Protein-Facilitated Folding of Group II Intron Ribozymes

Multiple studies hypothesize that DEAD-box proteins facilitate folding of the ai5γ group II intron. However, these conclusions are generally inferred from splicing kinetics, and not from direct monitoring of DEAD-box protein-facilitated folding of the intron. Using native gel electrophoresis and dimethyl sulfate structural probing, we monitored Mss-116-facilitated folding of ai5γ intron ribozymes and a catalytically active self-splicing RNA containing full-length intron and short exons. We found that the protein directly stimulates folding of these RNAs by accelerating formation of the compact near-native state. This process occurs in an ATP-independent manner, although ATP is required for the protein turnover. As Mss 116 binds RNA nonspecifically, most binding events do not result in the formation of the compact state, and ATP is required for the protein to dissociate from such nonproductive complexes and rebind the unfolded RNA. Results obtained from experiments at different concentrations of magnesium ions suggest that Mss 116 stimulates folding of ai5γ ribozymes by promoting the formation of unstable folding intermediates, which is then followed by a cascade of folding events resulting in the formation of the compact near-native state. Dimethyl sulfate probing results suggest that the compact state formed in the presence of the protein is identical to the near-native state formed more slowly in its absence. Our results also indicate that Mss 116 does not stabilize the native state of the ribozyme, but that such stabilization results from binding of attached exons.     

Construction of an Alpha Toxin Gene Knockout Mutant of Clostridium perfringens Type A by Use of a Mobile Group II Intron

In developing Clostridium perfringens as a safe vaccine vector, the alpha toxin gene (plc) in the bacterial chromosome must be permanently inactivated. Disrupting genes in C. perfringens by traditional mutagenesis methods is very difficult. Therefore, we developed a new strategy using group II intron-based Target-Tron technology to inactivate the plc gene in C. perfringens ATCC 3624. Western blot analysis showed no production of alpha toxin protein in the culture supernatant of the plc mutant. Advantages of this technology, such as site specificity, relatively high frequency of insertion, and introduction of no antibiotic resistance genes into the chromosome, could facilitate construction of other C. perfringens mutants.     

Many Independent Origins of trans Splicing of a Plant Mitochondrial Group II Intron

We examined the cis- vs. trans-splicing status of the mitochondrial group II intron nad1i728 in 439 species (427 genera) of land plants, using both Southern hybridization results (for 416 species) and intron sequence data from the literature. A total of 164 species (157 genera), all angiosperms, was found to have a trans-spliced form of the intron. Using a multigene land plant phylogeny, we infer that the intron underwent a transition from cis to trans splicing 15 times among the sampled angiosperms. In 10 cases, the intron was fractured between its 5 end and the intron-encoded matR gene, while in the other 5 cases the fracture occurred between matR and the 3 end of the intron. The 15 intron fractures took place at different time depths during the evolution of angiosperms, with those in Nymphaeales, Austrobaileyales, Chloranthaceae, and eumonocots occurring early in angiosperm evolution and those in Syringodium filiforme, Hydrocharis morsus-ranae, Najas, and Erodium relatively recently. The trans-splicing events uncovered in Austrobaileyales, eumonocots, Polygonales, Caryophyllales, Sapindales, and core Rosales reinforce the naturalness of these major clades of angiosperms, some of which have been identified solely on the basis of recent DNA sequence analyses.     

Mobile Phone Radiation Alters Proliferation of Hepatocarcinoma Cells

This study investigated the effects of intermittent exposure (15 min on, 15 min off for 1, 2, 3, or 4 h, at a specific absorption rate of 2 W/kg) to enhanced data rates for global system for mobile communication evolution-modulated radiofrequency radiation (RFR) at 900- and 1,800-MHz frequencies on the viability of the Hepatocarcinoma cells (Hep G2). Hep G2 cell proliferation was measured by a colorimetric assay based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. Cell injury was evaluated by analyzing the levels of lactate dehydrogenase (LDH) and glucose released from lysed cells into the culture medium. Morphological observation of the nuclei was carried out by 4′,6-diamidino-2-phenylindole (DAPI) staining using fluorescence microscopy. In addition, TUNEL assay was performed to confirm apoptotic cell death. It was observed that cell viability, correlated with the LDH and glucose levels, changed according to the frequency and duration of RFR exposure. Four-hour exposure produced more pronounced effects than the other exposure durations. 1,800-MHz RFR had a larger impact on cell viability and Hep G2 injury than the RFR at 900 MHz. Morphological observations also supported the biochemical results indicating that most of the cells showed irregular nuclei pattern determined by using the DAPI staining, as well as TUNEL assay which shows DNA damage especially in the cells after 4 h of exposure to 1,800-MHz RFR. Our results indicate that the applications of 900- and 1,800-MHz (2 W/kg) RFR cause to decrease in the proliferation of the Hep G2 cells after 4 h of exposure. Further studies will be conducted on other frequency bands of RFR and longer duration of exposure.     

