Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20_NLS mutant gene and examined polysome profile of cells that had been transfected with the S20_NLS gene. As a result, we observed the formation of recombinant 40S carried S20_NLS but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20_NLS in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20_NLS in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated.     

Deletion of L4 domains reveals insights into the importance of ribosomal protein extensions in eukaryotic ribosome assembly

Numerous ribosomal proteins have a striking bipartite architecture: a globular body positioned on the ribosomal exterior and an internal loop buried deep into the rRNA core. In eukaryotes, a significant number of conserved r-proteins have evolved extra amino- or carboxy-terminal tail sequences, which thread across the solvent-exposed surface. The biological importance of these extended domains remains to be established. In this study, we have investigated the universally conserved internal loop and the eukaryote-specific extensions of yeast L4. We show that in contrast to findings with bacterial L4, deleting the internal loop of yeast L4 causes severely impaired growth and reduced levels of large ribosomal subunits. We further report that while depleting the entire L4 protein blocks early assembly steps in yeast, deletion of only its extended internal loop affects later steps in assembly, revealing a second role for L4 during ribosome biogenesis. Surprisingly, deletion of the entire eukaryote-specific carboxy-terminal tail of L4 has no effect on viability, production of 60S subunits, or translation. These unexpected observations provide impetus to further investigate the functions of ribosomal protein extensions, especially eukaryote-specific examples, in ribosome assembly and function.     

Arginine methylation of ribosomal protein S3 affects ribosome assembly

The human ribosomal protein S3 (rpS3), a component of the 40S small subunit in the ribosome, is a known multi-functional protein with roles in DNA repair and apoptosis. We recently found that the arginine residue(s) of rpS3 are methylated by protein arginine methyltransferase 1 (PRMT1). In this paper, we confirmed the arginine methylation of rpS3 protein both in vitro and in vivo. The sites of arginine methylation are located at amino acids 64, 65 and 67. However, mutant rpS3 (3RA), which cannot be methylated at these sites, cannot be transported into the nucleolus and subsequently incorporated into the ribosome. Our results clearly show that arginine methylation of rpS3 plays a critical role in its import into the nucleolus, as well as in small subunit assembly of the ribosome.     

Saccharomyces cerevisiae ribosomal protein L26 is not essential for ribosome assembly and function

Ribosomal proteins play important roles in ribosome biogenesis and function. Here, we study the evolutionarily conserved L26 in Saccharomyces cerevisiae, which assembles into pre-60S ribosomal particles in the nucle(ol)us. Yeast L26 is one of the many ribosomal proteins encoded by two functional genes. We have disrupted both genes; surprisingly, the growth of the resulting rpl26 null mutant is apparently identical to that of the isogenic wild-type strain. The absence of L26 minimally alters 60S ribosomal subunit biogenesis. Polysome analysis revealed the appearance of half-mers. Analysis of pre-rRNA processing indicated that L26 is mainly required to optimize 27S pre-rRNA maturation, without which the release of pre-60S particles from the nucle(ol)us is partially impaired. Ribosomes lacking L26 exhibit differential reactivity to dimethylsulfate in domain I of 25S/5.8S rRNAs but apparently are able to support translation in vivo with wild-type accuracy. The bacterial homologue of yeast L26, L24, is a primary rRNA binding protein required for 50S ribosomal subunit assembly in vitro and in vivo. Our results underscore potential differences between prokaryotic and eukaryotic ribosome assembly. We discuss the reasons why yeast L26 plays such an apparently nonessential role in the cell.     

Analysis of nuclear targeting activities of transport signals in the human immunodeficiency virus Rev protein

The human immunodeficiency Rev protein shuttles between the nucleus and cytoplasm, while accumulating to high levels in the nucleus. Rev has a nuclear localization signal (NLS; AA 35-50) with an arginine-rich motif (ARM) that interacts with importin beta and a leucine-rich nuclear export signal (NES; AA 75-84) recognized by CRM1/exportin 1. Here we explore nuclear targeting activities of the transport signals of Rev. GFP tagging and quantitative fluorescence microscopy were used to study the localization behavior of Rev NLS/ARM mutants under conditions inhibiting the export of Rev. Rev mutant M5 was actively transported to the nucleus, despite its known failure to bind importin beta. Microinjection of transport substrates with Rev-NES peptides revealed that the Rev-NES has both nuclear import and export activities. Replacement of amino acid residues "PLER" (77-80) of the NES with alanines abolished bidirectional transport activity of the Rev-NES. These results indicate that both transport signals of Rev have nuclear import capabilities and that the Rev NLS has more than one nuclear targeting activity. This suggests that Rev is able to use various routes for nuclear entry rather than depending on a single pathway.     

