A mitochondrial group-I intron in fission yeast encodes a maturase and is mobile in crosses

The open reading frame in the first intron of the mitochondrial gene encoding subunit I of cytochrome c oxidase encodes a maturase and stimulates homologous recombination in Escherichia coli. In this paper, we demonstrate that this intron is mobile in crosses, indicating that it also encodes an endonuclease. This is the first report on an intron which possesses mobility and acts as a maturase.     

A group-I intron in the mitochondrial small subunit ribosomal RNA gene of Sclerotinia sclerotiorum

A 1 380-bp intervening sequence within the mitochondrial small subunit ribosomal RNA (mt SSU rRNA) gene of the fungus Sclerotinia sclerotiorum has been sequenced and identified as a group-I intron. This is the first report of an intron in the mt SSU rRNA gene. The intron shows close similarity in secondary structure to the subgroup-IC2 introns from Podospora (ND3i1, ND5i2, and COIi5) and Neurospora (ND5i1). The intron has an open reading frame (ORF) that encodes a putative protein of 420 amino acids which contains two copies of the LAGLI-DADG motif. The ORF belongs to a family of ORFs identified in Podospora (ND3i1, ND4Li1, ND4Li2, ND5i2, and COIi5) and Neurospora (ND5i1). The putative 420-aa polypeptide is also similar to a site-specific endonuclease in the chloroplast large subunit ribosomal RNA (LSU rRNA) gene of the green alga Chlamydomonas eugametos. In each clone of S. sclerotiorum examined, including several clones which were sampled over a 3-year period from geographically separated sites, all isolates either had the intron or lacked the intron within the mt SSU rRNA gene. Screening by means of Southern hybridization and PCR amplification detected the intron in the mt SSU rRNA genes of S. minor, S. trifoliorum and Sclerotium cepivorum, but not in other members of the Sclerotiniaceae, such as Botrytis anamorphs of Botryotinia spp., or in other ascomycetous and basidiomycetous fungi.     

Guinea pig S19 ribosomal protein as precursor of C5a receptor-directed monocyte-selective leukocyte chemotactic factor

Objective: To reveal the C5a receptor-mediated monocyte-selective chemoattraction of the homo-dimer of guinea pig S19 ribosomal protein (RP S19), and to study the topological relationship between the RP S19 and C5a receptor genes.Methods: cDNA cloning and nucleotide sequencing, leukocyte chemotaxis measurement, and fluorescent in situ hybridization (FISH) were performed in the guinea pig.Results: The amino acid sequence of the guinea pig RP S19 deduced from the cDNA nucleotide sequence was identical to the human protein. The dimer of a recombinant RP S19 attracted guinea pig monocytes but suppressed neutrophil chemotactic movement. Both effects were C5a receptor-mediated. In the FISH analysis, the signals denoting the guinea pig RP S19 gene and C5a receptor gene completely overlapped each other.Conclusions: The guinea pig RP S19 dimer possessed a dual ligand effect, agonistic to the monocyte C5a receptor and antagonistic to the neutrophil receptor. The RP S19 and C5a receptor genes co-localized on the same chromosome.Received 24 April 2004; returned for revision 14 June 2004; accepted by M. Katori 21 June 2004     

A mitochondrial frameshift-suppressor (+) of the yeast S. cerevisiae maps in the mitochondrial 15S rRNA locus

Summary The first case of a +1 extrageneic frameshift suppressor (MF1), mapping in the yeast mitochondrial 15S rRNA gene is reported. The suppressor was identified by genetic analyses in a leaky mitochondrial oxi1 frameshift mutant and the respective wild-type strain 777-3A of the yeast S. cerevisiae. This is in accordance with the finding that all mitochondrial frameshift mutants isolated from this strain tend to be leaky to a variable degree. MF1 does not suppress known nonsense mutations created by a direct basepair exchange in strain 777-3A. These mutants exhibit a non-leaky phenotype (Weiss-Brummer et al. 1984).     

