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1.  Trans-splicing and RNA editing of LSU rRNA in Diplonema mitochondria 
Nucleic Acids Research  2013;42(4):2660-2672.
Mitochondrial ribosomal RNAs (rRNAs) often display reduced size and deviant secondary structure, and sometimes are fragmented, as are their corresponding genes. Here we report a mitochondrial large subunit rRNA (mt-LSU rRNA) with unprecedented features. In the protist Diplonema, the rnl gene is split into two pieces (modules 1 and 2, 534- and 352-nt long) that are encoded by distinct mitochondrial chromosomes, yet the rRNA is continuous. To reconstruct the post-transcriptional maturation pathway of this rRNA, we have catalogued transcript intermediates by deep RNA sequencing and RT-PCR. Gene modules are transcribed separately. Subsequently, transcripts are end-processed, the module-1 transcript is polyuridylated and the module-2 transcript is polyadenylated. The two modules are joined via trans-splicing that retains at the junction ∼26 uridines, resulting in an extent of insertion RNA editing not observed before in any system. The A-tail of trans-spliced molecules is shorter than that of mono-module 2, and completely absent from mitoribosome-associated mt-LSU rRNA. We also characterize putative antisense transcripts. Antisense-mono-modules corroborate bi-directional transcription of chromosomes. Antisense-mt-LSU rRNA, if functional, has the potential of guiding concomitantly trans-splicing and editing of this rRNA. Together, these findings open a window on the investigation of complex regulatory networks that orchestrate multiple and biochemically diverse post-transcriptional events.
doi:10.1093/nar/gkt1152
PMCID: PMC3936708  PMID: 24259427
2.  The Fragmented Mitochondrial Ribosomal RNAs of Plasmodium falciparum 
PLoS ONE  2012;7(6):e38320.
Background
The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis.
Principal Findings
The identification of 14 additional small mitochondrial transcripts from P. falcipaurm and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome.
Significance
All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered.
doi:10.1371/journal.pone.0038320
PMCID: PMC3382252  PMID: 22761677
3.  Broad genomic and transcriptional analysis reveals a highly derived genome in dinoflagellate mitochondria 
BMC Biology  2007;5:41.
Background
Dinoflagellates comprise an ecologically significant and diverse eukaryotic phylum that is sister to the phylum containing apicomplexan endoparasites. The mitochondrial genome of apicomplexans is uniquely reduced in gene content and size, encoding only three proteins and two ribosomal RNAs (rRNAs) within a highly compacted 6 kb DNA. Dinoflagellate mitochondrial genomes have been comparatively poorly studied: limited available data suggest some similarities with apicomplexan mitochondrial genomes but an even more radical type of genomic organization. Here, we investigate structure, content and expression of dinoflagellate mitochondrial genomes.
Results
From two dinoflagellates, Crypthecodinium cohnii and Karlodinium micrum, we generated over 42 kb of mitochondrial genomic data that indicate a reduced gene content paralleling that of mitochondrial genomes in apicomplexans, i.e., only three protein-encoding genes and at least eight conserved components of the highly fragmented large and small subunit rRNAs. Unlike in apicomplexans, dinoflagellate mitochondrial genes occur in multiple copies, often as gene fragments, and in numerous genomic contexts. Analysis of cDNAs suggests several novel aspects of dinoflagellate mitochondrial gene expression. Polycistronic transcripts were found, standard start codons are absent, and oligoadenylation occurs upstream of stop codons, resulting in the absence of termination codons. Transcripts of at least one gene, cox3, are apparently trans-spliced to generate full-length mRNAs. RNA substitutional editing, a process previously identified for mRNAs in dinoflagellate mitochondria, is also implicated in rRNA expression.
Conclusion
The dinoflagellate mitochondrial genome shares the same gene complement and fragmentation of rRNA genes with its apicomplexan counterpart. However, it also exhibits several unique characteristics. Most notable are the expansion of gene copy numbers and their arrangements within the genome, RNA editing, loss of stop codons, and use of trans-splicing.
doi:10.1186/1741-7007-5-41
PMCID: PMC2151934  PMID: 17897476
4.  NSUN4 Is a Dual Function Mitochondrial Protein Required for Both Methylation of 12S rRNA and Coordination of Mitoribosomal Assembly 
PLoS Genetics  2014;10(2):e1004110.
Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m5C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.
