Seventeen copies of the human 37 kDa laminin receptor precursor/p40 ribosome-associated protein gene are processed pseudogenes arisen from retropositional events

A cDNA coding for a 37 kDa polypeptide has been identified in several species as both the potential precursor of the 67 kDa laminin receptor (37LRP) and a putative ribosome-associated protein (p40). Interestingly, increased expression of this polypeptide (37LRP/p40) is consistently observed in invasive and metastatic cancer cells and is associated with poor prognosis. Southern-blot analysis of human genomic DNA predicted multiple copies of the 37LRP/p40 gene. In this study, we report that the number of copies of this sequence in the human genome is 26 +/- 2. We have sequenced and analyzed 19 genomic clones corresponding to the 37LRP/p40 gene and found that they were all processed pseudogenes. They all lack intronic sequences and show multiple genetic alterations leading in some cases to the appearance of stop codons. Moreover, they all bear characteristic features of retroposons as the presence of a poly(A)-tail at their 3' end and short direct repeated flanking DNA sequences. None of the pseudogenes analyzed present cis-elements in their 5' flanking region such as TATA or GC boxes. Our date reveal that over 50% of the 37LRP/p40 gene copies are pseudogenes most probably generated by retropositional events. The finding of multiple pseudogenes for the 37LRP/p40 suggests that the accumulation of several copies of this gene might have given a survival advantage to the cell in the course of evolution.     

Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

Spb4p is a putative ATP-dependent RNA helicase that is required for synthesis of 60S ribosomal subunits. Polysome analyses of strains genetically depleted of Spb4p or carrying the cold-sensitive spb4-1 mutation revealed an underaccumulation of 60S ribosomal subunits. Analysis of pre-rRNA processing by pulse-chase labeling, northern hybridization, and primer extension indicated that these strains exhibited a reduced synthesis of the 25S/5.8S rRNAs, due to inhibition of processing of the 27SB pre-rRNAs. At later times of depletion of Spb4p or following transfer of the spb4-1 strain to more restrictive temperatures, the early pre-rRNA processing steps at sites A0, Al, and A2 were also inhibited. Sucrose gradient fractionation showed that the accumulated 27SB pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle. An HA epitope-tagged Spb4p is localized to the nucleolus and the adjacent nucleoplasmic area. On sucrose gradients, HA-Spb4p was found almost exclusively in rapidly sedimenting complexes and showed a peak in the fractions containing the 66S pre-ribosomes. We propose that Spb4p is involved directly in a late and essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase.     

The effect of sulfhydryl reagents upon the activity of 40S ribosomal subunits

p-Chloromercuribenzoate inhibited the poly (U)-dependent binding of the Phe-tRNA to the 40S ribosomal subunit but displayed no inhibitory effect on the binding of poly (U) to the ribosome. Other sulfhydryl reagents tested, like N-ethylmaleimide and iodoacetamide, did not affect the binding of Phe-tRNA to the small ribosomal subunit.     

Directed hydroxyl radical probing of 16S ribosomal RNA in ribosomes containing Fe(II) tethered to ribosomal protein S20

The 16S ribosomal RNA neighborhood of ribosomal protein S20 has been mapped, in both 30S subunits and 70S ribosomes, using directed hydroxyl radical probing. Cysteine residues were introduced at amino acid positions 14, 23, 49, and 57 of S20, and used for tethering 1-(p-bromoacetamidobenzyl)-Fe(II)-EDTA. In vitro reconstitution using Fe(II)-derivatized S20, together with the remaining small subunit ribosomal proteins and 16S ribosomal RNA (rRNA), yielded functional 30S subunits. Both 30S subunits and 70S ribosomes containing Fe(II)-S20 were purified and hydroxyl radicals were generated from the tethered Fe(II). Hydroxyl radical cleavage of the 16S rRNA backbone was monitored by primer extension. Different cleavage patterns in 16S rRNA were observed from Fe(II) tethered to each of the four positions, and these patterns were not significantly different in 30S and 70S ribosomes. Cleavage sites were mapped to positions 160-200, 320, and 340-350 in the 5' domain, and to positions 1427-1430 and 1439-1458 in the distal end of the penultimate stem of 16S rRNA, placing these regions near each other in three dimensions. These results are consistent with previous footprinting data that localized S20 near these 16S rRNA elements, providing evidence that S20, like S17, is located near the bottom of the 30S subunit.     

