuclear fractions (Fig. S1). The presence of DsRed-hMrt4 and wild-type hMrt4 in the ribosomal fraction indicates that the protein is integrated into pre-60S particles which are exported to the cytoplasm. The subcellular localization was further verified using transmission electron microscopy. Nano-gold immunostaining with specific anti-hMrt4 antibodies labeled the nucleolar compartment supporting the observations with the DsRed-hMrt4 fusion protein (Fig. S2). Additionally, to test whether the localization of hMrt4 is conserved, the yeast Mrt4 protein was analyzed in the mammalian system. The chimeric DsRed-yMrt4 protein displayed the same pattern of localization as observed for the human counterpart (Fig. 4C). 3.4. Subcellular localization of stalk elements Although several reports have indicated that the P0 protein may be found in the nucleus and its presence there was associated with so-called extra-ribosomal function (Yacoub et al., 1996; Frolov and Birchler, 1998; Borden et al., 1998) the nuclear localization of this protein is still controversial. In order to resolve this issue, we analyzed the subcellular localization of human P0 using specific antibodies which recognize P0 only, without cross-reactions with the P1/P2 proteins. In situ immunofluorescence showed that the steady-state signal comes from the cytoplasm (Fig. 5A). Additionally, Western blotting of cell fractions showed that the P0 protein is found in the cytoplasm (Fig. S1). These data were confirmed with transmission electron microscopy with anti-P0 antibodies, showing that P0 occupies exclusively the cytoplasmic compartment (Fig. S2). The subcellular localization of human P1/P2 proteins was also analyzed using specific antibodies; also in this case the cytoplasm was the only localization (Fig. 5A). It should be added that

behavior of P proteins is resistant to ActD and LMB. Neither compound affected P proteins cell distribution thus confirming that the P proteins are not actively transported to the nucleus (Figs. 7 and 8). Combined, these data show that all P-protein are found in the cytoplasm, this in turn indicates that they become incorporated into the ribosome only after its precursor leaves the nucleus. The stalk, apart from the P-proteins which form its main body, contains the L12 protein in the stalk base, together with the P0 protein. As it was shown earlier, the interplay between L12 and P0 is important in stalk formation (Garcia-Marcos et al., 2008), therefore knowing the cellular localization of L12 should provide information about the stalk assembly. To this end, localization of the GFP-L12 chimeric protein was studied. The protein was found in the nucleoli and in the cytoplasm of NIH3T3 cells (Fig. 5B). The nucleolar localization of L12 and its similar behavior to hMrt4 in the presence of LMB and ActD (Figs. 7 and 8) indicates that at an early step of pre-60S maturation this protein may functionally and/or structurally interact with Mrt4, because these two proteins share the same cellular compartment and are in close proximity by occupying the same region of 28S rRNA. 3.5. Mapping of hMrt4 nuclear localization signal Having observed nucleolar localization of hMrt4, we next ask which part of the protein is responsible for its nuclear import. Analysis of the yeast and human Mrt4 sequences with PSORT program (Nakai and Horton, 1999) revealed the presence of a bipartite nuclear localization signal (NLS) in the N-terminal part of the yeast and human proteins. The N-terminal extension with a predicted NLS is characteristic for Mrt4 proteins and is absent in P0 (Fig. 1). This information and the identification of an rRNAbinding site led us to design truncated mutants of hMrt4 to identify elements important for its nuclear import. We divided the protein in two parts: the N-terminal half harboring the rRNA-binding domain with a putative NLS (residues 1–120), and the C-terminal one (121–239) (Fig. 6A). The fusion protein GFP-hMrt41–120 had