Author Summary
Mitochondria perform a number of essential functions in the cell, including synthesis of ATP via the oxidative phosphorylation (OXPHOS) system. Normal mitochondrial function requires coordinated expression of two genomes: mitochondria's own genome (mtDNA), which encodes 13 respiratory chain subunits with essential structural and functional roles for the OXPHOS system, and the nuclear genome encoding the remaining ∼80 subunits. The mtDNA-encoded polypeptides are synthesized on mitochondrial ribosomes (mitoribosomes) located in the mitochondrial matrix. Biogenesis, maintenance and regulation of the complex mitochondrial translation apparatus are poorly understood despite its fundamental importance for cellular energy homeostasis. Here, we show that inactivation of the Nsun4 gene, encoding a mitochondrial m5C-methyltransferase, causes embryonic lethality, whereas tissue-specific disruption of Nsun4 in the heart causes cardiomyopathy with mitochondrial dysfunction. By performing sequencing of bisulfite-treated RNA we report that NSUN4 methylates C911 in 12S rRNA of the small ribosomal subunit. Surprisingly, NSUN4 can on its own perform this rRNA modification, whereas interaction with its partner protein MTERF4 is required for assembly of functional ribosomes. NSUN4 thus has dual roles in ribosome maturation and performs an important final quality control step to ensure that only mature mitoribosomal subunits are assembled into functional ribosomes.
doi:10.1371/journal.pgen.1004110
PMCID: PMC3916286  PMID: 24516400
5.  Rrp5p, Noc1p and Noc2p form a protein module which is part of early large ribosomal subunit precursors in S. cerevisiae 
Nucleic Acids Research  2012;41(2):1191-1210.
Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p–Noc1p–Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity.
doi:10.1093/nar/gks1056
PMCID: PMC3553968  PMID: 23209026
6.  cDNA cloning and gene characterization of the mitochondrial large subunit (LSU) rRNA from the liver fluke Fasciola hepatica. Evidence of heterogeneity in the fluke mitochondrial genome. 
Nucleic Acids Research  1988;16(14B):7001-7012.
A cDNA clone that encodes the large subunit of mitochondrial ribosomal RNA (LSU rRNA) from the liver fluke F. hepatica was isolated and characterized. This RNA molecule is polyadenylated at the 3' end and represents 10% of the poly A+RNA in adult F. hepatica. Fluke LSU rRNA has significant sequence homology to mosquito mitochondria LSU rRNA and is more closely related to the mitochondrial rRNA of hermaphroditic than dioecious trematodes. Mitochondrial DNA constitutes approximately 10% of the total cellular DNA of adult flukes. This percentage is lower in non-embryonated eggs as are the levels of LSU rRNA indicating eggs have lower metabolic activity. Analysis of transcription and the number of mitochondrial genomes in S. mansoni shows that the LSU rRNA is more abundant in females than in males. Restriction endonuclease analysis of the fluke mitochondrial LSU rRNA genes suggests the presence of heterogeneous repeated copies in the mitochondrial genome or heterogeneity among individual genomes of mitochondria.
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PMCID: PMC338347  PMID: 3405756
7.  Transcriptional Down-Regulation and rRNA Cleavage in Dictyostelium discoideum Mitochondria during Legionella pneumophila Infection 
PLoS ONE  2009;4(5):e5706.
Bacterial pathogens employ a variety of survival strategies when they invade eukaryotic cells. The amoeba Dictyostelium discoideum is used as a model host to study the pathogenic mechanisms that Legionella pneumophila, the causative agent of Legionnaire's disease, uses to kill eukaryotic cells. Here we show that the infection of D. discoideum by L. pneumophila results in a decrease in mitochondrial messenger RNAs, beginning more than 8 hours prior to detectable host cell death. These changes can be mimicked by hydrogen peroxide treatment, but not by other cytotoxic agents. The mitochondrial large subunit ribosomal RNA (LSU rRNA) is also cleaved at three specific sites during the course of infection. Two LSU rRNA fragments appear first, followed by smaller fragments produced by additional cleavage events. The initial LSU rRNA cleavage site is predicted to be on the surface of the large subunit of the mitochondrial ribosome, while two secondary sites map to the predicted interface with the small subunit. No LSU rRNA cleavage was observed after exposure of D. discoideum to hydrogen peroxide, or other cytotoxic chemicals that kill cells in a variety of ways. Functional L. pneumophila type II and type IV secretion systems are required for the cleavage, establishing a correlation between the pathogenesis of L. pneumophila and D. discoideum LSU rRNA destruction. LSU rRNA cleavage was not observed in L. pneumophila infections of Acanthamoeba castellanii or human U937 cells, suggesting that L. pneumophila uses distinct mechanisms to interrupt metabolism in different hosts. Thus, L. pneumophila infection of D. discoideum results in dramatic decrease of mitochondrial RNAs, and in the specific cleavage of mitochondrial rRNA. The predicted location of the cleavage sites on the mitochondrial ribosome suggests that rRNA destruction is initiated by a specific sequence of events. These findings suggest that L. pneumophila specifically disrupts mitochondrial protein synthesis in D. discoideum during the course of infection.
doi:10.1371/journal.pone.0005706
PMCID: PMC2683564  PMID: 19492077
8.  Influence of an Oyster Reef on Development of the Microbial Heterotrophic Community of an Estuarine Biofilm 
Applied and Environmental Microbiology  2004;70(11):6834-6845.