Agonist and antagonist dual effect of the cross-linked S19 ribosomal protein dimer in the C5a receptor-mediated respiratory burst reaction of phagocytic leukocytes

Objective: To examine the behavior of the crosslinked dimer of S19 ribosomal protein (RP S19), a natural C5a receptor ligand, in the C5a receptor-mediated respiratory burst reaction of monocytes and neutrophils.Methods: The respiratory burst reaction of leukocytes was quantitatively observed by continuous spectrophotometric measurement of the reduction of a water-soluble tetrazolium salt, WST-1.Results: The RP S19 dimer induced the respiratory burst of monocytes, but not of neutrophils. Furthermore, in neutrophils, the RP S19 dimer inhibited the reaction induced by C5a, but did not affect the formyl-Met-Leu-Phe-induced reaction. The dimer of a deletion mutant at the C-terminal portion of the RP S19 induced a respiratory burst reaction similar to the one induced by C5a, both in monocytes and neutrophils. Inversely, a chimeric fusion protein between C5a and RPS19, consisting of the addition of the 12 C-terminal amino acid residues of RP S19 after the C-terminal Arg⁷⁴ of the C5a molecule, behaved similarly to the RP S19 dimer.Conclusions: The RP S19 dimer works as an agonist and antagonist of the C5a receptor in the monocyte and the neutrophil respiratory burst reactions, respectively. The switch moiety between the antagonist and agonist of the RP S19 dimer for the C5a receptor in the induction of respiratory burst of phagocytes localizes at the C-terminal region of RP S19.Received 27 August 2004; returned for revision 22 October 2004; accepted by M. Katori 28 October 2004     

Identification of mutations in the ribosomal protein L5 (RPL5) and ribosomal protein L11 (RPL11) genes in Czech patients with Diamond‐Blackfan anemia

Diamond‐Blackfan anemia (DBA) is a congenital red blood cell aplasia that is usually diagnosed during early infancy. Apart from defects in red blood cell maturation, the disorder is also associated with various physical anomalies in 40% of patients. Mutations in the ribosomal protein (RP) S19 are found in 25% of patients, while mutations in other proteins of the small ribosomal subunit—RPS17 and RPS24—have been found in a fraction of patients. Recently, mutations in RPL5, RPL11, and RPL35a of the large ribosomal subunit have also been reported in several DBA patients. Here, we present the identification of mutations in the RPL5 and RPL11 genes in patients from the Czech DBA Registry. Mutations in RPL5 were identified in eight patients from 6 out of 28 families (21.4%), and mutations in RPL11 in two patients from 2 out of 28 families (7.1%). Interestingly, all 10 patients with either an RPL5 or RPL11 mutation exhibited one or more physical anomalies; specifically, thumb anomalies (flat thenar) were always present, while no such anomaly was observed in seven patients with an RPS19 mutation. Moreover, 9 out of 10 patients with either an RPL5 or RPL11 mutation were born small for gestational age (SGA) compared to 3 out of 7 patients from the RPS19‐mutated group. These observations may suggest that mutations, at least in RPL5, seem to generally have more profound impact on fetal development than mutations in RPS19. Since RPL5 and RPL11, together with RPL23, are also involved in the MDM2‐mediated p53 pathway regulation, we also screened the RPL23 gene for mutations; however, no mutations were identified. Hum Mutat 0, 1–7, 2009. © 2009 Wiley‐Liss, Inc.     

Roles of the ribosomal protein S19 dimer and the C5a receptor in pathophysiological functions of phagocytic leukocytes