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N-terminal half of hMrt4. Next, the localization of fusion proteins with deletion of a part or the whole predicted bipartite NLS (hMrt411–239 , hMrt420–239 , hMrt450–239 ) was studied. Surprisingly, in all these cases we saw incrustation of the nucleolus with the fusion proteins, but a high level of florescence was observed in the cytoplasm as well, indicating that the N-terminal extension characteristic for hMrt4 with the predicted NLS is not absolutely required for nuclear import, but may have only a minor influence (Fig. 6B). Further truncation of the protein showed that a sequence required for the protein targeting to the nucleolus is present in the region between residues 50 and 79, because its removal (GFPhMrt480–239 ) abolished the incrustation of the nucleolus by the hybrid protein without impeding its import into the nucleoplasm (Fig. 6B and Fig. S3). Unexpectedly, at the same time export of the hybrid protein to the cytoplasm was completely abolished. This shows that upon disruption of the rRNA-biding domain located in the region 1–79, the hybrid protein is no longer integrated into pre60S, its export with maturating 60S is hampered, and therefore it remains in the nucleoplasm. The latter observation indicated that an additional NLS must be preset within the region 80–120. The only sequence resembling an NLS in this region is the tetrapeptide 94 KRLR97 . To verify this assumption, we fused regions 80–120 or 94–97 to the GFP reporter protein. Indeed, hybrid proteins with those amino acid regions were imported into the nucleus (Fig. S4), showing that the short tetrapetide may contribute to the nuclear import of the Mrt4 protein, however, less efficiently in comparison to a control strong NLS from SV40 large T antigen (Kalderon et al., 1984). 3.6. hMrt4 in ribosome biogenesis

Fig. 3. Complementation analysis of yeast P0 protein by various forms of hMrt4. (A) Complementation of yeast deletion strain mrt4 (Mat˛; his31; leu20; met150; ura30; YKL009w::kanMX4) with human Mrt4 protein. Cells carrying the indicated plasmids were spotted in serial 10-fold dilutions onto YPD plates and incubated at 30 ◦ C; BY4741—wild-type strain. (B) Immunodetection of hMrt4 hybrid proteins in mutant yeast cells using specific anti-hMrt4 and anti-P0 antibodies. (C) Growth of yeast mutants expressing the hybrid proteins. All mutants were derived from C12 yeast strain (MAT˛, his31; leu20; lys20; ura30; rpp0::KanMX4, (CEN6/ARSH4, URA3, PMET25 -EGFP-RPP0)), and protein constructs were expressed from pCM184 vector (CEN4/ARS1, TRP1, PCYC1/tetO ). Yeast cells were cultivated to OD600 = 0.1 at 30 ◦ C and serially diluted by a factor of 10. Aliquots of each dilution were applied to FOA plates and cultivated at 30 ◦ C for 5 days.

a distribution pattern similar to that of the whole DsReD-hMrt4 molecule, whereas the fusion containing the C-terminal part of hMrt4 only (GFP-hMrt4121–239 ) was exclusively present in the cytoplasm (Fig. 6B). This result shows that the element(s) responsible for the nuclear/nucleolar localization is indeed localized in the

The involvement of hMrt4 in ribosome biogenesis and its behavior in the cell were analyzed in the presence of two compounds, actinomycin D (ActD) and leptomycin B (LMB). At low concentrations, ActD is a specific inhibitor of RNA polymerase I (Perry and Kelley, 1970), and blocks rRNA synthesis, while LMB blocks nuclear export of pre-60S to the cytoplasm (Ho et al., 2000). Therefore, very early ribosome maturation events are hampered by ActD and very late ones by LMB. Upon addition of ActD, hMrt4 was retained in the nucleoplasm with concomitant disappearance of nucleoli; withdrawal of ActD restored the nucleolar localization of hMrt4 (Fig. 7A). Earlier (see Section 3.5) we showed that GFPtagged hMrt480–239 , where rRNA-binding domain was disrupted, similarly showed nucleoplasmic localization. Taken together, those results clearly show that the nucleolar accumulation of hMrt4 is rRNA-dependent. When the rRNA is absent (as caused by ActD action) or the rRNA-binding domain of hMrt4 is disrupted (as in the GFP-tagged hMrt480–239 construct), the protein is no longer addressed to the nucleolus. ActD did not exert any effect on the localization of P0 and P1/P2 proteins, underscoring the fact that the P-proteins are not imported into the nucleus (Fig. 7B). Another protein which forms the base of the stalk, L12, was miss-localized in the nucleoplasm upon ActD treatment, similarly to Mrt4 (Fig. 7B). Several known trans-acting factors were also analyzed under the same conditions, such as Fib, Nmd3, Rlp24 and Ebp1, and additionally the L9 protein. In all the cases, the effect of ActD was as expected, where shuttle factors (Nmd3, Rlp24) were retained in the nucleoplasm, whereas Ebp1, regarded as a nuclear export adaptor, remained in the cytoplasm. Fib localization was not sensitive to ActD and protein retained in the nucleolus. L9, a structural ribosomal protein was in the nucleoplasm (Fig. S5). Interesting information was brought by the analysis with LMB; under conditions where Nmd3, Rlp24, L9 were miss-localized in the nucleus and Ebp1 localized in the cytoplasm (Fig. S5), hMrt4 accumulated in the nucleus, similarly to Nmd3, Rlp24 and L9 (Fig. 8A). It should be noted, however, that the behavior of hMrt4 was