We characterized microbial biofilm communities developed over two very closely located but distinct benthic habitats in the Pensacola Bay estuary using two complementary cultivation-independent molecular techniques. Biofilms were grown for 7 days on glass slides held in racks 10 to 15 cm over an oyster reef and an adjacent muddy sand bottom. Total biomass and optical densities of dried biofilms showed dramatic differences for oyster reef versus non-oyster reef biofilms. This study assessed whether the observed spatial variation was reflected in the heterotrophic prokaryotic species composition. Genomic biofilm DNA from both locations was isolated and served as a template to amplify 16S rRNA genes with universal eubacterial primers. Fluorescently labeled PCR products were analyzed by terminal restriction fragment length polymorphism, creating a genetic fingerprint of the composition of the microbial communities. Unlabeled PCR products were cloned in order to construct a clone library of 16S rRNA genes. Amplified ribosomal DNA restriction analysis was used to screen and define ribotypes. Partial sequences from unique ribotypes were compared with existing database entries to identify species and to construct phylogenetic trees representative of community structures. A pronounced difference in species richness and evenness was observed at the two sites. The biofilm community structure from the oyster reef setting had greater evenness and species richness than the one from the muddy sand bottom. The vast majority of the bacteria in the oyster reef biofilm were related to members of the γ- and δ-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium -Bacteroides cluster, and the phyla Planctomyces and Holophaga-Acidobacterium. The same groups were also present in the biofilm harvested at the muddy sand bottom, with the difference that nearly half of the community consisted of representatives of the Planctomyces phylum. Total species richness was estimated to be 417 for the oyster reef and 60 for the muddy sand bottom, with 10.5% of the total unique species identified being shared between habitats. The results suggest dramatic differences in habitat-specific microbial diversity that have implications for overall microbial diversity within estuaries.
doi:10.1128/AEM.70.11.6834-6845.2004
PMCID: PMC525144  PMID: 15528551
9.  Tertiary Endosymbiosis in Two Dinotoms Has Generated Little Change in the Mitochondrial Genomes of Their Dinoflagellate Hosts and Diatom Endosymbionts 
PLoS ONE  2012;7(8):e43763.
Background
Mitochondria or mitochondrion-derived organelles are found in all eukaryotes with the exception of secondary or tertiary plastid endosymbionts. In these highly reduced systems, the mitochondrion has been lost in all cases except the diatom endosymbionts found in a small group of dinoflagellates, called ‘dinotoms’, the only cells with two evolutionarily distinct mitochondria. To investigate the persistence of this redundancy and its consequences on the content and structure of the endosymbiont and host mitochondrial genomes, we report the sequences of these genomes from two dinotoms.
Methodology/Principal Findings
The endosymbiont mitochondrial genomes of Durinskia baltica and Kryptoperidinium foliaceum exhibit nearly identical gene content with other diatoms, and highly conserved gene order (nearly identical to that of the raphid pennate diatom Fragilariopsis cylindrus). These two genomes are differentiated from other diatoms' by the fission of nad11 and by an insertion within nad2, in-frame and unspliced from the mRNA. Durinskia baltica is further distinguished from K. foliaceum by two gene fusions and its lack of introns. The host mitochondrial genome in D. baltica encodes cox1 and cob plus several fragments of LSU rRNA gene in a hugely expanded genome that includes numerous pseudogenes, and a trans-spliced cox3 gene, like in other dinoflagellates. Over 100 distinct contigs were identified through 454 sequencing, but intact full-length genes for cox1, cob and the 5′ exon of cox3 were present as a single contig each, suggesting most of the genome is pseudogenes. The host mitochondrial genome of K. foliaceum was difficult to identify, but fragments of all the three protein-coding genes, corresponding transcripts, and transcripts of several LSU rRNA fragments were all recovered.
Conclusions/Significance
Overall, the endosymbiont and host mitochondrial genomes in the two dinotoms have changed surprisingly little from those of free-living diatoms and dinoflagellates, irrespective of their long coexistence side by side in dinotoms.
doi:10.1371/journal.pone.0043763
PMCID: PMC3423374  PMID: 22916303
10.  A Structural Model for the Large Subunit of the Mammalian Mitochondrial Ribosome 
Journal of molecular biology  2006;358(1):193-212.
Protein translation is essential for all forms of life and is conducted by a macromolecular complex, the ribosome. Evolutionary changes in protein and RNA sequences can affect the three-dimensional organization of structural features in ribosomes in different species. The most dramatic changes occur in animal mitochondria, whose genomes have been significantly reduced and altered. The RNA component of the mitochondrial ribosome (mitoribosome) is reduced in size, with a compensatory increase in protein content. Until recently, it was unclear how these changes affect the three-dimensional structure of the mitoribosome. Here we present a structural model of the large subunit (LSU) of the mammalian mitoribosome developed by combining molecular modeling techniques with cryo-electron microscopic (cryo-EM) studies. The model contains 93% of the mitochondrial rRNA (mito-rRNA) sequence and 16 mitochondrial ribosomal proteins (MRPs) in the large subunit of the mitoribosome. Despite the smaller mitochondrial rRNA, the spatial positions of RNA domains known to be directly involved in protein synthesis are essentially the same as in Bacterial and Archaeal ribosomes. However, the dramatic reduction in rRNA content necessitates evolution of unique structural features to maintain connectivity between RNA domains. The smaller rRNA sequence also limits the likelihood of tRNA binding at E-site of the mitoribosome, and correlates with the reduced size of D- and T-loops in some animal mitochondrial tRNAs, suggesting co-evolution of mitochondrial rRNA and tRNA structures.
