A rice (Oryza sativa L.) cDNA encodes a protein sequence homologous to the eukaryotic ribosomal 5S RNA-binding protein

A rice (Oryza sativa L.) cDNA clone coding for the cytoplasmic ribosomal protein L5, which associates with 5 S rRNA for ribosome assembly, was cloned and its nucleotide sequence was determined. The primary structure of rice L5, deduced from the nucleotide sequence, contains 294 amino acids and has intriguing features some of which are also conserved in other eucaryotic homologues. These include: four clusters of basic amino acids, one of which may serve as a nucleolar localization signal; three repeated amino acid sequences; the conservation of glycine residues. This protein was identified as the nuclear-encoded cytoplasmic ribosomal protein L5 of rice by sequence similarity to other eucaryotic ribosomal 5 S RNA-binding proteins of rat, chicken, Xenopus laevis, and Saccharomyces cerevisiae. Rice L5 shares 51 to 62% amino acid sequence identity with the homologues. A group of ribosomal proteins from archaebacteria including Methanococcus vanniellii L18 and Halobacterium cutirubrum L13, which are known to be associated with 5 S rRNA, also related to rice L5 and the other eucaryotic counterparts, suggesting an evolutionary relationship in these ribosomal 5 S RNA-binding proteins.     

Phosphorylation of ribosomal protein L5 by protein kinase CKII decreases its 5S rRNA binding activity.

We have recently reported that ribosomal protein L5 associates with the beta subunit of protein kinase CKII (CKII) (Kim, J.-M., Cha, J. -Y., Marshak, D. R., and Bae, Y.-S. (1996) Biochem. Biophys. Res. Commun. 226, 180-186). In this study, we demonstrate that CKII is able to catalyze the phosphorylation of the human L5 protein in vitro, which results in a decrease in 5S rRNA binding activity. Phosphoamino acid analysis indicated that the phosphorylation occurs on serine residues. Sequence analysis of cyanogen bromide-digested phosphopeptides and analysis of L5 deletion mutants indicates that the main phosphorylated residues are located within two fragments corresponding of residues 142-200 and residues 272-297 of the human L5. Based on our present results, we suggest that the phosphorylation of L5 by CKII is one of the mechanisms that regulates nucleolar targeting of 5S rRNA and/or ribosome assembly in the cell.     

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies.

We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.     

Structure and function in the herpes simplex virus 1 RNA-binding protein U(s)11: mapping of the domain required for ribosomal and nucleolar association and RNA binding in vitro.

The herpes simplex virus 1 US11 protein is an RNA-binding regulatory protein that specifically and stably associates with 60S ribosomal subunits and nucleoli and is incorporated into virions. We report that US11/ beta-galactosidase fusion protein expressed in bacteria bound to rRNA from the 60S subunit and not the 40S subunit. This binding reflects the specificity of ribosomal subunit association. Analyses of deletion mutants of the US11 gene showed that specific RNA binding activity, nucleolar localization, and association with 60S ribosomal subunits were found to map to the amino acid sequences of the carboxyl terminus of US11 protein, suggesting that these activities all reflect specific binding of US11 to large subunit rRNA. The carboxyl-terminal half of the protein consists of a regular tripeptide repeat of the sequence RXP and constitutes a completely novel RNA-binding domain. All of the mutant US11 proteins could be incorporated into virus particles, suggesting that the signal for virion incorporation either is at the amino-terminal four amino acids or is redundant in the protein.     

The primary structure of chicken ribosomal protein L5.

The nucleotide sequence of a cDNA for chicken ribosomal protein L5, which is considered to associate with 5S rRNA, was determined. The cDNA is 975 bp long. The deduced protein has 297 amino acids and has a molecular mass of 34,090 Da. A comparative analysis of the amino acid sequences of chicken L5 and its homologous proteins revealed an extremely conserved region which contains a cluster of basic amino acids.     

Mutual induced fit binding of Xenopus ribosomal protein L5 to 5S rRNA

A library of random mutations in Xenopus ribosomal protein L5 was generated by error-prone PCR and used to delineate the binding domain for 5S rRNA. All but one of the amino acid substitutions that affected binding affinity are clustered in the central region of the protein. Several of the mutations are conservative substitutions of non-polar amino acid residues that are unlikely to form energetically significant contacts to the RNA. Thermal denaturation, monitored by circular dichroism (CD), indicates that L5 is not fully structured and association with 5S rRNA increases the t(m) of the protein by 16 degrees C. L5 induces changes in the CD spectrum of 5S rRNA, establishing that the complex forms by a mutual induced fit mechanism. Deuterium exchange reveals that a considerable amount of L5 is unstructured in the absence of 5S rRNA. The fluorescence emission of W266 provides evidence for structural changes in the C-terminal region of L5 upon binding to 5S rRNA; whereas, protection experiments demonstrate that the N terminus remains highly sensitive to protease digestion in the complex. Analysis of the amino acid sequence of L5 by the program PONDR predicts that the N and C-terminal regions of L5 are intrinsically disordered, but that the central region, which contains three essential tyrosine residues and other residues important for binding to 5S rRNA, is likely to be structured. Initial interaction of the protein with 5S rRNA likely occurs through this region, followed by induced folding of the C-terminal region. The persistent disorder in the N-terminal domain is possibly exploited for interactions between the L5-5S rRNA complex and other proteins.     