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Fig. 4. Cellular localization of hMrt4 protein in NIH3T3 cells. hMrt4 was analyzed as a fusion with the fluorescent protein DsRed in transiently transfected NIH3T3 cells. (A) Distribution of DsRed-hMrt4 in fixed cells, 24, 48 and 72 h after transfection. Panel on the left shows red fluorescence of the hybrid proteins, in the center Hoechst-stained nucleus, on the right contrast phase view of the whole cell. (B) Co-transfection with DsRed-hMrt4 and nucleolar specific marker, fibrillarin fused to fluorescent protein YFP (Fib-YFP): red fluorescence, DsRed-hMrt4; green fluorescence, Fib-YFP; yellow, merged images of DsRed-hMrt4 and Fib-YFP. Inset, merged picture of Hoechst staining of nucleus and phase contrast cell image. (C) Distribution of yeast yMrt4 protein fused to DsRed (DsRed-yMrt4). Cells were fixed 24 h post-transfection. Nucleus stained with Hoechst and the cell in phase contrast are also shown. Bars, 40 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

heterogeneous and some cells displayed cytoplasmic accumulation; P0 and P1/P2 localization was unaffected by LMB and they remained in the cytoplasm (Fig. 8B), once again showing that these proteins occupy the cytoplasm. Another stalk element, L12 ribosomal protein, accumulated in the nucleoplasm, similarly to L9 (Fig. 8B).

4. Discussion The advent of proteomic analyzes of the pre-ribosomal particles in the turn of the century have provided an integrated view of ribosome maturation, including identification of numerous transacting factors involved in this process. Among them, a group of

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Fig. 5. Localization of ribosomal stalk constituents in NIH3T3 cells. (A) Immunodetection of human P0 and P1/P2 proteins with primary antibodies specific for the human P0 or P1/P2 proteins. Secondary antibodies were conjugated to Alexa-fluor 488. Nucleus stained with Hoechst and cells in phase contrast are also shown; (B) Localization of human L12 protein fused with green fluorescent protein (L12-GFP) after transient transfection. Bars, 40 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

factors similar to ribosomal proteins, described as ribosomal-like, were identified, including Mrt4, the ribosomal P0-like protein, the subject of this study (Lee and Baserga, 1999; Dunbar et al., 2000; Saveanu et al., 2003). The ribosomal-like proteins are supposed to bind to pre-rRNA, later to be replaced by the ultimate ribosomal counterpart on the mature ribosome. Mrt4 was initially identified in yeast cells and connected to mRNA turnover (Zuk et al., 1999), later on its presence on the pre-60S subunit was reported (Andersen et al., 2005), and very recently yeast Mrt4 has been directly linked to pre-60S maturation (Lo et al., 2009; Kemmler et al., 2009; Rodriguez-Mateos et al., 2009b). A comparison of the primary structures of Mrt4 and P0 proteins showed two highly conserved regions. The N-terminal domain of Mrt4 resembles the rRNA-binding domain found in bacterial and eukaryal L10 and P0 proteins, and the C-terminal domain similar to the internal domain of P0 responsible for interaction with translation factors. Additionally, in the mammalian Mrt4 protein, a cluster of acidic amino acids is present at the very C-terminus, resembling that found in P-proteins. As shown before, the N-terminal domains of prokaryotic and eukaryal/archeal L10/P0 proteins can be functionally swapped (Santos and Ballesta, 2005; Uchiumi et al., 1999). It was therefore likely that the N-terminal domain of Mrt4 could functionally replace the 26S rRNA-binding domain of P0. To verify this conjecture, we constructed a chimeric protein (hMrt41–118 -P0103–312 ), in which the rRNA-binding domain of the yeast P0 protein was replaced by the homologous part of human Mrt4. A P0 null yeast mutant expressing the fusion protein was viable, but displayed a slow growth phenotype, and the hMrt41–118 P0103–312 protein was integrated into the ribosome. One should keep in mind that P0 is the central organizer of GAC, which in turn