doi:10.1016/j.jmb.2006.01.094
PMCID: PMC3495566  PMID: 16510155
11.  Characterization of fragmented mitochondrial ribosomal RNAs of the colorless green alga Polytomella parva 
Nucleic Acids Research  2003;31(2):769-778.
We have identified previously in mitochondrial DNA of the colorless, chlorophycean, green algal taxon, Polytomella parva, potential coding regions for four small subunit (SSU) and eight large subunit (LSU) rRNA fragments. In this study with P.parva, we isolated RNA from a mitochondrial-enriched preparation, characterized the 12 mitochondrial rRNA transcripts by either northern blot analysis or chemical sequencing and performed secondary structure modeling of the SSU and LSU rRNA sequences. The results show the following features about the mitochondrial SSU and LSU rRNAs of P.parva: (i) they are considerably shorter than their homologs from other green algae, although the main domains typical of conventional rRNAs are conserved; (ii) the rRNA fragmentation pattern is most similar to that of Chlamydomonas reinhardtii among green algae that have been characterized; (iii) three nucleotides are missing from the normally highly conserved GTPase center of the LSU rRNA; and (iv) post-transcriptional modification of the 3′-terminal region of the SSU rRNA is unusual in that it has the ‘eubacterial’ 3-methyluridine (corresponding to m3U at Escherichia coli 16S rRNA position 1498) but lacks the more highly conserved modifications at two adjacent A residues (corresponding to N6,N6-dimethyladenosine at E.coli 16S rRNA positions 1518 and 1519). This is the first report of the characterization by direct sequencing of fragmented mitochondrial rRNAs from a green alga.
PMCID: PMC140509  PMID: 12527787
12.  Methylated regions of hamster mitochondrial ribosomal RNA: structural and functional correlates. 
Nucleic Acids Research  1981;9(2):323-337.
The positions of post-transcriptionally methylated residues within hamster mitochondrial ribosomal RNA have been established. Comparisons with other mitochondrial rRNA, and with bacterial, eucaryotic and chloroplast rRNA show that the methylated regions i) are comprised of conserved primary sequences and/or secondary structures and ii) are situated at the subunit interface of the ribosome. The comparative analyses also reveal that the ribose-methylated sequence UmGmU of hamster mitochondrial large ribosomal subunit (LSU1) RNA lies in a universally conserved hairpin loop which contains a putative puromycin-reactive nucleotide. The "UmGmU hairpin" is within 100 nucleotides of two chloramphenicol-resistance residues of LSU RNA. We present a secondary structure for this region which is conserved in LSU RNAs. This structure allows physical juxtaposition of the three antibiotic-interacting loci and thus defines RNA components of the ribosomal-binding site for the 3'-terminus of aminoacyl-tRNA.
Images
PMCID: PMC326695  PMID: 6782552
13.  rRNA Maturation in Yeast Cells Depleted of Large Ribosomal Subunit Proteins 
PLoS ONE  2009;4(12):e8249.
The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein – rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5′ end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.
doi:10.1371/journal.pone.0008249
PMCID: PMC2788216  PMID: 20011513
14.  "Tandem duplication-random loss" is not a real feature of oyster mitochondrial genomes 
BMC Genomics  2009;10:84.
Duplications and rearrangements of coding genes are major themes in the evolution of mitochondrial genomes, bearing important consequences in the function of mitochondria and the fitness of organisms. Yu et al. (BMC Genomics 2008, 9:477) reported the complete mt genome sequence of the oyster Crassostrea hongkongensis (16,475 bp) and found that a DNA segment containing four tRNA genes (trnK1, trnC, trnQ1 and trnN), a duplicated (rrnS) and a split rRNA gene (rrnL5') was absent compared with that of two other Crassostrea species. It was suggested that the absence was a novel case of "tandem duplication-random loss" with evolutionary significance. We independently sequenced the complete mt genome of three C. hongkongensis individuals, all of which were 18,622 bp and contained the segment that was missing in Yu et al.'s sequence. Further, we designed primers, verified sequences and demonstrated that the sequence loss in Yu et al.'s study was an artifact caused by placing primers in a duplicated region. The duplication and split of ribosomal RNA genes are unique for Crassostrea oysters and not lost in C. hongkongensis. Our study highlights the need for caution when amplifying and sequencing through duplicated regions of the genome.
doi:10.1186/1471-2164-10-84
PMCID: PMC2652495  PMID: 19228377
15.  Primary and secondary structures of Escherichia coli MRE 600 23S ribosomal RNA. Comparison with models of secondary structure for maize chloroplast 23S rRNA and for large portions of mouse and human 16S mitochondrial rRNAs 
Nucleic Acids Research  1981;9(17):4303-4324.