Identification of an unconventional nuclear localization signal in human ribosomal protein S2

Ribosomal proteins must be imported into the nucleus after being synthesized in the cytoplasm. Since the rpS2 amino acid sequence does not contain a typical nuclear localization signal, we used deletion mutant analysis and rpS2-beta-galactosidase chimeric proteins to identify the nuclear targeting domains in rpS2. Nuclear rpS2 is strictly localized in the nucleoplasm and is not targeted to the nucleoli. Subcellular localization analysis of deletion mutants of rpS2-beta-galactosidase chimeras identified a central domain comprising 72 amino acids which is necessary and sufficient to target the chimeric beta-galactosidase to the nucleus. The nuclear targeting domain shares no significant similarity to already characterized nuclear localization signals in ribosomal proteins or other nuclear proteins. Although a Nup153 fragment containing the importinbeta binding site fused to VP22 blocks nuclear import of rpS2-beta-galactosidase fusion proteins, nuclear uptake of rpS2 could be mediated by several import receptors since it binds to importinalpha/beta and transportin.     

Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly

Assembly of bacterial 30S ribosomal subunits requires structural rearrangements to both its 16S rRNA and ribosomal protein components. Ribosomal protein S4 nucleates 30S assembly and associates rapidly with the 5′ domain of the 16S rRNA. In vitro, transformation of initial S4–rRNA complexes to long-lived, mature complexes involves refolding of 16S helix 18, which forms part of the decoding center. Here we use targeted mutagenesis of Geobacillus stearothermophilus S4 to show that remodeling of S4–rRNA complexes is perturbed by ram alleles associated with reduced translational accuracy. Gel mobility shift assays, SHAPE chemical probing, and in vivo complementation show that the S4 N-terminal extension is required for RNA binding and viability. Alanine substitutions in Y47 and L51 that interact with 16S helix 18 decrease S4 affinity and destabilize the helix 18 pseudoknot. These changes to the protein–RNA interface correlate with no growth (L51A) or cold-sensitive growth, 30S assembly defects, and accumulation of 17S pre-rRNA (Y47A). A third mutation, R200A, over-stabilizes the helix 18 pseudoknot yet results in temperature-sensitive growth, indicating that complex stability is finely tuned by natural selection. Our results show that early S4–RNA interactions guide rRNA folding and impact late steps of 30S assembly.     

L22 ribosomal protein and effect of its mutation on ribosome resistance to erythromycin

The ribosomal protein L22 is a core protein of the large ribosomal subunit interacting with all domains of the 23S rRNA. The triplet Met82-Lys83-Arg84 deletion in L22 from Escherichia coli renders cells resistant to erythromycin which is known as an inhibitor of the nascent peptide chain elongation. The crystal structure of the Thermus thermophilus L22 mutant with equivalent triplet Leu82-Lys83-Arg84 deletion has been determined at 1.8A resolution. The superpositions of the mutant and the wild-type L22 structures within the 50S subunits from Haloarcula marismortui and Deinococcus radiodurans show that the mutant beta-hairpin is bent inward the ribosome tunnel modifying the shape of its narrowest part and affecting the interaction between L22 and 23S rRNA. 23S rRNA nucleotides of domain V participating in erythromycin binding are located on the opposite sides of the tunnel and are brought to those positions by the interaction of the 23S rRNA with the L22 beta-hairpin. The mutation in the L22 beta-hairpin affects the orientation and distances between those nucleotides. This destabilizes the erythromycin-binding "pocket" formed by 23S rRNA nucleotides exposed at the tunnel surface. It seems that erythromycin, while still being able to interact with one side of the tunnel but not reaching the other, is therefore unable to block the polypeptide growth in the drug-resistant ribosome.     

Loc1p is required for efficient assembly and nuclear export of the 60S ribosomal subunit

Loc1p is an exclusively nuclear dsRNA-binding protein that affects the asymmetric sorting of ASH1 mRNA to daughter cells in Saccharomyces cerevisiae. In addition to the role in cytoplasmic RNA localization, Loc1p is a constituent of pre-60S ribosomes. Cells devoid of Loc1p display a defect in the synthesis of 60S ribosomal subunits, resulting in “half-mer” polyribosomes. Previously, we reported that Loc1p is located throughout the entire nucleus; however, upon closer inspection we discovered that Loc1p is enriched in the nucleolus consistent with a role in 60S ribosome biogenesis. Given that Loc1p is an RNA-binding protein and presumably functions in the assembly of 60S ribosomal subunits, we investigated if Loc1p has a role in rRNA processing and nuclear export of 60S subunits. Analysis of pre-rRNA processing revealed that loc1Δ cells exhibit gross defects in 25S rRNA synthesis, specifically a delay in processing at sites A0, A1 and A2 in 35S pre-rRNA. Furthermore, loc1Δ cells exhibit nuclear export defects for 60S ribosomal subunits, again, consistent with a role for Loc1p in the assembly of 60S ribosomal subunits. It is attractive to hypothesize that the two phenotypes associated with loc1Δ cells, namely altered ASH1 mRNA localization and ribosome biogenesis, are not mutually exclusive, but that ribosome biogenesis directly impacts mRNA localization.     