is vital for ribosome function; it seems hardly likely that this crucial part of the ribosome could be organized in a wrong way, in the case of hMrt41–118 -P0103–312 protein. This indicates that the rRNAbinding domain of hMrt4 is able to interact with the rRNA region responsible for P0 integration, and suggests that the Mrt4 occupies the same region of rRNA in pre-60S. This suggestion is supported by the very recent data showing that the yeast Mrt4 interacts with 26S rRNA, and that the yMrt4 and P0 proteins are unable simultaneously bind to pre-60S (Rodriguez-Mateos et al., 2009a). We also found that hMrt4 can partially complement the lack of yMrt4, indicating that their function was preserved throughout the evolution, confirming a recent observation (Lo et al., 2009). The sequence similarity between hMrt4 and P0 is not limited to the rRNA-binding domain, but extends to a region that in P0 is responsible for translation factor-binding, and also includes a short C-terminal cluster of acidic amino acids which are indispensible for P0 function. Thus, we checked whether hMrt4 could substitute for the yeast P0 on the ribosome. Despite this sequence similarity, wild-type hMrt4 could not support yeast growth in the absence of yeast P0. What is more, hybrid proteins in which the P domain of P0 was added to hMrt4 were also unable to complement the lack of P0. These results show that despite their similarity, hMrt4 and P0 play different roles—one in ribosome assembly and the other in the functioning of the mature ribosome. This functional divergence is reflected in the cellular distribution of the two proteins. While Mrt4 is mainly localized to the nucleolus, P0 and its P1/P2 protein partners are primarily cytoplasmic. However, it should be mentioned that human P0 has also been reported in the nuclear compartment (Borden et al., 1998), presumably in connection with an extra-ribosomal function, and our previous work also showed presence of a small fraction of P0

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Fig. 6. Subcellular distribution of truncated hMrt4. (A) Scheme of truncated variants of hMrt4 as hybrid proteins with GFP. Wild-type hMrt41–239 , and truncated mutants: hMrt41–120 , hMrt4121–239 , hMrt411–239 , hMrt420–239 , hMrt450–239 , hMrt480–239 . (B) Intracellular distribution of truncated mutants of hMrt4 fused to GFP in NIH3T3 cells 24 h after transient transfection. Center, nucleus stained with Hoechst, cells in phase contrast are shown to the right panel. Bars, 40 ␮m.

in the nucleus, but interestingly without its P1/P2 protein partners (Tchorzewski et al., 2003). Theses inconsistencies are probably caused by different specificity of antibodies used for the two analyses. Previously, human auto-antibodies from individuals suffering from an autoimmune disease were used. Currently, using rabbit anti-P0 antibodies, we could not detect P0 in the nucleus. Appar-