We determined 90% of the primary structure of E.coli MRE 600 23S rRNA by applying the sequencing gel technique to products of T1, S1, A and Naja oxiana nuclease digestion. Eight cistron heterogeneities were detected, as well as 16 differences with the published sequence of a 23S rRNA gene of an E.coli K12 strain. The positions of 13 post-transcriptionally modified nucleotides and of single-stranded, double-stranded and subunit surface regions of E.coli 23S rRNA were identified. Using these experimental results and by comparing the sequences of E.coli 23S rRNA, maize chloro. 23S rRNA and mouse and human mit 16S rRNAs, we built models of secondary structure for the two 23S rRNAs and for large portions of the two mit rRNAs. The structures proposed for maize chloroplast and E.coli 23S rRNAs are very similar, consisting of 7 domains closed by long-range base-pairings. In the mitochondrial 16S rRNAs, 3 of these domains are strongly reduced in size and have a very different primary structure compared to those of the 23S rRNAs. These domains were previously found to constitute a compact area in the E.coli 50S subunits. The conserved domains do not belong to this area and contain almost all the modified nucleotides. The most highly conserved domain, 2042-2625, is probably part of the ribosomal A site. Finally, our study strongly suggests that in cytoplasmic ribosomes the 3′-end of 5.8S rRNA is basepaired with the 5′-end of 26S rRNA. This confirms the idea that 5.8S RNA is the counterpart of the 5′-terminal region of prokaryotic 23S rRNA.
PMCID: PMC327436  PMID: 6170936
16.  Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae 
PLoS ONE  2013;8(7):e68412.
During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.
doi:10.1371/journal.pone.0068412
PMCID: PMC3707915  PMID: 23874617
17.  Molecular characterization and mRNA expression of two key enzymes of hypoxia-sensing pathways in eastern oysters Crassostrea virginica (Gmelin): Hypoxia-inducible factor α (HIF-α) and HIF-prolyl hydroxylase (PHD) 
Oxygen homeostasis is crucial for development, survival and normal function of all metazoans. A family of transcription factors called hypoxia-inducible factors (HIF) is critical in mediating the adaptive responses to reduced oxygen availability. The HIF transcription factor consists of a constitutively expressed β subunit and an oxygen-dependent α subunit; the abundance of the latter determines the activity of HIF and is regulated by a family of O2- and Fe2+-dependent enzymes prolyl hydroxylases (PHDs). Currently very little is known about the function of this important pathway and the molecular structure of its key players in hypoxia-tolerant intertidal mollusks including oysters, which are among the animal champions of anoxic and hypoxic tolerance and thus can serve as excellent models to study the role of HIF cascade in adaptations to oxygen deficiency. We have isolated transcripts of two key components of the oxygen sensing pathway - the oxygen-regulated HIF-α subunit and PHD - from an intertidal mollusk, the eastern oyster Crassostrea virginica, and determined the transcriptional responses of these two genes to anoxia, hypoxia and cadmium (Cd) stress. HIF-α and PHD homologs from eastern oysters C. virginica show significant sequence similarity and share key functional domains with the earlier described isoforms from vertebrates and invertebrates. Phylogenetic analysis shows that genetic diversification of HIF and PHD isoforms occurred within the vertebrate lineage indicating functional diversification and specialization of the oxygen-sensing pathways in this group, which parallels situation observed for many other important genes. HIF-α and PHD homologs are broadly expressed at the mRNA level in different oyster tissues and show transcriptional responses to prolonged hypoxia in the gills consistent with their putative role in oxygen sensing and the adaptive response to hypoxia. Similarity in amino acid sequence, domain structure and transcriptional responses between HIF-α and PHD homologs from oysters and other invertebrate and vertebrate species implies the highly conserved functions of these genes throughout the evolutionary history of animals, in accordance with their critical role in oxygen sensing and homeostasis.
doi:10.1016/j.cbd.2010.10.003
PMCID: PMC3102143  PMID: 21106446
Oxygen sensing; hypoxia-inducible factor; evolution; expression; intertidal mollusk
18.  Apoptosis-like programmed cell death induces antisense ribosomal RNA (rRNA) fragmentation and rRNA degradation in Leishmania 
Cell Death and Differentiation  2012;19(12):1972-1982.