Monocytes and neutrophils, the major phagocytic leukocytes, migrate to inflammatory sites by sensing chemoattractants such as anaphylatoxin C5a with membrane receptors such as C5a receptor. Upon stimulation, the leukocytes increase cytoplasmic Ca(2+) concentration and generate radical oxygen species. These leukocytes have different functions in inflammation. Neutrophils migrate more rapidly and induce vascular plasma leakage upon infiltration. Monocytes infiltrate tissue more slowly but have superior capacities of phagocytosis and antigen presentation. There must be mechanisms to separately recruit the leukocyte species at an inflammatory site. Ribosomal protein S19 (RP S19) is a component of ribosome. During apoptosis, RP S19 is dimerized and obtains a ligand capacity to C5a receptor. The RP S19 dimer attracts monocytes to phagocytically clear the apoptotic cells that released the dimer molecules. The phagocytic monocytes/macrophages then translocate to regional lymph nodes and present apoptotic cell-derived antigens. Oppositely, the RP S19 dimer inhibits C5a-induced neutrophil migration and promotes apoptosis of neutrophils via the C5a receptor. The RP S19 dimer seems to prevent excessive tissue destruction induced by neutrophils. Skp is a molecular chaperon of Gram-negative bacteria. Skp also attracts monocytes and neutrophils as a ligand of C5a receptor. However, it promotes neither cytoplasmic Ca(2+) enhancement nor radical oxygen generation.     

Role of blood ribosomal protein S19 in coagulum resorption: A study using Gln137Glu‐ribosomal protein S19 gene knock‐in mouse

Sera of human, guinea pig or mouse contain a strong monocyte chemoattractant capacity that is attributed to the ribosomal protein S19 (RP S19) oligomers generated during blood coagulation. In contrast, sera prepared from Gln137Glu‐RP S19 gene knock‐in mice contained negligible chemoattractant capacity. When coagula that had been pre‐formed from the blood of both the wild type and knock‐in mice were intraperitoneally inserted into host mice, after 3 days of recovery, the knock‐in mouse coagula remained larger than the wild type mouse coagula. The wild type mouse coagula were covered by multiple macrophage layers at the surface and were infiltrated inside by macrophages. Knock‐in mouse coagula exhibited less macrophage involvement. When coagula of knock‐in mice and coagula of knock‐in mice containing C5a/RP S19, an artificial substitute of the RP S19 oligomers, were intraperitoneally inserted as pairs, the C5a/RP S19 containing coagulum was more rapidly absorbed, concomitant with increased macrophage involvement. Finally, when the knock‐in mouse and wild type mouse coagula pairs were inserted into mice in which macrophages had been depleted using clodronate liposome, the size difference of recovered coagula was reversed. These results indicate the importance of the RP S19 oligomer‐induced macrophage recruitment in coagulum resorption.     

A group I intron in the chloroplast large subunit rRNA gene of Chlamydomonas eugametos encodes a double-strand endonuclease that cleaves the homing site of this intron

Summary During interspecific crosses between Chlamydomonas eugametos and Chlamydomonas moewusii, an optional group I intron of 955 base pairs (CeLSU· 5) in the C. eugametos chloroplast large subunit rRNA gene undergoes a duplicative transposition event which is associated with frequent co-conversion of flanking cpDNA sequences. In the present study, we show that the basic protein of 218 amino acids encoded by CeLSU· 5 could mediate the phenomenon of intron transposition, also called intron homing. We overexpressed the ORF specifying this protein in E. coli using expression vectors that contain a C. moewusii cpDNA sequence encompassing the intron homing site. The expression product was found to exhibit a double-strand DNA endonuclease activity that is specific for the homing site. This activity was detected in vivo by self-linearization of the expression plasmids.     

Two co-existing mechanisms account for the large-scale deletions of mitochondrial DNA in Podospora anserina that involve the 5′ border of a group-II intron

A degenerative syndrome associated with the accumulation of site-specific deletions within mitochondrial chromosomes occurs in strains of Podospora anserina carrying the AS1-4 nuclear mutation. The site-specific deletion event has been assumed to result from the transposition of a group-II intron (intron α) behind an IBS motif, followed by recombination between the two intron repeats. We show here that a number of distinct deletions can accumulate in AS1-4 strains. Most of them are present in low amounts in wild-type cells where they are only detectable in PCR experiments. The deletions can be divided into two classes. In class I, intron α is joined to an IBS motif. In class II, the intron is not joined to an IBS site, it can be truncated or contain a few upstream exonic nucleotides; some junctions carry non-templated nucleotides. These results indicate that at least two mechanisms are involved in the generation of large-scale mitochondrial deletions in Podospora. One of them seems to be based on the transposition properties of the group-II α intron, the other one on illegitimate recombination. We propose that these two mechanisms use DNA double-strand breaks occurring within the 5′ region of intron α. Received: 15 May / 18 August 1998     

Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase,a key mediator of mTOR function

Current understanding of the mechanisms by which cell growth is regulated lags significantly behind our knowledge of the complex processes controlling cell cycle progression. Recent studies suggest that the mammalian target of rapamycin (mTOR) pathway is a key regulator of cell growth via the regulation of protein synthesis. The key mTOR effectors of cell growth are eukaryotic initiation factor 4E-binding protein 1 (4EBP-1) and the ribosomal protein S6 kinase (S6K). Here we will review the current models for mTOR dependent regulation of ribosome function and biogenesis as well as its role in coordinating growth factor and nutrient signaling to facilitate homeostasis of cell growth and proliferation. We will place particular emphasis on the role of S6K1 signaling and will highlight the points of cross talk with other key growth control pathways. Finally, we will discuss the impact of S6K signaling and the consequent feedback regulation of the PI3K/Akt pathway on disease processes including cancer.     

Involvement of regional neutrophil apoptosis promotion by ribosomal protein S19 oligomers in resolution of experimental acute inflammation

Isolated peripheral neutrophils spontaneously underwent apoptosis in association with extra‐cellular liberation of the monocyte‐attracting ribosomal protein S19 (RP S19) oligomers. This apoptosis was prevented by the simultaneous presence of anti‐RP S19 antibodies or of a C5a receptor antagonist, but was promoted by supplementing extrinsic RP S19 oligomers. Transformed HL‐60 cells to over‐produce Gln137Asn‐mutant RP S19 were differentiated to neutrophil‐like cells. The neutrophil‐like cells gained resistance against the spontaneous apoptosis concomitant with the generation of non‐functional RP S19 oligomers. When the neutrophil‐like cells were intradermally transplanted into mice, the mutant RP S19‐producing neutrophils persisted for a long period of time, whereas wild‐type RP S19‐producing neutrophils underwent apoptosis and were promptly cleared by infiltrated macrophages. When an experimental pleurisy was introduced by injecting carrageenan into the pleural cavity of mice, the inflammation spread slightly to lung parenchyma. When antibodies neutralizing the RP S19 oligomers were simultaneously administrated with carrageenan, the neutrophil infiltration in the lung parenchymal lesion become more severe, occurring as alveolar septal destruction and hemorrhage concomitant with an augmented neutrophil number in the pleural exudate. These results indicate the importance of the RP S19 oligomers and the C5a receptor in neutrophil clearance and acute inflammation resolution.     

The 5S rRNA Gene Clusters Have a Defined Orientation Toward the Nucleolus in Petunia Hybrida and Crepis Capillaris

The 3D localization of the 5S ribosomal RNA genes was studied in cells of the cortex zone of roots in the plant species Petunia hybrida inbred line V26 and in Crepis capillaris. The analysis was carried out on interphase nuclei (both species) and on prophase nuclei (C. capillaris). The 5S ribosomal RNA genes were detected by fluorescence in-situ hybridization and 3D images were obtained by confocal scanning laser microscopy. In both plant species, the 5S ribosomal genes were localized at the short arm of chromosome 2, which, in both plants, also possesses a satellite at its end. Statistical and visual analysis of interphase nuclei showed that: (1) there is a preference for an association of the 5S rRNA gene clusters of the two homologous chromosomes, and (2) the 5S rRNA gene clusters in both species had a preserved spatial position within the interphase nucleus and they tended to be polarized with respect to their neighbouring cells (i.e. a relic telophase orientation). Moreover, tracing of the chromosomal segment between the 5S loci and the active NOR revealed that the homologous chromosomes during early/mid prophase were aligned and that they entered the nucleolus side by side, at least for these chromosome segments. We interpret our data to mean that location of 5S rRNA near the nucleolus favours their functioning in ribosome biogenesis.     

