Characterization of the rRNA-encoding genes and transcripts, and a group-I self-splicing intron in Pneumocystis carinii

Lin, Hua; Niu, Manette T.; Yoganathan, Thillainathan; Buck, Gregory A.

doi:10.1016/0378-1119(92)90268-t

Cited by 28 publications

(24 citation statements)

References 27 publications

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“…3Ј end radiolabeled precursor. Such structural heterogeneity is commonly seen for pre-rRNA transcripts and ribozymes (21,(25)(26)(27)(28). Attempts to produce more homogeneous solutions by using published protocols (26,27) did not alter these results (data not shown).…”

Section: Discussionmentioning

confidence: 55%

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In vitro suicide inhibition of self-splicing of a group I intron from Pneumocystis carinii by an N 3′ → P 5′ phosphoramidate hexanucleotide

Testa

Gryaznov

Turner

1999

Proc. Natl. Acad. Sci. U.S.A.

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Binding enhancement by tertiary interactions is a strategy that takes advantage of the higher order folding of functionally important RNAs to bind short nucleic acid-based compounds tightly and more specifically than possible by simple base pairing. For example, tertiary interactions enhance binding of specific hexamers to a group I intron ribozyme from the opportunistic pathogen Pneumocystis carinii by 1,000-to 100,000-fold relative to binding by only base pairing. One such hexamer, d(AnTnGnAnCn)rU, contains an N3 3 P5 phosphoramidate deoxysugar-phosphate backbone (n) that is resistant to chemical and enzymatic decay. Here, it is shown that this hexamer is also a suicide inhibitor of the intron's self-splicing reaction in vitro. The hexamer is ligated in trans to the 3 exon of the precursor, producing dead-end products. At 4 mM Mg 2؉ , the fraction of trans-spliced product is greater than normally spliced product at hexamer concentrations as low as 200 nM. This provides an additional level of specificity for compounds that can exploit the catalytic potential of complexes with RNA targets.Most human therapeutics have been discovered by screening natural products. Synthetic organic chemistry has made it possible to synthesize such natural products and derivatives thereof in large quantities, thus broadening the range of compounds that can be used clinically (1-3). Synthetic methodology coupled with the outpouring of protein structural information has also allowed rational design of completely new therapeutic compounds (4,5). Similarly, the recent explosion in nucleic acid sequence information is providing a knowledge base for structure-based targeting of RNA. The first generation of such therapeutics consists of antisense nucleic acids that bind mRNA through Watson-Crick base pairing and thereby regulate translation (6, 7). Because of the long sequences employed, typically 15-20 nucleotides, potential disadvantages include high cost of synthesis (8) and lack of specificity (9). Cost of synthesis can be reduced and specificity increased by designing short antisense agents whose binding to RNA targets is enhanced by tertiary interactions (10, 11). Here we show that one such hexanucleotide can also be a suicide inhibitor (12, 13) of RNA function. This provides an additional design principle for increasing specificity of compounds that can exploit the catalytic potential of complexes with RNA targets.Our model system is a large-subunit ribosomal RNA (rRNA) precursor from the opportunistic pathogen Pneumocystis carinii, which is a common cause of death in immunocompromised patients (14, 15). The rRNA precursor contains a group I self-splicing intron (10, 16) that provides a potential therapeutic target (16, 17) because self-splicing is required for assembly of active ribosomes (18). Hexamers that mimic this intron's 5Ј exon can bind to the catalytic core of a ribozyme derived from the intron as much as 100,000-fold more tightly than expected if the hexamers bound by simple base pairing (10). Much of this bindi...

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Section: Discussionmentioning

confidence: 55%

“…carinii is one of a class of mammalian opportunistic pathogens that contain conserved group I introns (21). The functional importance of introns combined with their absence in mammalian hosts makes them potentially important targets for pharmacological intervention (17,22).…”

Section: Discussionmentioning

confidence: 99%

In vitro suicide inhibition of self-splicing of a group I intron from Pneumocystis carinii by an N 3′ → P 5′ phosphoramidate hexanucleotide

Testa

Gryaznov

Turner

1999

Proc. Natl. Acad. Sci. U.S.A.

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“…It has previously been demonstrated that pentamidine acts on an unidentified mitochondrial target to inhibit the glycerol growth of S. cerevisiae (Ludewig et al+, 1994)+ Our results show that pentamidine inhibits yeast mitochondrial translation potently and specifically+ This effect and inhibition of nuclear group I intron splicing in C. albicans (Miletti & Leibowitz, 2000) are two apparent direct effects of pentamidine on yeast cells+ Because P. carinii is a pathogenic fungus related to S. cerevisiae and C. albicans, the mechanism of pentamidine action on P. carinii may also involve interference with mitochondrial translation and nuclear group I intron splicing (Edman et al+, 1988;Sogin & Edman, 1989;Lin et al+, 1992;Liu et al+, 1994;Liu & Leibowitz, 1993)+…”