The exosome subunit Rrp43p is required for the efficient maturation of 5.8S, 18S and 25S rRNA

The Saccharomyces cerevisiae protein Rrp43p co-purifies with four other 3'-->5' exoribonucleases in a complex that has been termed the exosome. Rrp43p itself is similar to prokaryotic RNase PH. Individual exosome subunits have been implicated in the 3' maturation of the 5.8S rRNA found in 60S ribosomes and the 3' degradation of mRNAs. However, instead of being deficient in 60S ribosomes, Rrp43p-depleted cells were deficient in 40S ribosomes. Pulse-chase and steady-state northern analyses of pre-RNA and rRNA levels revealed a significant delay in the synthesis of both 25S and 18S rRNAs, accompanied by the stable accumulation of 35S and 27S pre-rRNAs and the under-accumulation of 20S pre-rRNA. In addition, Rrp43p-depleted cells accumulated a 23S aberrant pre-rRNA and a fragment excised from the 5' ETS. Therefore, in addition to the maturation of 5.8S rRNA, Rrp43p is required for the maturation 18S and 25S rRNA.     

Interaction of the sarcin/ricin domain of 23 S ribosomal RNA with proteins L3 and L6

We investigated interaction of an RNA domain covering the target site of alpha-sarcin and ricin (sarcin/ricin domain) of Escherichia coli 23 S rRNA with ribosomal proteins. RNA fragments comprising residues 2630-2788 (Tox-1) and residues 2640-2774 (Tox-2) of 23 S rRNA were transcribed in vitro and used to analyze the binding proteins by gel shift and filter binding. Protein L6 bound to both Tox-1 (Kd: 0.31 microM) and Tox-2 (Kd: 0.18 microM), and L3 bound only to Tox-1 (Kd: 0.069 microM) in a solution containing 10 mM MgCl2 and 175 mM KCl at 0 degreesC. Footprinting studies were performed using the chemical probe dimethyl sulfate on full-length 23 S rRNA. Binding of L6 protected a single base, A-2757, and strongly enhanced reactivity of C-2752. A direct role of A-2757 in the L6 binding was verified by site-directed mutagenesis; replacements of A-2757 with G and C impaired the L6 binding. On the other hand, binding of L3 protected A-2632, A-2634, A-2635, A-2675, A-2726, A-2733, A-2749, and A-2750. Interestingly, binding of L6 and L3 together protected additional bases A-2657, A-2662, C-2666, and C-2667 in the sarcin/ricin loop, in addition to A-2740, A-2741, A-2748, A-2753, A-2764, A-2765, and A-2766 in the other stem-loop. This appears to be due to cooperative interaction of L3 and L6 with the RNA. The results are discussed with respect to conformational modulation of the sarcin/ricin domain by the protein binding.     

The Orc4p and Orc5p subunits of the Xenopus and human origin recognition complex are related to Orc1p and Cdc6p