ently, the rabbit antibodies do not recognize the nuclear form of P0, possibly due to steric hindrance, or amount of nuclear P0 is below detection threshold. This observation raises an interesting possibility in the light of three very recent publications which showed that P0 might join the pre-60S in the cytoplasm and/or in the nucleus (Lo et al., 2009; Kemmler et al., 2009; Rodriguez-Mateos et al., 2009b). Consequently, the presence of different forms of P0 in the cytoplasm and in the nucleus may lead to two alternative pathways of stalk assembling on the pre-60S. In contrast to the P-proteins, the steady-state distribution of another stalk protein, L12, was nucleolar/cytoplasmic, resembling the behavior of other ribosomal proteins (Jakel and Gorlich, 1998). As it was shown before, there is a tight interplay between proteins which form the base of the stalk (Rosendahl and Douthwaite, 1995), and especially L11/L12 enhance the binding of L10/P0 to rRNA (Iben and Draper, 2008). Therefore, L12 together with Mrt4 may represent important elements in the rRNA structure formation of the so-called thiostrepton loop, involved in the stalk binding. Having localized the hMrt4 protein mainly in the nucleolus, next we addressed the mechanism of its nuclear import. It is generally accepted that import to the nucleus is directed by nuclear localization signal (NLS) recognized by importins (Jans et al., 2000). In the case of ribosomal proteins many examples of typical (Annilo et al., 1998; Timmers et al., 1999; Russo et al., 1997) and atypical NLSs (Timmers et al., 1999; Stuger et al., 2000) have been reported. In contrast to the nuclear import, the mechanism by which proteins accumulate in the nucleolus is poorly understood. Specific nucleolar targeting signals (NuLS) composed of basic amino acid clusters are present in retroviral proteins, but they are not generally found in nucleolar proteins (Hatanaka, 1990). Since the nucleolus is not a membrane-isolated structure, it is presumed that nucleolar accumulation occurs via interactions with established nucleolar components, such as rRNA (Olson and Dundr, 2005). For ribosomal proteins the nucleolar localization signal could be their rRNA-binding domain, which should not be considered as a localization signal sensu strict (Houmani and Ruf, 2009). Interestingly, examination of the Mrt4 sequence showed that in the N-terminal extension of the Mrt4 proteins, absent in the ribosomal protein P0, a putative NLS can be found 4 KRDKKVSLTKTAKK18 (Fig. 1), which could represent a bipartite NLS sequence with two clusters of basic amino acids separated by seven residues (Dingwall et al., 1988). The first part of the sequence, 4 KRDKK8 , could even represent a classical monopartite NLS fitting the consensus (KR/KXR/K) detected in a significant number of NLSs (Chelsky et al., 1989). However, our deletion experiment showed that this particular sequence is not required for nuclear import, because the GFP-Mrt420–239 fusion protein lacking this putative NLS was still able to enter the nucleus, although its cytoplasmic abundance was enhanced in comparison with that seen for a full-length construct. Deletion of the first 80 amino acids abolished the nucleolar localization without, however, affecting the nuclear import of hMrt4, indicating that with the rRNA-binding domain disrupted the protein is no longer capable of nucleolar accumulation. This observation suggests that the mechanism responsible for the nucleolar targeting of Mrt4 is similar to that found for a number of ribosomal proteins and relies on an interaction with rRNA. Deletion of an additional 40 residues completely abolished the nuclear import of the hybrid protein, indicating that within the region of amino acids 81–120 an additional NLS is present. The only sequence within this region resembling a NLS is the tetrapeptide 94 KRLR97 (underlined in Fig. 1A). Fusion of the 81–120 region to the reporter protein GFP showed that a NLS is located in this polypeptide indeed, and the predicted tetrapeptide was confirmed to be a functional NLS, because the hybrid construct Mrt494–97 -GFP was imported into the nucleus. The steady-state distribution of the hMrt4 protein, primarily nucleolar with cytoplasmic incrustation, raises the question

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Fig. 7. Localization of hMrt4 and ribosomal stalk constituents in NIH3T3 cells after ActD treatment. Eighteen hours after transfection cells were transferred for 16 h into fresh medium without or with actinomycin D (50 ng/ml ActD). (A) Subcellular distribution of fusion protein DsRed-hMrt4. For recovery experiment, cells were washed from ActDcontaining medium and fresh medium was added. (B) Subcellular distribution of ribosomal stalk constituents. Cells were fixed/permeabilized with 4% formaldehyde/acetone and localization of P0 and P1/P2 was performed by indirect immunofluorescence with specific primary antibodies against the human P0 or P1/P2 proteins, followed by secondary antibody conjugated to Alexa-fluor 488. The L12 protein was analyzed as L12-GFP fusion construct and visualized in the same way as for hMrt4. Nucleus stained with Hoechst and cells in phase contrast are also shown. Inset (c), control experiment, without ActD treatment. Bars, 40 ␮m.