Involvement of cross-linked ribosomal protein S19 oligomers and C5a receptor in definitive erythropoiesis

We performed a series of experiments under a working hypothesis that cross-linked oligomers of ribosomal protein S19 (RP S19) play an essential role in definitive erythropoiesis as a ligand of the C5a receptor of erythroblasts and macrophages. We found molecules functionally and immunologically indistinguishable from RP S19 oligomers in the extracellular fluid of porcine and guinea pig bone marrow. When an increased hematopoietic state was induced in guinea pigs by bloodletting, the bone marrow RP S19 oligomer concentration was concomitantly increased. However, when the RP S19 oligomers were immunologically neutralized or the C5a receptor was pharmacologically antagonized, hyper-erythropoiesis induced by bloodletting was prevented and the anemic state was retarded in guinea pigs. When the RP S19 oligomers were neutralized in mice after bloodletting, the reactive hyper proliferation of erythroblasts in the spleen was prevented. Proerythroblasts and erythroblasts prepared by bone marrow aspiration from healthy individuals were found to express significant levels of the C5a receptor and type 2 transglutaminase genes. Majority of erythroblasts in cord blood of healthy newborns bore the C5a receptor. Taken together, these results support our hypothesis.     

The roles of a ribosomal protein S19 polymer in a mouse model of carrageenan-induced acute pleurisy

C5-deficient mice usually present moderate neutrophil activation during the initiation phase of acute inflammation. Conversely, C5a receptor (C5aR)-deficient mice show unusually excessive activation of neutrophils. We identified the ribosomal protein S19 (RP S19) polymer, which is cross-linked at Lys122 and Gln137 by transglutaminases in apoptotic neutrophils, as a second C5aR ligand during the resolution phase of acute inflammation. The RP S19 polymer promotes apoptosis via the neutrophil C5aR and phagocytosis via the macrophage C5aR. To confirm the roles of the RP S19 polymer, we employed a carrageenan-induced acute pleurisy mouse model using C57BL/6J mice with a knock-in of the Gln137Glu mutant RP S19 gene and replaced the RP S19 polymer with either an S-tagged C5a/RP S19 recombinant protein or the RP S19^122–145 peptide monomer and dimer (as functional C5aR agonists/antagonists) and the RP S19^122–145 peptide trimer (as a functional C5aR antagonist). Neutrophils and macrophages were still present in the thoracic cavities of the knock-in mice at 24 h and 7 days after carrageenan injection, respectively. Knock-in mice showed structural organization and severe hemorrhaging from the surrounding small vessels of the alveolar walls in the lung parenchyma. In contrast to the RP S19^122–145 peptide monomer and trimer, the simultaneous presence of S-tagged C5a/RP S19 and the RP S19^122–145 peptide dimer completely improved the physiological and pathological acute inflammatory cues. The RP S19 polymer, especially the dimer, appears to play a role at the resolution phase of carrageenan-induced acute pleurisy in C57BL/6J model mice.     

Molecular mechanism of monocyte predominant infiltration in chronic inflammation: mediation by a novel monocyte chemotactic factor, S19 ribosomal protein dimer

A novel monocyte chemotactic factor, a cross-linked homodimer of S19 ribosomal protein (RP S19) was initially isolated from a rheumatoid arthritis synovial lesion. The RP S19 dimer causes the monocyte specific chemotaxis in vitro and the monocyte predominant infiltration in vivo, via its agonistic and antagonistic effects on the C5a receptors of monocytes and polymorphonuclear leukocytes, respectively. The agonistic effect is attributed to the similarity of regional structures between RP S19 and C5a, the complement C5-derived leukocyte chemotactic factor, although overall homology of the amino acid sequence between these molecules is only 4%. The antagonistic effect depends upon the C-terminal portion of RP S19. The RP S19 dimer is produced and released by apoptotic cells, and this dimer recruits monocytes from the circulation to the apoptotic lesion. The infiltrated monocytes/macrophages engulf the apoptotic cells, translocate to regional lymph nodes via lymphatics and present the antigenic information of the apoptotic cells to the T cell repertoire. In this manner, the apoptotic cell clearance system connects to the acquired immune system. The innate and acquired immune mechanisms, mediated by the RP S19 dimer, participate in the pathology of inveterate chronic inflammation such as rheumatoid arthritis.     