The location of origins of DNA replication within the Saccharomyces cerevisiae genome is primarily determined by the origin recognition complex (ORC) interacting with specific DNA sequences. The analogous situation in vertebrate cells is far less clear, although ORC subunits have been identified in several vertebrate organisms including Xenopus laevis. Monoclonal antibodies were raised against Xenopus Orc1p and used for single-step immunoaffinity purification of the entire ORC from an egg extract. Six polypeptides ( approximately 110, 68, 64, 48, 43, and 27 kDa) copurified with Xenopus Orc1p. Protein sequencing also showed the 64-kDa protein to be the previously identified Xenopus Orc2p. Microsequencing of the 43- and 48-kDa proteins that copurified with Orc1p and Orc2p led to their identification as the Orc4p and Orc5p subunits, respectively. Peptide sequences from the 43-kDa protein also allowed the isolation of cDNAs encoding the Xenopus, mouse, and human ORC4 subunits. Human ORC5 was also cloned; its sequence displayed extensive homology to both Drosophila and yeast ORC5. Surprisingly, comparison of the amino acid sequences of Orc1p, Orc4p, and Orc5p suggests that they are structurally related to each other and to the replication initiation protein, Cdc6p. Finally, we present the sequence of the putative Xenopus and human Orc3p.     

Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1

Nin1p, a component of the 26S proteasome of Saccharomyces cerevisiae, is required for activation of Cdc28p kinase at the G1-S-phase and G2-M boundaries. By exploiting the temperature-sensitive phenotype of the nin1-1 mutant, we have screened for genes encoding proteins with related functions to Nin1p and have cloned and characterized two new multicopy suppressors, SUN1 and SUN2, of the nin1-1 mutation. SUN1 can suppress a null nin1 mutation, whereas SUN2, an essential gene, does not. Sun1p is a 268-amino acid protein which shows strong similarity to MBP1 of Arabidopsis thaliana, a homologue of the S5a subunit of the human 26S proteasome. Sun1p binds ubiquitin-lysozyme conjugates as do S5a and MBP1. Sun2p (523 amino acids) was found to be homologous to the p58 subunit of the human 26S proteasome. cDNA encoding the p58 component was cloned. Furthermore, expression of a derivative of p58 from which the N-terminal 150 amino acids had been removed restored the function of a null allele of SUN2. During glycerol density gradient centrifugation, both Sun1p and Sun2p comigrated with the known proteasome components. These results, as well as other structural and functional studies, indicate that both Sun1p and Sun2p are components of the regulatory module of the yeast 26S proteasome.     

The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing

The 26 S proteasome is the central protease involved in ubiquitin-mediated protein degradation and fulfills vital regulatory functions in eukaryotes. The proteolytic core of the complex is the 20 S proteasome, a cylindrical particle with two outer rings each made of 7 different alpha-type subunits and two inner rings made of 7 different beta-type subunits. In the archaebacterial 20 S proteasome ancestor proteolytically active sites reside in the 14 uniform beta-subunits. Their N-terminal threonine residues, released by precursor processing, perform the nucleophilic attack for peptide bond hydrolysis. By directed mutational analysis of 20 S proteasomal beta-type proteins of Saccharomyces cerevisiae, we identified three active site-carrying subunits responsible for different peptidolytic activities as follows: Pre3 for post-glutamyl hydrolyzing, Pup1 for trypsin-like, and Pre2 for chymotrypsin-like activity. Double mutants harboring only trypsin-like or chymotrypsin-like activity were viable. Mutation of two potentially active site threonine residues in the Pre4 subunit excluded its catalytic involvement in any of the three peptidase activities. The generation of different, incompletely processed forms of the Pre4 precursor in active site mutants suggested that maturation of non-active proteasomal beta-type subunits is exerted by active subunits and occurs in the fully assembled particle. This trans-acting proteolytic activity might also account for processing intermediates of the active site mutated Pre2 subunit, which was unable to undergo autocatalytic maturation.     

Microsporidia are related to Fungi: evidence from the largest subunit of RNA polymerase II and other proteins

We have determined complete gene sequences encoding the largest subunit of the RNA polymerase II (RBP1) from two Microsporidia, Vairimorpha necatrix and Nosema locustae. Phylogenetic analyses of these and other RPB1 sequences strongly support the notion that Microsporidia are not early-diverging eukaryotes but instead are specifically related to Fungi. Our reexamination of elongation factors EF-1alpha and EF-2 sequence data that had previously been taken as support for an early (Archezoan) divergence of these amitochondriate protists show such support to be weak and likely caused by artifacts in phylogenetic analyses. These EF data sets are, in fact, not inconsistent with a Microsporidia + Fungi relationship. In addition, we show that none of these proteins strongly support a deep divergence of Parabasalia and Metamonada, the other amitochondriate protist groups currently thought to compose early branches. Thus, the phylogenetic placement among eukaryotes for these protist taxa is in need of further critical examination.     