Site-Specific,Insertional Inactivation of incA in Chlamydia trachomatis Using a Group II Intron

Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that has until more recently remained recalcitrant to genetic manipulation. However, the field still remains hindered by the absence of tools to create selectable, targeted chromosomal mutations. Previous work with mobile group II introns demonstrated that they can be retargeted by altering DNA sequences within the intron’s substrate recognition region to create site-specific gene insertions. This platform (marketed as TargeTron™, Sigma) has been successfully employed in a variety of bacteria. We subsequently modified TargeTron™ for use in C. trachomatis and as proof of principle used our system to insertionally inactivate incA, a chromosomal gene encoding a protein required for homotypic fusion of chlamydial inclusions. C. trachomatis incA::GII(bla) mutants were selected with ampicillin and plaque purified clones were then isolated for genotypic and phenotypic analysis. PCR, Southern blotting, and DNA sequencing verified proper GII(bla) insertion, while continuous passaging in the absence of selection demonstrated that the insertion was stable. As seen with naturally occurring IncA⁻ mutants, light and immunofluorescence microscopy confirmed the presence of non-fusogenic inclusions in cells infected with the incA::GII(bla) mutants at a multiplicity of infection greater than one. Lack of IncA production by mutant clones was further confirmed by Western blotting. Ultimately, the ease of retargeting the intron, ability to select for mutants, and intron stability in the absence of selection makes this method a powerful addition to the growing chlamydial molecular toolbox.     

Updated Progress on Group II Intron Splicing Factors in Plant Chloroplasts

Group II introns are large catalytic RNAs (ribozymes) in the bacteria and organelle genomes of several lower eukaryotes. Many critical photosynthesis-related genes in the plant chloroplast genome also contain group II introns, and their splicing is critical for chloroplast biogenesis and photosynthesis processes. The structure of chloroplast group II introns was altered during evolution, resulting in the loss of intron self-splicing. Therefore, the assistance of protein factors was required for their splicing processes. As an increasing number of studies focus on the mechanism of chloroplast intron splicing; many new nuclear-encoded splicing factors that are involved in the chloroplast intron splicing process have been reported. This report reviewed the research progress of the updated splicing factors found to be involved in the splicing of chloroplast group II introns. We discuss the main problems that remain in this research field and suggest future research directions.     

Evolutionary Dynamics of the mS952 Intron: A Novel Mitochondrial Group II Intron Encoding a LAGLIDADG Homing Endonuclease Gene

Examination of the mitochondrial small subunit ribosomal RNA (rns) gene of five species of the fungal genus Leptographium revealed that the gene has been invaded at least once at position 952 by a group II intron encoding a LAGLIDADG homing endonuclease gene. Phylogenetic analyses of the intron and homing endonuclease sequences indicated that each element in Leptographium species forms a single clade and is closely related to the group II intron/homing endonuclease gene composite element previously reported at position 952 of the mitochondrial rns gene of Cordyceps species and of Cryphonectria parasitica. The results of an intron survey of the mt rns gene of Leptographium species superimposed onto the phylogenetic analysis of the host organisms suggest that the composite element was transmitted vertically in Leptographium lundbergii. However, its stochastic distribution among strains of L. wingfieldii, L. terebrantis, and L. truncatum suggests that it has been horizontally transmitted by lateral gene transfer among these species, although the random presence of the intron may reflect multiple random loss events. A model is proposed describing the initial invasion of the group II intron in the rns gene of L. lundbergii by a LAGLIDADG homing endonuclease gene and subsequent evolution of this gene to recognize a novel DNA target site, which may now promote the mobility of the intron and homing endonuclease gene as a composite element.     

The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility

Background

The a2 mating type locus gene lga2 is critical for uniparental mitochondrial DNA inheritance during sexual development of Ustilago maydis. Specifically, the absence of lga2 results in biparental inheritance, along with efficient transfer of intronic regions in the large subunit rRNA gene between parental molecules. However, the underlying role of the predicted LAGLIDADG homing endonuclease gene I-UmaI located within the group II intron LRII1 has remained unresolved.