The primary structure of rat ribosomal protein S5. A ribosomal protein present in the rat genome in a single copy.

The amino acid sequence of the rat 40 S ribosomal subunit protein S5 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed by the determination, directly from the protein, of 17 residues near the NH2 terminus. S5 has 204 amino acids; the molecular weight is 22,863. The protein designated S5a has the same amino acid sequence as S5 except that it lacks the NH2-terminal 5 residues. It is not known whether the conversion of a portion of S5 to S5a is physiological or fortuitous. The mRNA for S5 has about 820 nucleotides. Hybridization of the S5 cDNA to digests of nuclear DNA indicates that the rat genome has only a single copy of the gene; this is in distinction to the mouse and human genomes which have three to six copies of the S5 gene. Rat ribosomal protein S5 is related to the eubacteria, the arachaebacteria, and the chloroplast family of S7 ribosomal proteins. There is a peptide of 16 residues at the carboxyl terminus of S5 that is highly conserved in 18 species spanning the three kingdoms and chloroplasts.     

On the interaction of ribosomal protein L5 with 5S rRNA

Ribosomal protein L5, a 5S rRNA binding protein in the large subunit, is composed of a five-stranded antiparallel beta-sheet and four alpha-helices, and folds in a way that is topologically similar to the ribonucleprotein (RNP) domain [Nakashima et al., RNA 7, 692-701, 20011. The crystal structure of ribosomal protein L5 (BstL5) from Bacillus stearothermophilus suggests that a concave surface formed by an anti-parallel beta-sheet and long loop structures are strongly involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurred at beta-strands and loop structures in BstL5. The mutation of Lys33 at the beta 1-strand caused a significant reduction in 5S rRNA binding. In addition, the Arg92, Phe122, and Glu134 mutations on the beta2-strand, the alpha3-beta4 loop, and the beta4-beta5 loop, respectively, resulted in a moderate decrease in the 5S rRNA binding affinity. In contrast, mutation of the conserved residue Pro65 at the beta2-strand had little effect on the 5S rRNA binding activity. These results, taken together with previous results, identified Lys33, Asn37, Gln63, and Thr90 on the beta-sheet structure, and Phe77 at the beta2-beta3 loop as critical residues for the 5S rRNA binding. The contribution of these amino acids to 5S rRNA binding was further quantitatively evaluated by surface plasmon resonance (SPR) analysis by the use of BIAcore. The results showed that the amino acids on the beta-sheet structure are required to decrease the dissociation rate constant for the BstL5-5S rRNA complex, while those on the loops are to increase the association rate constant for the BstL5-5S rRNA interaction.     

Subcellular distribution of ADAR1 isoforms is synergistically determined by three nuclear discrimination signals and a regulatory motif

ADAR1 is an RNA-specific adenosine deaminase that edits RNA sequences. We have demonstrated previously that different ADAR1 isoforms are induced during acute inflammation. Here we show that the mouse ADAR1 isoforms are differentially localized in cellular compartments and that their localization is controlled by several independent signals. Nuclear import of the full-length ADAR1 is predominantly regulated by a nuclear localization signal at the C terminus (NLS-c), which consists of a bipartite basic amino acid motif plus the last 39 residues of ADAR1. Deletion of the NLS-c causes the truncated ADAR1 protein to be retained in the cytoplasm. The addition of this sequence to pyruvate kinase causes the cytoplasmic protein to be localized within the nucleus. The localization of nuclear ADAR1 is determined by a dynamic balance between the nucleolar binding activity of the nucleolar localization signal (NoLS) in the middle of the protein and the exporting activity of the nuclear exporter signal (NES) near the N terminus. The NoLS consists of a typical monopartite cluster of basic residues followed by the third double-stranded RNA-binding domain. These signals act independently; however, NES function can be completely silenced by the NLS-c when a regulatory motif within the catalytic domain and the NoLS are deleted. Thus, the intracellular distribution of the various ADAR1 isoforms is determined by NLS-c, NES, NoLS, and a regulatory motif.     