A ribosomal protein gene cluster is encoded in the mitochondrial DNA of Dictyostelium discoideum: UGA termination codons and similarity of gene order to Acanthamoeba castellanii

We sequenced a region of about 14.5 kb downstream from the ribosomal protein L11 gene (rpl11) in the mitochondrial DNA (54±2 kb) of the cellular slime mold Dictyostelium discoideum. Sequence analysis revealed that eleven ribosomal protein genes and six open reading frames (ORFs) formed a cluster arranged in the order: rpl11–orf189–rps12–rps7–rpl2–rps19–orf425–orf1740–rpl16–rpl14–orf188–rps14–rps8–rpl6–rps13–orf127–orf796. This order was very similar to that of homologous genes in Acanthamoeba castellanii mitochondrial DNA. The N-terminal region of ORF425 and the C-terminal region of ORF1740 had partial similarities to the S3 ribosomal protein of other organisms. The termination codons of rpl16 and orf188 were UGA, which has not hitherto been found in genes encoded in D. discoideum mitochondrial DNA. Received: 28 August 1997 / 2 January 1998     

Nucleotide-sequence analysis indicates that a DNA plasmid in a diseased isolate of Ophiostoma novo-ulmi is derived by recombination between two long repeat sequences in the mitochondrial large subunit ribosomal RNA gene

The nucleotide sequence of a mitochondrial plasmid (2234 bp) in a diseased isolate of Ophiostoma novo-ulmi, and sequences of the mitochondrial DNA that overlap and flank the plasmid end-points, have been determined. The plasmid was shown to be derived from the O. novo-ulmi mitochondrial large subunit ribosomal RNA gene and contained most of intron 1, the whole of exon 2, and probably the first part of intron 2. Within intron 1 there is an open reading frame with the potential to encode a 323 amino-acid polypeptide which contained dodecapeptide sequences typical of RNA maturases and DNA endonucleases. The endpoints of the plasmid in the mtDNA were located within two 90-bp direct imperfect repeat sequences, one of which comprised the last 7 bp of exon 1 and the first 83 bp of intron 1 whilst the other comprised the last 7 bp of exon 2 and the first 83 bp of intron 2. It is proposed that the Ld plasmid was generated by intramolecular recombination between these two repeats with the crossover point probably within the last 15 bp.     

Characterization of a novel open reading frame,urf a,in the mitochondrial genome of fission yeast: Correlation of urf a mutations with a mitochondrial mutator phenotype and a possible role of frameshifting in urf a expression

Summary Between the genes for tRNA^gln and tRNA^ile an open reading frame of 227 amino acids has been identified which is unique among known mitochondrial genomes and which has been termed urf a (Lang et al. 1983; Kornrumpf et al. 1984). It uses the mitochondrial genetic code, i.e., it contains a TGA codon, whereas all other protein-encoding genes, and all but one intronic open reading frame, use the standard genetic code (UGG for tryptophan). A previous paper has demonstrated that mutator strains show an increased formation of mitochondrial drug-resistant and respiration-deficient mutants (including deletions). In this paper we show that the mutator activity is correlated with mutations in urf a. A detailed analysis of one urf a mutant is presented (ana ^r -6), where the deletion of an A residue leads to a frameshift mutation and consequently to premature termination of the putative protein. The phenotype of colonies originating from a single mutant clone varies from no growth up to full growth on non-fermentable substrate. This phenomenon of phenotypic segregation can be explained by the ability of the cell to perform translational frameshifting. A detailed analysis of the DNA sequence and the putative urf a protein will be presented and a possible function of the protein will be discussed.Dedicated to Professor Fritz Kaudewitz on the occasion of his 70th birthday on March 11, 1991.