The isolation of eukaryotic ribosomal proteins. The purification and characterization of the 40 S ribosomal subunit proteins S2, S3, S4, S5, S6, S7, S8, S9, S13, S23/S24, S27, and S28

The proteins of the small subunit of rat liver ribosomes were separated into five groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5 (Collatz, E., Lin, A., St?ffler, G., Tsurugi, K., and Wool, I.G., (1976) J. Biol. Chem. 251, 1808-1816). From the several groups, 12 proteins (S2,S3, S4, S5, S6, S7, S8, S9, S13, S23/S24, S27, and S28) wereisolated by ion exchange chromatography on carboxymethylcellulose, by chromatography on sulfopropyl-Sephadex, and by gel filtration through Sephadex G-75. The amount of protein obtained varied from 1 to 9 mg depending on the number of steps required for the preparation; several proteins had no detectable contamination and the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyazrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined.     

Major groove binding of the tRNA/mRNA complex to the 16 S ribosomal RNA decoding site

We propose a detailed three-dimensional model, with atomic detail, for the structure of the Escherichia coli 16 S rRNA decoding site in a complex with mRNA and the A and P-site tRNAs. Model building began with four primary assumptions: (1) A and P-site tRNA conformations are identical with those seen in the tRNA crystal structure; (2) A and P-site tRNAs adopt an S-type orientation upon binding mRNA in the ribosome; (3) A1492 and A1493 bind non-specifically to the mRNA through a series of hydrogen bonds; and (4) C1400 lies in close proximity to the P-site tRNA wobble base in order to satisfy a UV-induced photocrosslink formed between the two residues. We have models with both major groove and minor groove binding of the tRNA/mRNA complex to the decoding site RNA, and conclude that major groove binding is more likely. Both classes of models maintain structural features reported in the NMR structure of the A-site region of the decoding site RNA with bound paromomycin. We also present models for the tRNA/mRNA complex bound to the decoding site RNA in the presence of the aminoglycoside paromomycin. We discuss possible mechanisms for ribosomal proof reading and antibiotic disruption of this proofreading.     

Rpp1, an essential protein subunit of nuclear RNase P required for processing of precursor tRNA and 35S precursor rRNA in Saccharomyces cerevisiae

The gene for an essential protein subunit of nuclear RNase P from Saccharomyces cerevisiae has been cloned. The gene for this protein, RPP1, was identified by virtue of its homology with a human scleroderma autoimmune antigen, Rpp30, which copurifies with human RNase P. Epitope-tagged Rpp1 can be found in association with both RNase P RNA and a related endoribonuclease, RNase MRP RNA, in immunoprecipitates from crude extracts of cells. Depletion of Rpp1 in vivo leads to the accumulation of precursor tRNAs with unprocessed 5' and 3' termini and reveals rRNA processing defects that have not been described previously for proteins associated with RNase P or RNase MRP. Immunoprecipitated complexes cleave both yeast precursor tRNAs and precursor rRNAs.     