Characterization of the nuclear export signal in the coronavirus infectious bronchitis virus nucleocapsid protein

The nucleocapsid (N) protein of infectious bronchitis virus (IBV) localizes to the cytoplasm and nucleolus and contains an eight-amino-acid nucleolar retention motif. In this study, a leucine-rich nuclear export signal (NES) (291-LQLDGLHL-298) present in the C-terminal region of the IBV N protein was analyzed by using alanine substitution and deletion mutagenesis to investigate the relative contributions that leucine residues make to nuclear export and where these residues are located on the structure of the IBV N protein. The analysis indicated that Leu296 and Leu298 are required for efficient nuclear export of the protein. Structural information indicated that both of these amino acids are available for interaction with protein complexes involved in this process. However, export of N protein from the nucleus/nucleolus was not inhibited by leptomycin B treatment, indicating that N protein nuclear export is independent of the CRM1-mediated export pathway.     

Requirement for C-terminal extension to the RNA binding domain for efficient RNA binding by ribosomal protein L2

Ribosomal protein L2 is a primary 23S rRNA binding protein in the large ribosomal subunit. We examined the contribution of the N- and C-terminal regions of Bacillus stearothermophilus L2 (BstL2) to the 23S rRNA binding activity. The mutant desN, in which the N-terminal 59 residues of BstL2 were deleted, bound to the 23S rRNA fragment to the same extent as wild type BstL2, but the mutation desC, in which the C-terminal 74 amino acid residues were deleted, abolished the binding activity. These observations indicated that the C-terminal region is involved in 23S rRNA binding. Subsequent deletion analysis of the C-terminal region found that the C-terminal 70 amino acids are required for efficient 23S rRNA binding by BstL2. Furthermore, the surface plasmon resonance analysis indicated that successive truncations of the C-terminal residues increased the dissociation rate constants, while they had little influence on association rate constants. The result indicated that reduced affinities of the C-terminal deletion mutants were due only to higher dissociation rate constants, suggesting that the C-terminal region primarily functions by stabilizing the protein L2-23S rRNA complex.     

The primary structure of rat ribosomal protein L7. The presence near the amino terminus of L7 of five tandem repeats of a sequence of 12 amino acids

The covalent structure of rat ribosomal protein L7 was determined in part from the sequence of nucleotides in a recombinant cDNA and in part from the sequence of amino acids in portions of the protein. The complementary analyses supplemented and confirmed each other. Ribosomal protein L7 contains 258 amino acids and has a molecular weight of 30,040. The protein has an unusual and striking structural feature near the NH2 terminus: five tandem repeats of a sequence of 12 residues. Rat L7 appears to be related to ribosomal protein L7 from the moderate halophile Vibrio costicola and perhaps to L30 from Bacillus stearothermophilus, to L7 from the moderate halophile NRCC 41227, and to L22 from Nicotinia tobaccum chloroplast. In addition, there is a sequence of 24 amino acids in rat protein L7 that may be related to segments of the same number of residues in Escherichia coli ribosomal proteins S10, S15, L9, and L22.     

Identification and functional analysis of the nuclear localization signals of ribosomal protein L25 from Saccharomyces cerevisiae

The regions of the large subunit ribosomal protein L25 from Saccharomyces cerevisiae responsible for nuclear localization of the protein were identified by constructing fusion genes encoding various segments of L25 linked to the amino terminus of beta-galactosidase. Indirect immunofluorescence of yeast cells expressing the fusions demonstrated that amino acid residues 1 to 17 as well as 18 to 41 of L25 promote import of the reporter protein into the nucleus. Both nuclear localization signal (NLS) sequences appear to consist of two distinct functional parts: one showed relatively weak nuclear targeting activity, whereas the other considerably enhances this activity but does not promote nuclear import by itself. Microinjection of in vitro prepared intact and N-terminally truncated L25 into Xenopus laevis oocytes demonstrated that the region containing the two NLS sequences is indeed required for efficient nuclear localization of the ribosomal protein. This conclusion was confirmed by complementation experiments using a yeast strain that conditionally expresses wild-type L25. The latter experiments also indicated that amino acid residues 1 to 41 of L25 are required for full functional activity of yeast 60 S ribosomal subunits. Yeast cells expressing forms of L25 that lack this region are viable, but show impaired growth and a highly abnormal cell morphology.     