Few natural antisense (as) RNAs have been reported as yet in the unicellular protozoan Leishmania. Here, we describe that Leishmania produces natural asRNAs complementary to all ribosomal RNA (rRNA) species. Interestingly, we show that drug-induced apoptosis-like programmed cell death triggers fragmentation of asRNA complementary to the large subunit gamma (LSU-γ) rRNA, one of the six 28S rRNA processed fragments in Leishmania. Heat and oxidative stress also induce fragmentation of asrRNA, but to a lesser extent. Extensive asrRNA cleavage correlates with rRNA breakdown and translation inhibition. Indeed, overexpression of asLSU-γ rRNA accelerates rRNA degradation upon induction of apoptosis. In addition, we provide mechanistic insight into the regulation of apoptosis-induced asrRNA fragmentation by a 67 kDa ATP-dependent RNA helicase of the DEAD-box subfamily. This helicase binds both sense (s)LSU-γ and asLSU-γ rRNAs, and appears to have a key role in protecting rRNA from degradation by preventing asrRNA cleavage and thus cell death. Remarkably, the asrRNA fragmentation process operates not only in trypanosomatid protozoa but also in mammals. Our findings uncover a novel mechanism of regulation involving asrRNA fragmentation and rRNA breakdown, that is triggered by apoptosis and conditions of reduced translation under stress, and seems to be evolutionary conserved.
doi:10.1038/cdd.2012.85
PMCID: PMC3504711  PMID: 22767185
Leishmania; stress; apoptosis-like programmed cell death; antisense rRNA fragmentation; rRNA degradation; ATP-dependent DEAD-box helicase
19.  Microsporidian Encephalitozoon cuniculi, a unicellular eukaryote with an unusual chromosomal dispersion of ribosomal genes and a LSU rRNA reduced to the universal core. 
Nucleic Acids Research  1998;26(15):3513-3520.
Microsporidia are eukaryotic parasites lacking mitochondria, the ribosomes of which present prokaryote-like features. In order to better understand the structural evolution of rRNA molecules in microsporidia, the 5S and rDNA genes were investigated in Encephalitozoon cuniculi . The genes are not in close proximity. Non-tandemly arranged rDNA units are on every one of the 11 chromosomes. Such a dispersion is also shown in two other Encephalitozoon species. Sequencing of the 5S rRNA coding region reveals a 120 nt long RNA which folds according to the eukaryotic consensus structural shape. In contrast, the LSU rRNA molecule is greatly reduced in length (2487 nt). This dramatic shortening is essentially due to truncation of divergent domains, most of them being removed. Most variable stems of the conserved core are also deleted, reducing the LSU rRNA to only those structural features preserved in all living cells. This suggests that the E.cuniculi LSU rRNA performs only the basic mechanisms of translation. LSU rRNA phylogenetic analysis with the BASEML program favours a relatively recent origin of the fast evolving microsporidian lineage. Therefore, the prokaryote-like ribosomal features, such as the absence of ITS2, may be derived rather than primitive characters.
PMCID: PMC147740  PMID: 9671812
20.  Complete mitochondrial DNA sequence of oyster Crassostrea hongkongensis-a case of "Tandem duplication-random loss" for genome rearrangement in Crassostrea? 
BMC Genomics  2008;9:477.
Background
Mitochondrial DNA sequences are extensively used as genetic markers not only for studies of population or ecological genetics, but also for phylogenetic and evolutionary analyses. Complete mt-sequences can reveal information about gene order and its variation, as well as gene and genome evolution when sequences from multiple phyla are compared. Mitochondrial gene order is highly variable among mollusks, with bivalves exhibiting the most variability. Of the 41 complete mt genomes sequenced so far, 12 are from bivalves. We determined, in the current study, the complete mitochondrial DNA sequence of Crassostrea hongkongensis. We present here an analysis of features of its gene content and genome organization in comparison with two other Crassostrea species to assess the variation within bivalves and among main groups of mollusks.
Results
The complete mitochondrial genome of C. hongkongensis was determined using long PCR and a primer walking sequencing strategy with genus-specific primers. The genome is 16,475 bp in length and contains 12 protein-coding genes (the atp8 gene is missing, as in most bivalves), 22 transfer tRNA genes (including a suppressor tRNA gene), and 2 ribosomal RNA genes, all of which appear to be transcribed from the same strand. A striking finding of this study is that a DNA segment containing four tRNA genes (trnk1, trnC, trnQ1 and trnN) and two duplicated or split rRNA gene (rrnL5' and rrnS) are absent from the genome, when compared with that of two other extant Crassostrea species, which is very likely a consequence of loss of a single genomic region present in ancestor of C. hongkongensis. It indicates this region seem to be a "hot spot" of genomic rearrangements over the Crassostrea mt-genomes. The arrangement of protein-coding genes in C. hongkongensis is identical to that of Crassostrea gigas and Crassostrea virginica, but higher amino acid sequence identities are shared between C. hongkongensis and C. gigas than between other pairs. There exists significant codon bias, favoring codons ending in A or T and against those ending with C. Pair analysis of genome rearrangements showed that the rearrangement distance is great between C. gigas-C. hongkongensis and C. virginica, indicating a high degree of rearrangements within Crassostrea. The determination of complete mt-genome of C. hongkongensis has yielded useful insight into features of gene order, variation, and evolution of Crassostrea and bivalve mt-genomes.