Methodology/Principal Findings

We have investigated the enzymatic activity of I-UmaI in vitro based on expression of a tagged full-length and a naturally occurring mutant derivative, which harbors only the N-terminal LAGLIDADG domain. This confirmed Mg²⁺-dependent endonuclease activity and cleavage at the LRII1 insertion site to generate four base pair extensions with 3′ overhangs. Specifically, I-UmaI recognizes an asymmetric DNA sequence with a minimum length of 14 base pairs (5′-GACGGGAAGACCCT-3′) and tolerates subtle base pair substitutions within the homing site. Enzymatic analysis of the mutant variant indicated a correlation between the activity in vitro and intron homing. Bioinformatic analyses revealed that putatively functional or former functional I-UmaI homologs are confined to a few members within the Ustilaginales and Agaricales, including the phylogenetically distant species Lentinula edodes, and are linked to group II introns inserted into homologous positions in the LSU rDNA.

Conclusions/Significance

The present data provide strong evidence that intron homing efficiently operates under conditions of biparental inheritance in U. maydis. Conversely, uniparental inheritance may be critical to restrict the transmission of mobile introns. Bioinformatic analyses suggest that I-UmaI-associated introns have been acquired independently in distant taxa and are more widespread than anticipated from available genomic data.     

Gene Targeting in Gram-Negative Bacteria by Use of a Mobile Group II Intron (“Targetron”) Expressed from a Broad-Host-Range Vector

Mobile group II introns (“targetrons”) can be programmed for insertion into virtually any desired DNA target with high frequency and specificity. Here, we show that targetrons expressed via an m-toluic acid-inducible promoter from a broad-host-range vector containing an RK2 minireplicon can be used for efficient gene targeting in a variety of gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. Targetrons expressed from donor plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies of 1 to 58% of screened colonies in the E. coli lacZ, P. aeruginosa pqsA and pqsH, and A. tumefaciens aopB and chvI genes. The development of this broad-host-range system for targetron expression should facilitate gene targeting in many bacteria.     

Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b₅, and NADH/FAD-dependent cytochrome b₅ reductase. mARC proteins share a significant degree of homology to the molybdenum cofactor-binding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo- or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.     

Initiation of DNA Replication in Cells of Higher Eukaryotes

In this review, the problems concerning initiation of DNA replication in higher eukaryotes are discussed, with special emphasis on the methods of replication origin mapping and biological tests for the activity of DNA replication origins in higher eukaryotes. Protein factors interacting with replication origins are considered in detail. The main events of replication initiation in higher eukaryotes are briefly analyzed. New data on the control of replication timing of large genomic regions are discussed.     