Section: Pentamidine Inhibition Of Mitochondrial Translation Explainsmentioning

confidence: 99%

“…Since its introduction, pentamidine has been used for the treatment and prophylaxis of infections by Pneumocystis carinii (Ivády & Páldy, 1957;Hughes, 1991) and other eukaryotic microbial pathogens+ However, due to the inability to culture P. carinii from patients (Sloand et al+, 1993), the mechanism of action of pentamidine on this organism remains unknown (reviewed by Queener, 1995)+ In vitro, pentamidine has been shown to inhibit topoisomerases from P. carinii (Dykstra & Tidwell, 1991)+ In addition, pentamidine is known to bind in the minor groove of duplex DNA (Cory et al+, 1992;Nunn et al+, 1994)+ Because gene sequence analysis has shown P. carinii to be a fungus related to Saccharomyces cerevisiae (Edman et al+, 1988), this latter organism is a useful model for studying the mechanism of pentamidine antimicrobial activity (Ludewig et al+, 1994)+ Pentamidine appears to act on a mitochondrial target in S. cerevisiae, because growth inhibition was observed at 200-fold lower pentamidine concentrations on nonfermentable glycerol medium than on a fermentable carbon source+ However, the actual target of this inhibition was not mitochondrial DNA (mtDNA) maintenance, because petite mutants were generated only by much higher concentrations of pentamidine than are needed to inhibit growth on glycerol (Ludewig et al+, 1994)+ Furthermore, pentamidine inhibits the growth of yeast at much lower concentrations than are required to uncouple oxidative phosphorylation (Ludewig et al+, 1994)+ Such uncoupling has been noted in vitro in rat liver mitochondria, and may explain the toxicity of this drug to animals (Moreno, 1996)+ Pneumocystis carinii harbors group I introns in its rRNA genes (Edman et al+, 1988;Liu et al+, 1992;Liu & Leibowitz, 1993)+ Model transcripts containing these introns self-splice in vitro (Sogin & Edman, 1989;Lin et al+, 1992;Liu et al+, 1992;Liu & Leibowitz, 1993), and their splicing is inhibited by pentamidine (Liu & Leibowitz, 1993Liu et al+, 1994)+ Pentamidine inhibits the in vitro splicing catalyzed by these ribozymes noncompetitively relative to the guanosine cofactor, with total inhibition being achieved at concentrations of 250 mM (Liu et al+, 1994)+ However, the inability to grow clinical isolates of P. carinii in culture has precluded determination of whether this inhibition accounts for the anti-Pneumocystis activity...…”

Section: Introductionmentioning

confidence: 99%

Pentamidine inhibits mitochondrial intron splicing and translation in Saccharomyces cerevisiae

Zhang

Bell

Perlman

et al. 2000

RNA

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Pentamidine inhibits in vitro splicing of nuclear group I introns from rRNA genes of some pathogenic fungi and is known to inhibit mitochondrial function in yeast. Here we report that pentamidine inhibits the self-splicing of three group I and two group II introns of yeast mitochondria. Comparison of yeast strains with different configurations of mitochondrial introns (12, 5, 4, or 0 introns) revealed that strains with the most introns were the most sensitive to growth inhibition by pentamidine on glycerol medium. Analysis of blots of RNA from yeast strains grown in raffinose medium in the presence or absence of pentamidine revealed that the splicing of seven group I and two group II introns that have intron reading frames was inhibited by the drug to varying extents. Three introns without reading frames were unaffected by the drug in vivo, and two of these were inhibited in vitro, implying that the drug affects splicing by acting directly on RNA in vitro, but on another target in vivo. Because the most sensitive introns in vivo are the ones whose splicing depends on a maturase encoded by the intron reading frames, we tested pentamidine for effects on mitochondrial translation. We found that the drug inhibits mitochondrial but not cytoplasmic translation in cells at concentrations that inhibit mitochondrial intron splicing. Therefore, pentamidine is a potent and specific inhibitor of mitochondrial translation, and this effect explains most or all of its effects on respiratory growth and on in vivo splicing of mitochondrial introns.

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“…In eukaryotic nuclear genomes, group-I introns are found exclusively in the rDNA genes, and most of them are located in the SSU rDNA gene of a range of organisms (Damberger and Gutell, 1994), including fungi-mainly parasitic or symbiotic-such as Ustilago maydis (deWachter et al, 1992), Pseudohalonectria lignicola (Chen et al, 1996), Hymenoscyphus ericae (Egger et al, 1995), Phialophora americana, and Cenococcum geophilum (Rogers et al, 1993). Considerably fewer are the reported cases where group-I introns have been identified in the nuclear LSU rDNA genes, e.g., Pneumocystis carinii (Lin et al, 1992;Liu and Leibowitz, 1993), Beauveria brongniartii (Neuveglise and Brygoo, 1994;Neuveglise et al, 1997), Physarum flaviconum and P. polycephalum (Vader et al, 1994;Nomiyama et al, 1986), and Gaumanomyces graminis (Tan and Wong, 1996;Tan, 1997). In many cases the introns appear in a limited number of discrete DNA sequence positions, as reported for the nuclear SSU rDNA genes (Gargas et al, 1995) or the LSU rDNA genes (Neuveglise et al, 1997;De Jonckheere and Brown, 1998).…”

confidence: 96%

Identification of Group-I Introns at Three Different Positions within the 28S rDNA Gene of the Entomopathogenic Fungus Metarhizium anisopliae var. anisopliae

Mavridou

Cannone

Typas

2000

Fungal Genetics and Biology

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Characterization of the rRNA-encoding genes and transcripts, and a group-I self-splicing intron in Pneumocystis carinii

Cited by 28 publications

References 27 publications

In vitro suicide inhibition of self-splicing of a group I intron from Pneumocystis carinii by an N 3′ → P 5′ phosphoramidate hexanucleotide

In vitro suicide inhibition of self-splicing of a group I intron from Pneumocystis carinii by an N 3′ → P 5′ phosphoramidate hexanucleotide

Pentamidine inhibits mitochondrial intron splicing and translation in Saccharomyces cerevisiae

Identification of Group-I Introns at Three Different Positions within the 28S rDNA Gene of the Entomopathogenic Fungus Metarhizium anisopliae var. anisopliae

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