regarding the mechanism of such localization. The ActD experiment and the effect of the N-terminal deletions in hMrt4 (GFP-hMrt480–239 ) suggest that the nucleolar localization of Mrt4 is rRNA-dependent. The dual localization indicates that Mrt4 may have a nuclear-cytoplasmic shuttling capacity, because deletion of N-terminal domain or inhibition of rRNA synthesis blocks the cyto-

plasmic accumulation of hMrt4, and it seems reasonable to propose that the protein may leave the nucleus only when bound to the pre-60S. A direct involvement of hMrt4 in the 60S maturation was further confirmed by the effect of LMB treatment, where hMrt4 accumulated in the nucleus; the nuclear accumulation of hMrt4 was incomplete and variable from cell to cell but increased sig-

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Fig. 8. Localization of hMrt4 and ribosomal stalk constituents in NIH3T3 cells after LMB treatment. The effects of LMB on subcellular localization of analyzed proteins were recorded 16 h after treatment with 2.5 nM LMB: (A) LMB-dependent distribution of fusion protein DsRed-hMrt4; (B) distribution of ribosomal stalk constituents. Visualization was performed as described in Fig. 7; Insets (c), control without LMB. Bars, 40 ␮m.

nificantly with extended incubation times. By acting on the Crm1 nuclear export adaptor, LMB blocks the last nuclear step of pre-60S maturation, suggesting that hMrt4 accompanies pre-60S until the very last phase of nuclear pre-60S maturation. The experiments with ActD and LMB both indicated that hMrt4 actively takes part in the nuclear events associated with ribosome maturation. However, as indicated, the picture of LMB-treated cells was not homogeneous, and in some small pool of cells a signal from hMrt4 was observed in the cytoplasm, indicating that hMrt4 could be involved in cytoplasmic events as well. An involvement of hMrt4 in cytoplasmic maturation events of pre-60S is also suggested by the results where hMrt4 was found in the cytoplasmic ribosomal fraction. Taking together all these results, one may conclude that the hMrt4 protein is a trans-acting factor, integrated into the pre-60S in the nucleus, but with a shuttling capacity, probably leaving the pre60S subunit after its export to the cytoplasm. It should be stressed here that our results on hMrt4 combined with those of three most recent studies on yeast Mrt4 (Kemmler et al., 2009; Lo et al., 2009; Rodriguez-Mateos et al., 2009b) document the functional conservation of these proteins. In conclusion, we have shown that the hMrt4 and ribosomal P0 proteins are related in their rRNA-binding domain, but have

divergent functions. hMrt4 most likely binds to the same site as P0 on the rRNA, but is not able to functionally complement a lack of P0. The primary site of hMrt4 action is the nucleolus, but it is also present in the cytoplasm. P0, with cytoplasmic localization, replaces Mrt4 at one of the final steps of 60S subunit maturation, as shown very recently (Kemmler et al., 2009; Lo et al., 2009; Rodriguez-Mateos et al., 2009b). However, the question arises, why is the process of stalk assembly on the pre-60S particle so involved? The answer to this question is probably related to the crucial function of the stalk. This structure represents the major part of the GTPase-associated center and has a pivotal role in the ribosome-mediated stimulation of translation factor-dependent GTP hydrolysis at all steps of translation. Since the stalk is critical for proper execution of the complex process of translation, this suggests two, not mutually exclusive explanations for the stepwise assembly of the stalk. First, it is likely that upon entrance of pre-60S to the cytoplasm, addition of the P0 protein may trigger a cascade of fine-tuning processes of pre-60S maturation dependent on a group of GTP/ATP-ases modulating the pre-ribosome structure. Second, the replacement of Mrt4 by P0 may serve as a final control step ensuring that only fully mature functional ribosome enters translation.

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