Molecular characterization of soybean ribosomal protein S13 targeted to the nucleus

We isolated the full-length genomic clone of STLI25 encoding RPS13 (ribosomal protein S13) in soybean. Genomic DNA structure of SLTI25 is similar to that of Arabidopsis ribosomal protein S13. RNA expression of SLTI25 was induced by salt, ABA, or wounding stress, but reduced by dehydration stress. To determine the subcellular localization of the gene product fused to GFP, we were able to confirm that SLTI25-GFP was restricted to the nucleus. By domain swapping analysis, it was shown that C-terminal region of SLTI25 with a putative nucleus localization signal was necessary and sufficient for nucleus targeting of the fusion protein in plants. This new findings provide an evidence that SLTI25 is targeted to the nucleus for the ribosome subunit assembly in plants. Published in Russian in Fiziologiya Rastenii, 2009, vol. 56, No. 3, pp. 445–452 This text was submitted by the authors in English. These authors contributed equally to the work.     

The uL10 protein,a component of the ribosomal P-stalk,is released from the ribosome in nucleolar stress

The ribosomal uL10 protein, formerly known as P0, is an essential element of the ribosomal GTPase-associated center responsible for the interplay with translational factors during various stages of protein synthesis. In eukaryotic cells, uL10 binds two P1/P2 protein heterodimers to form a pentameric P-stalk, described as uL10-(P1-P2)₂, which represents the functional form of these proteins on translating ribosomes. Unlike most ribosomal proteins, which are incorporated into pre-ribosomal particles during early steps of ribosome biogenesis in the nucleus, P-stalk proteins are attached to the 60S subunit in the cytoplasm. Although the primary role of the P-stalk is related to the process of translation, other extraribosomal functions of its constituents have been proposed, especially for the uL10 protein; however, the list of its activities beyond the ribosome is still an open question. Here, by the combination of biochemical and advanced fluorescence microscopy techniques, we demonstrate that upon nucleolar stress induction the uL10 protein accumulates in the cytoplasm of mammalian cells as a free, ribosome-unbound protein. Importantly, using a novel approach, FRAP-AC (FRAP after photoConversion), we have shown that the ribosome-free pool of uL10 represents a population of proteins released from pre-existing ribosomes. Taken together, our data indicate that the presence of uL10 on the ribosomes is affected in stressed cells, thus it might be considered as a regulatory element responding to environmental fluctuations.     

Regulation of subcellular localization of the antiproliferative protein Tob by its nuclear export signal and bipartite nuclear localization signal sequences

Tob, a member of the Tob and BTG antiproliferative protein family, plays an important role in many cellular processes including cell proliferation. In this study, we have addressed molecular mechanisms regulating subcellular localization of Tob. Treatment with leptomycin B, an inhibitor of nuclear export signal (NES) receptor, resulted in a change in subcellular distribution of Tob from its pan-cellular distribution to nuclear accumulation, indicating the existence of NES in Tob. Our results have then identified an N-terminal region (residues 2-14) of Tob as a functional NES. They have also shown that Tob has a functional, bipartite nuclear localization signal (NLS) in residues 18-40. Thus, Tob is shuttling between the nucleus and the cytoplasm by its NES and NLS. To examine a possible relationship between subcellular distribution of Tob and its function, we exogenously added a strong NLS sequence or a strong NES sequence or both to Tob. The obtained results have demonstrated that the strong NLS-added Tob has a much weaker activity to inhibit cell cycle progression from G0/G1 to S phase. These results suggest that cytoplasmic localization or nucleocytoplasmic shuttling is important for the antiproliferative function of Tob.     

The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities

Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.     