The participation of 5S rRNA in the co-translational formation of a eukaryotic 5S ribonucleoprotein complex

The eukaryotic ribosomal 5S RNA–protein complex (5S rRNP) is formed by a co-translational event that requires 5S rRNA binding to the nascent peptide chain of eukaryotic ribosomal protein L5. Binding between 5S rRNA and the nascent chain is specific: neither the 5S rRNA nor the nascent chain of L5 protein can be substituted by other RNAs or other ribosomal proteins. The region responsible for binding 5S rRNA is located at positions 35–50 with amino acid sequence RLVIQDIKNKYNTPKYRM. Eukaryotic 5S rRNA binds a nascent chain having this sequence, but such binding is not substantive enough to form a 5S-associated RNP complex, suggesting that 5S rRNA binding to the nascent chain is amino acid sequence dependent and that formation of the 5S rRNP complex is L5 protein specific. Microinjection of 5S rRNP complex into the cytoplasm of Xenopus oocytes results in both an increase in the initial rate and also in the extent of net nuclear import of L5. This suggests that the 5S rRNP complex enhances nuclear transport of L5. We propose that 5S rRNA plays a chaperone-like role in folding of the nascent chain of L5 and directs L5 into a 5S rRNP complex for nuclear entry.     

Requirement for C-terminal Extension to the RNA Binding Domain for Efficient RNA Binding by Ribosomal Protein L2

Ribosomal protein L2 is a primary 23S rRNA binding protein in the large ribosomal subunit. We examined the contribution of the N- and C-terminal regions of Bacillus stearothermophilus L2 (BstL2) to the 23S rRNA binding activity. The mutant desN, in which the N-terminal 59 residues of BstL2 were deleted, bound to the 23S rRNA fragment to the same extent as wild type BstL2, but the mutation desC, in which the C-terminal 74 amino acid residues were deleted, abolished the binding activity. These observations indicated that the C-terminal region is involved in 23S rRNA binding. Subsequent deletion analysis of the C-terminal region found that the C-terminal 70 amino acids are required for efficient 23S rRNA binding by BstL2. Furthermore, the surface plasmon resonance analysis indicated that successive truncations of the C-terminal residues increased the dissociation rate constants, while they had little influence on association rate constants. The result indicated that reduced affinities of the C-terminal deletion mutants were due only to higher dissociation rate constants, suggesting that the C-terminal region primarily functions by stabilizing the protein L2-23S rRNA complex.     

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding.     

Tus,an E. coli Protein,Contains Mammalian Nuclear Targeting and Exporting Signals

Shuttling of proteins between nucleus and cytoplasm in mammalian cells is facilitated by the presence of nuclear localization signals (NLS) and nuclear export signals (NES), respectively. However, we have found that Tus, an E. coli replication fork arresting protein, contains separate sequences that function efficiently as NLS and NES in mammalian cell lines, as judged by cellular location of GFP-fusion proteins. The NLS was localized to a short stretch of 9 amino acids in the carboxy-terminus of Tus protein. Alterations of any of these basic amino acids almost completely abolished the nuclear targeting. The NES comprises a cluster of leucine/hydrophobic residues located within 21 amino acids at the amino terminus of Tus. Finally, we have shown that purified GFP-Tus fusion protein or GFP-Tus NLS fusion protein, when added to the culture media, was internalized very efficiently into mammalian cells. Thus, Tus is perhaps the first reported bacterial protein to possess both NLS and NES, and has the capability to transduce protein into mammalian cells.     

The bovine immunodeficiency virus Rev protein: identification of a novel nuclear import pathway and nuclear export signal among retroviral Rev/Rev-like proteins

The Rev protein is essential for the replication of lentiviruses. Rev is a shuttling protein that transports unspliced and partially spliced lentiviral RNAs from the nucleus to the cytoplasm via the nucleopore. To transport these RNAs, the human immunodeficiency virus type 1 (HIV-1) Rev uses the karyopherin β family importin β and CRM1 proteins that interact with the Rev nuclear localization signal (NLS) and nuclear exportation signal (NES), respectively. Recently, we reported the presence of new types of bipartite NLS and nucleolar localization signal (NoLS) in the bovine immunodeficiency virus (BIV) Rev protein. Here we report the characterization of the nuclear import and export pathways of BIV Rev. By using an in vitro nuclear import assay, we showed that BIV Rev is transported into the nucleus by a cytosolic and energy-dependent importin α/β classical pathway. Results from glutathione S-transferase (GST) pulldown assays that showed the binding of BIV Rev with importins α3 and α5 were in agreement with those from the nuclear import assay. We also identified a leptomycin B-sensitive NES in BIV Rev, which indicates that the protein is exported via CRM1 like HIV-1 Rev. Mutagenesis experiments showed that the BIV Rev NES maps between amino acids 109 to 121 of the protein. Remarkably, the BIV Rev NES was found to be of the cyclic AMP (cAMP)-dependent protein kinase inhibitor (PKI) type instead of the HIV-1 Rev type. In summary, our data showed that the nuclear import mechanism of BIV Rev is novel among Rev proteins characterized so far in lentiviruses.