Effects of potassium chloride concentration on protein content and polyphenylalanine synthesizing capability of 40S ribosomal subunits from canine pancreas

Ribosomal small subunits from canine pancreas were used to survey the effects of potassium chloride in the concentration range from 0.4 to 1.25 M. When combined with 60S particles, the treated 40S subunits showed no significant change in phenylalanine incorporating activity until exposure to 0.95 M KCl. Decreases in protein content of the subunits were observed at high ionic strengths. Attempts to separate dissociated ribosomal protein and the remaining core particles treated with 1.25 M KCl by centrifugation of the salt-treated particles though a 40% sucrose cushion led to the observation that ribosomal subparticles isolated in this manner retained full phenylalanine incorporating activity, whereas centrifugation through other solutions resulted in inactive or less active particles. Experiments were performed to elucidate the mechanism by which the 40% sucrose cushion was stabilizing the high salt treated 40S subunits. Two-dimensional gel electrophoresis of the proteins of the various particles isolated in the study was performed. The active 40S particles contained 23-31 protein spots. The isolation of fully active 40S subunits with fewer proteins than previously reported should simplify elucidating their role in the function of the small subunit.     

TrkB and TrkC signaling are required for maturation and synaptogenesis of hippocampal connections

Recent studies have suggested a role for neurotrophins in the growth and refinement of neural connections, in dendritic growth, and in activity-dependent adult plasticity. To unravel the role of endogenous neurotrophins in the development of neural connections in the CNS, we studied the ontogeny of hippocampal afferents in trkB (-/-) and trkC (-/-) mice. Injections of lipophilic tracers in the entorhinal cortex and hippocampus of newborn mutant mice showed that the ingrowth of entorhinal and commissural/associational afferents to the hippocampus was not affected by these mutations. Similarly, injections of biocytin in postnatal mutant mice (P10-P16) did not reveal major differences in the topographic patterns of hippocampal connections. In contrast, quantification of biocytin-filled axons showed that commissural and entorhinal afferents have a reduced number of axon collaterals (21-49%) and decreased densities of axonal varicosities (8-17%) in both trkB (-/-) and trkC (-/-) mice. In addition, electron microscopic analyses showed that trkB (-/-) and trkC (-/-) mice have lower densities of synaptic contacts and important structural alterations of presynaptic boutons, such as decreased density of synaptic vesicles. Finally, immunocytochemical studies revealed a reduced expression of the synaptic-associated proteins responsible for synaptic vesicle exocytosis and neurotransmitter release (v-SNAREs and t-SNAREs), especially in trkB (-/-) mice. We conclude that neither trkB nor trkC genes are essential for the ingrowth or layer-specific targeting of hippocampal connections, although the lack of these receptors results in reduced axonal arborization and synaptic density, which indicates a role for TrkB and TrkC receptors in the developmental regulation of synaptic inputs in the CNS in vivo. The data also suggest that the genes encoding for synaptic proteins may be targets of TrkB and TrkC signaling pathways.     

Two regions of the Escherichia coli 16S ribosomal RNA are important for decoding stop signals in polypeptide chain termination

Two regions of the 16S rRNA, helix 34, and the aminoacyl site component of the decoding site at the base of helix 44, have been implicated in decoding of translational stop signals during the termination of protein synthesis. Antibiotics specific for these regions have been tested to see how they discriminate the decoding of UAA, UAG, and UGA by the two polypeptide chain release factors (RF-1 and RF-2). Spectinomycin, which interacts with helix 34, stimulated RF-1 dependent binding to the ribosome and termination. It also stimulated UGA dependent RF-2 termination at micromolar concentrations but inhibited UGA dependent RF-2 binding at higher concentrations. Alterations at position C1192 of helix 34, known to confer spectinomycin resistance, reduced the binding of f[3H]Met-tRNA to the peptidyl-tRNA site. They also impaired termination in vitro, with both factors and all three stop codons, although the effect was greater with RF-2 mediated reactions. These alterations had previously been shown to inhibit EF-G mediated translocation. As perturbations in helix 34 effect both termination and elongation reactions, these results indicate that helix 34 is close to the decoding site on the bacterial ribosome. Several antibiotics, hygromycin, neomycin and tetracycline, specific for the aminoacyl site, were shown to inhibit the binding and function of both RFs in termination with all three stop codons in vitro. These studies indicate that decoding of all stop signals is likely to occur at a similar site on the ribosome to the decoding of sense codons, the aminoacyl site, and are consistent with a location for helix 34 near this site.