Leucyl-tRNA Synthetase-dependent and -independent Activation of a Group I Intron

Leucyl-tRNA synthetase (LeuRS) is an essential RNA splicing factor for yeast mitochondrial introns. Intracellular experiments have suggested that it works in collaboration with a maturase that is encoded within the bI4 intron. RNA deletion mutants of the large bI4 intron were constructed to identify a competently folded intron for biochemical analysis. The minimized bI4 intron was active in RNA splicing and contrasts with previous proposals that the canonical core of the bI4 intron is deficient for catalysis. The activity of the minimized bI4 intron was enhanced in vitro by the presence of the bI4 maturase or LeuRS.Although the aminoacyl-tRNA synthetases (aaRSs)⁶ are best known for their role in protein synthesis, many have functionally expanded and are essential to a wide range of other cellular activities that are unrelated to tRNA aminoacylation (1). The class I aaRSs, leucyl- (LeuRS or NAM2) and tyrosyl-tRNA synthetase (TyrRS or CYT-18) are required for RNA splicing of cognate group I introns in the mitochondria of certain lower eukaryotes (2). In yeast, processing of two related group I introns called bI4 and aI4α (Fig. 1) from the cob and cox1α genes, respectively, require yeast mitochondrial LeuRS (3, 4). Likewise, expression of Neurospora crassa mitochondrial genes, such as those for the large ribosomal RNA, is dependent on TyrRS for excising group I introns (5).Open in a separate windowFIGURE 1.Predicted secondary structures of the bI4 and aI4α group I introns. The secondary structure of the canonical core was based on previous predictions (19). Solid bold lines indicate linear connectivities of the nucleic acid strand with arrowheads oriented in the 5′ to 3′ direction. The dashed lines represent putative tertiary interactions. Dotted lines with numbers identify insertions where secondary structures were ambiguous. Arrows in the P1 and P9 domain show splice sites, whereas boxed nucleotides are paired regions.LeuRS facilitates RNA splicing in concert with a bI4 maturase that is encoded within the bI4 intron. Genetic investigations showed that an inactivated bI4 maturase resulting in deficient splicing activity of the bI4 and aI4α group I introns can be rescued by a suppressor mutation of LeuRS to restore mitochondrial respiration (4, 6). In addition, the splicing defect can be compensated by a mutant aI4α DNA endonuclease that is closely related to the bI4 maturase (7, 8).Previously, we used intracellular three-hybrid assays to demonstrate that LeuRS and bI4 maturase can independently bind to the bI4 intron and stimulate RNA splicing activity in the non-physiological yeast nucleus compartment (9). RNA-dependent two-hybrid assays also supported that the bI4 intron could simultaneously bind both the bI4 maturase and LeuRS. In this case, the RNA was co-expressed with LeuRS and bI4 maturase that was fused to either LexA or B42 to generate a two-hybrid response. This suggested that the bI4 intron was bridging these two protein splicing factors. In either the RNA-dependent two-hybrid or three-hybrid assays, bI4 intron splicing occurred only in the presence of LeuRS or bI4 maturase or both.We hypothesized that the bI4 maturase and LeuRS bind to distinct sites of the bI4 intron to form a ternary complex and promote efficient splicing activity. However, the functional basis of the collaboration between these two splicing cofactors or how either of them promotes RNA splicing remains unclear.We sought to characterize the respective splicing roles of the bI4 maturase and LeuRS via biochemical investigations. Previous attempts to develop an in vitro splicing assay for the bI4 intron or its closely related aI4α intron have failed (10, 11). It was hypothesized that the long length of the bI4 intron (∼1600 nucleotides) and its highly A:U-rich content (∼80%) hindered RNA folding in vitro as well as stabilization of its competent structure.Efforts to produce an active form of the bI4 intron have relied on building chimeric group I introns by interchanging RNA domains with the more stable Tetrahymena thermophila group I intron (11). Based on these results, it was proposed that the catalytic core of the bI4 group I intron was inherently defective (11). In this case, the group I intron would be expected to be completely dependent on its protein splicing factors similar to the bI3 intron that relies on the bI3 maturase and Mrs1 for activity (12). Thus, it was hypothesized that the bI4 maturase and/or LeuRS splicing factors aided the bI4 group I intron by targeting its core region to compensate for these deficiencies.We focused our efforts on re-designing the bI4 intron to develop a minimized molecule that might be competent for splicing. Because both the bI4 and aI4α group I introns rely on the bI4 maturase and LeuRS for their splicing activity, we compared their secondary structures to identify and eliminate peripheral regions outside of their catalytic cores. A small active derivative of the bI4 intron, comprised of just 380 nucleotides primarily from the canonical core, was generated. Thus, we show that, in and of itself, the canonical core of this group I intron is competent for splicing. Both the bI4 maturase and LeuRS enhance the splicing activity of the minimized bI4 intron. However, it is possible that protein-dependent splicing of the bI4 intron represents an intermediate evolutionary step in which the RNA activity is becoming increasingly dependent on its protein splicing factors.     

Inhibition of Fatty Acid Metabolism Reduces Human Myeloma Cells Proliferation

Multiple myeloma is a haematological malignancy characterized by the clonal proliferation of plasma cells. It has been proposed that targeting cancer cell metabolism would provide a new selective anticancer therapeutic strategy. In this work, we tested the hypothesis that inhibition of β-oxidation and de novo fatty acid synthesis would reduce cell proliferation in human myeloma cells. We evaluated the effect of etomoxir and orlistat on fatty acid metabolism, glucose metabolism, cell cycle distribution, proliferation, cell death and expression of G1/S phase regulatory proteins in myeloma cells. Etomoxir and orlistat inhibited β-oxidation and de novo fatty acid synthesis respectively in myeloma cells, without altering significantly glucose metabolism. These effects were associated with reduced cell viability and cell cycle arrest in G0/G1. Specifically, etomoxir and orlistat reduced by 40–70% myeloma cells proliferation. The combination of etomoxir and orlistat resulted in an additive inhibitory effect on cell proliferation. Orlistat induced apoptosis and sensitized RPMI-8226 cells to apoptosis induction by bortezomib, whereas apoptosis was not altered by etomoxir. Finally, the inhibitory effect of both drugs on cell proliferation was associated with reduced p21 protein levels and phosphorylation levels of retinoblastoma protein. In conclusion, inhibition of fatty acid metabolism represents a potential therapeutic approach to treat human multiple myeloma.