Conclusion
The mt-genome of C. hongkongensis shares some similarity with, and interesting differences to, other Crassostrea species and bivalves. The absence of trnC and trnN genes and duplicated or split rRNA genes from the C. hongkongensis genome is a completely novel feature not previously reported in Crassostrea species. The phenomenon is likely due to the loss of a segment that is present in other Crassostrea species and was present in ancestor of C. hongkongensis, thus a case of "tandem duplication-random loss (TDRL)". The mt-genome and new feature presented here reveal and underline the high level variation of gene order and gene content in Crassostrea and bivalves, inspiring more research to gain understanding to mechanisms underlying gene and genome evolution in bivalves and mollusks.
doi:10.1186/1471-2164-9-477
PMCID: PMC2576254  PMID: 18847502
21.  Analysis of Ribosomal Protein Gene Structures: Implications for Intron Evolution  
PLoS Genetics  2006;2(3):e25.
Many spliceosomal introns exist in the eukaryotic nuclear genome. Despite much research, the evolution of spliceosomal introns remains poorly understood. In this paper, we tried to gain insights into intron evolution from a novel perspective by comparing the gene structures of cytoplasmic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs), which are held to be of archaeal and bacterial origin, respectively. We analyzed 25 homologous pairs of CRP and MRP genes that together had a total of 527 intron positions. We found that all 12 of the intron positions shared by CRP and MRP genes resulted from parallel intron gains and none could be considered to be “conserved,” i.e., descendants of the same ancestor. This was supported further by the high frequency of proto-splice sites at these shared positions; proto-splice sites are proposed to be sites for intron insertion. Although we could not definitively disprove that spliceosomal introns were already present in the last universal common ancestor, our results lend more support to the idea that introns were gained late. At least, our results show that MRP genes were intronless at the time of endosymbiosis. The parallel intron gains between CRP and MRP genes accounted for 2.3% of total intron positions, which should provide a reliable estimate for future inferences of intron evolution.
Synopsis
Genes in eukaryotes are usually intervened by extra bits of DNA sequence, called introns, that have to be removed after the genes are transcribed into RNA. Why do introns exist in eukaryotic genes? What is the reason for the increased intron density in higher eukaryotes? There is much that is not known about introns. This research tries to clarify the evolutionary process by which introns arose by comparing the gene structures of two types of ribosomal proteins; one in cytoplasm and the other in mitochondria of the cell. Since cytoplasm and mitochondria are of archaeal and bacterial origin, respectively, cytoplasmic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) are believed to diverge at the same time with the divergence of archaea and bacteria. Thus, a comparative analysis of CRP and MRP genes may reveal whether introns already existed at the last common ancestor of archaea and bacteria (introns-early) or whether they emerged late (introns-late). The results make it clear, at least, that all of the introns in MRP genes were gained during the course of eukaryotic evolution and therefore lend more support to the introns-late theory.
doi:10.1371/journal.pgen.0020025
PMCID: PMC1386722  PMID: 16518464
22.  Abundant 5S rRNA-Like Transcripts Encoded by the Mitochondrial Genome in Amoebozoa ▿ † 
Eukaryotic Cell  2010;9(5):762-773.
5S rRNAs are ubiquitous components of prokaryotic, chloroplast, and eukaryotic cytosolic ribosomes but are apparently absent from mitochondrial ribosomes (mitoribosomes) of many eukaryotic groups including animals and fungi. Nevertheless, a clearly identifiable, mitochondrion-encoded 5S rRNA is present in Acanthamoeba castellanii, a member of Amoebozoa. During a search for additional mitochondrial 5S rRNAs, we detected small abundant RNAs in other members of Amoebozoa, namely, in the lobose amoeba Hartmannella vermiformis and in the myxomycete slime mold Physarum polycephalum. These RNAs are encoded by mitochondrial DNA (mtDNA), cosediment with mitoribosomes in glycerol gradients, and can be folded into a secondary structure similar to that of bona fide 5S rRNAs. Further, in the mtDNA of another slime mold, Didymium nigripes, we identified a region that in sequence, potential secondary structure, and genomic location is similar to the corresponding region encoding the Physarum small RNA. A mtDNA-encoded small RNA previously identified in Dictyostelium discoideum is here shown to share several characteristics with known 5S rRNAs. Again, we detected genes encoding potential homologs of this RNA in the mtDNA of three other species of the genus Dictyostelium as well as in a related genus, Polysphondylium. Taken together, our results indicate a widespread occurrence of small, abundant, mtDNA-encoded RNAs with 5S rRNA-like structures that are associated with the mitoribosome in various amoebozoan taxa. Our working hypothesis is that these novel small abundant RNAs represent radically divergent mitochondrial 5S rRNA homologs. We posit that currently unrecognized 5S-like RNAs may exist in other mitochondrial systems in which a conventional 5S rRNA cannot be identified.
doi:10.1128/EC.00013-10
PMCID: PMC2863963  PMID: 20304999
23.  Nucleotide sequence and evolution of the 18S ribosomal RNA gene in maize mitochondria. 
Nucleic Acids Research  1984;12(16):6629-6644.