The nuclear localization signal of mitotic kinesin-like protein Mklp-1: effect on Mklp-1 function during cytokinesis

The mitotic kinesin-like protein (Mklp-1) localizes in the nucleus during interphase due to the presence of nuclear localization signal(s) [NLS(s)] within its sequence. Here, we mapped two NLSs to be 899SRKRRSST906 and 949KRKKP953 in the tail domain of Mklp-1, and showed that ectopic expression of a mutant Mklp-1 without the NLSs leads to cell cycle arrest at cytokinesis, indicating that the NLSs are necessary for Mklp-1 to execute its normal function during cell division. Furthermore, mutation of two serine residues in the first NLS to aspartic acid, which mimics phosphorylation, attenuated its nuclear localization function, suggesting that the function of this NLS might be regulated by phosphorylation.     

A molecular dynamics study of the C-terminal fragment of the L7/L12 ribosomal protein

The crystallographic dimer of the C-terminal fragment (CTF) of the L7/L12 ribosomal protein has been subjected to molecular dynamics (MD) simulations. A 90 picosecond (ps) trajectory for the protein dimer, 19 water molecules and two counter ions has been calculated at constant temperature. Effects of intermolecular interactions on the structure and dynamics have been studied. The exact crystallographic symmetry is lost and the atomic fluctuations differ from one monomer to the other. The average MD structure is more stable than the X-ray one, as judged by accessible surface area and energy calculations. Crystal (non-dimeric) interactions have been simulated in another 40 ps trajectory by using harmonic restraints to represent intermolecular hydrogen bonds. The conformational changes with respect ot the X-ray structure are then virtually suppressed.The unrestrained dimer trajectory has been scanned for cooperative motions involving secondary structure elements. The intrinsic collective motions of the monomer are transmitted via intermolecular contacts to the dimer structure.The existence of a stable dimeric form of CTF, resembling the crystallographic one, has been documented. At the cost of fairly small energy expenditure the dimer has considerable conformational flexibility. This flexibility may endow the dimer with some functional potential as an energy transducer.     

The C-terminal fragment of ribosomal protein S15 is located in the decoding site of the human ribosome

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

Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide the first evidence of the active nuclear import of CERKL and suggest that the identified NLS might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus.     

Mapping of the nuclear localization signals in open reading frame 2 protein from porcine circovirus type 1

Porcine circovirus type 1 （PCV1） contains two major open reading frames encoding the replication-associated proteins and the major structural capsid （Cap） protein. PCV1 Cap has an N-terminus carrying several potential monopartite or bipartite nuclear localization signals （NLS）. The contribution of these partially overlapping motifs to nuclear importing was identified by expression of mutated PCVI Cap versions fused to enhanced green fluorescent protein （EGFP）. The Cterminus truncated PCV1 Cap-EGFP was localized in nuclei of PK-15 cells similar to the wild-type PCV1 Cap-EGFP, whereas truncation of the N-terminus rendered the fusion protein distributed into cytoplasm, indicating that the nuclear import of PCV1 Cap was efficiently mediated by its N-terminal region. Substitutions of basic residues in stretches 9RR- RR12 or the right part of 25RRPYLAHPAFRNRYRWRRK43 resulted in a diffused distribution of the fusion protein in both nuclei and cytoplasm, indicating that the two NLSs were responsible for restricted nuclear targeting of PCV1 Cap.     

Nucleolar binding sequences of the ribosomal protein S6e family reside in evolutionary highly conserved peptide clusters

Proteomic analyses of the nucleolus have revealed almost 700 functionally diverse proteins implicated in ribosome biogenesis, nucleolar assembly, and regulation of vital cellular processes. However, this nucleolar inventory has not unveiled a specific consensus motif necessary for nucleolar binding. The ribosomal protein family characterized by their basic nature should exhibit distinct binding sequences that enable interactions with the rRNA precursor molecules facilitating subunit assembly. We succeeded in delineating 2 minimal nucleolar binding sequences of human ribosomal protein S6 by fusing S6 cDNA fragments to the 5' end of the LacZ gene and subsequently detecting the intracellular localization of the beta-galactosidase fusion proteins. Nobis1 (nucleolar binding sequence 1), comprising of 4 highly conserved amino acid clusters separated by glycine or proline, functions independently of the 3 authentic nuclear localization signals (NLSs). Nobis2 consists of 2 conserved peptide clusters and requires the authentic NLS2 in its native context. Similarly, we deduced from previous publications that the single Nobis of ribosomal protein S25 is also highly conserved. The functional protein domain organization of the ribosomal protein S6e family consists of 3 modules: NLS, Nobis, and the C-terminal serine cluster of the phosphorylation sites. This modular structure is evolutionary conserved in vertebrates, invertebrates, and fungi. Remarkably, nucleolar binding sequences of small and large ribosomal proteins reside in peptide clusters conserved over millions of years.