The nucleotide sequence of the gene coding for the 18S ribosomal RNA of maize mitochondria has been determined and a model for the secondary structure is proposed. Dot matrix analysis has been used to compare the extent and distribution of sequence similarities of the entire maize mitochondrial 18S rRNA sequence with that of 15 other small subunit rRNA sequences. The mitochondrial gene shows great similarity to the eubacterial sequences and to the maize chloroplast, and less similarity to mitochondrial rRNA genes in animals and fungi. We propose that this similarity is due to a slow rate of nucleotide divergence in plant mtDNA compared to the mtDNA of animals. Sequence comparisons indicate that the evolution of the maize mitochondrial 18S, chloroplast 16S and nuclear 17S ribosomal genes have been essentially independent, in spite of evidence for DNA transfer between organelles and the nucleus.
PMCID: PMC320101  PMID: 6089124
24.  Molecular evolution of rDNA in early diverging Metazoa: First comparative analysis and phylogenetic application of complete SSU rRNA secondary structures in Porifera 
Background
The cytoplasmic ribosomal small subunit (SSU, 18S) ribosomal RNA (rRNA) is the most frequently-used gene for molecular phylogenetic studies. However, information regarding its secondary structure is neglected in most phylogenetic analyses. Incorporation of this information is essential in order to apply specific rRNA evolutionary models to overcome the problem of co-evolution of paired sites, which violates the basic assumption of the independent evolution of sites made by most phylogenetic methods. Information about secondary structure also supports the process of aligning rRNA sequences across taxa. Both aspects have been shown to increase the accuracy of phylogenetic reconstructions within various taxa.
Here, we explore SSU rRNA secondary structures from the three extant classes of Phylum Porifera (Grant, 1836), a pivotal, but largely unresolved taxon of early branching Metazoa. This is the first phylogenetic study of poriferan SSU rRNA data to date that includes detailed comparative secondary structure information for all three sponge classes.
Results
We found base compositional and structural differences in SSU rRNA among Demospongiae, Hexactinellida (glass sponges) and Calcarea (calcareous sponges). We showed that analyses of primary rRNA sequences, including secondary structure-specific evolutionary models, in combination with reconstruction of the evolution of unusual structural features, reveal a substantial amount of additional information. Of special note was the finding that the gene tree topologies of marine haplosclerid demosponges, which are inconsistent with the current morphology-based classification, are supported by our reconstructed evolution of secondary structure features. Therefore, these features can provide alternative support for sequence-based topologies and give insights into the evolution of the molecule itself. To encourage and facilitate the application of rRNA models in phylogenetics of early metazoans, we present 52 SSU rRNA secondary structures over the taxonomic range of Porifera in a database, along with some basic tools for relevant format-conversion.
Conclusion
We demonstrated that sophisticated secondary structure analyses can increase the potential phylogenetic information of already available rDNA sequences currently accessible in databases and conclude that the importance of SSU rRNA secondary structure information for phylogenetic reconstruction is still generally underestimated, at least among certain early branching metazoans.
doi:10.1186/1471-2148-8-69
PMCID: PMC2289807  PMID: 18304338
25.  Systematically fragmented genes in a multipartite mitochondrial genome 
Nucleic Acids Research  2010;39(3):979-988.
Arguably, the most bizarre mitochondrial DNA (mtDNA) is that of the euglenozoan eukaryote Diplonema papillatum. The genome consists of numerous small circular chromosomes none of which appears to encode a complete gene. For instance, the cox1 coding sequence is spread out over nine different chromosomes in non-overlapping pieces (modules), which are transcribed separately and joined to a contiguous mRNA by trans-splicing. Here, we examine how many genes are encoded by Diplonema mtDNA and whether all are fragmented and their transcripts trans-spliced. Module identification is challenging due to the sequence divergence of Diplonema mitochondrial genes. By employing most sensitive protein profile search algorithms and comparing genomic with cDNA sequence, we recognize a total of 11 typical mitochondrial genes. The 10 protein-coding genes are systematically chopped up into three to 12 modules of 60–350 bp length. The corresponding mRNAs are all trans-spliced. Identification of ribosomal RNAs is most difficult. So far, we only detect the 3′-module of the large subunit ribosomal RNA (rRNA); it does not trans-splice with other pieces. The small subunit rRNA gene remains elusive. Our results open new intriguing questions about the biochemistry and evolution of mitochondrial trans-splicing in Diplonema.
doi:10.1093/nar/gkq883
PMCID: PMC3035467  PMID: 20935050

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