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PubMed1989

Molecular biology of Leishmania.

Leishmania is a trypanosomatid protozoa with a digenetic life cycle. Sandflies inject promastigotes, the free living form present in their salivary glands, into mammals where the parasite colonizes macrophages, transforming into intracellular amastigotes. The cycle is completed when during a blood meal the insect ingests infected macrophages, the amastigotes are released in the gut where they transform back into promastigotes. Leishmania has to adapt to the changing life conditions, from free-living forms in the poikilothermic insect vector to obligatory intracellular parasite in the homeothermic mammalian host. It also has to adapt to the acidic pH of the macrophage's phagolysosome where amastigotes multiply. The adaptative response of Leishmania includes morphological, physiological, and biochemical changes. Promastigotes can be grown in culture medium. Studies of changes taking place during adaptation have been facilitated by the establishment of in vitro conditions that allow the transformation of amastigotes into promastigotes and vice versa. The system is well suited for studying regulation of gene expression during adaptative differentiation. Some mechanisms of mRNA processing are unique to these protozoa: trans-splicing and RNA editing. Several genes that are differentially expressed in the two stages have been studied. No obvious cis regulatory motifs have been found in the DNA.

Amino Acid Sequence↗

PubMed1991

RNA-mediated transfer of the gene coxII from the mitochondrion to the nucleus during flowering plant evolution.

The gene coxII, normally present in the mitochondrion, was functionally transferred to the nucleus during flowering plant evolution. coxII transfer is estimated to have occurred between 60 and 200 million years ago, whereas loss of coxII from the mitochondrion occurred much more recently, being restricted to a single genus of legumes. Most legumes have coxII in both the nucleus and the mitochondrion; however, no evidence is found for simultaneous coxII expression in both compartments. The nuclear coxII sequence more closely resembles edited mitochondrial coxII transcripts than the genes encoding these RNAs. Hence, gene transfer appears to have involved reverse transcription of an edited RNA intermediate. The nuclear gene contains an intron at the junction of the transit peptide sequence and the mature protein-coding sequence; exon shuffling may have played a role in assembling a functional coxII gene in the nucleus.

Amino Acid Sequence↗

PubMed2002

The evolutionary history of kinetoplastids and their kinetoplasts.

Despite extensive phylogenetic analysis of small subunit ribosomal RNA (SSUrRNA) genes, the deep-level relationships among kinetoplastids remain poorly understood, limiting our grasp of their evolutionary history, especially the origins of their bizarre mitochondrial genome organizations. In this study we examine the SSUrRNA data in the light of a new marker--cytoplasmic heat shock protein 90 (hsp90) sequences. Our phylogenetic analyses divide kinetoplastids into four main clades. Clades 1-3 include the various bodonid kinetoplastids. Trypanosomatids comprise the fourth clade. SSUrRNA analyses give vastly different and poorly supported positions for the root of the kinetoplastid tree, depending on the out-group and analysis method. This is probably due to the extraordinary length of the branch between kinetoplastids and any out-group. In contrast, almost all hsp90 analyses place the root between clade 1 (including Dimastigella, Rhynchomonas, several Bodo spp., and probably Rhynchobodo) and all other kinetoplastids. Maximum likelihood and maximum likelihood distance analyses of hsp90 protein and second codon-position nucleotides place trypanosomatids adjacent to Bodo saltans and Bodo cf. uncinatus (clade 3), as (weakly) do SSUrRNA analyses. Hsp90 first codon- plus second codon-position nucleotide analyses return a slightly different topology. We show that this may be an artifact caused, in part, by the different evolutionary behavior of first- and second-codon positions. This study provides the most robust evidence to date that trypanosomatids are descended from within bodonids and that B. saltans is a close relative of trypanosomatids. A total reevaluation of the high-level systematics within kinetoplastids is needed. We confirm that the interlocking network organization of kinetoplast DNA seen in trypanosomatids is a derived condition within kinetoplastids but suggest that open-conformation minicircles may have arisen early in kinetoplastid evolution. Further understanding of the evolution of kinetoplast structure and RNA editing is hampered by a paucity of data from basal (i.e., clade 1) bodonids.

Animals↗

PubMed2003

Radiation of extant marsupials after the K/T boundary: evidence from complete mitochondrial genomes.

The complete mitochondrial (mt) genomes of five marsupial species have been sequenced. The species represent all three South American orders (Didelphimorphia, Paucituberculata, and Microbiotheria). Phylogenetic analysis of this data set indicates that Didelphimorphia is a basal marsupial lineage followed by Paucituberculata. The South American microbiotherid Dromiciops gliroides (monito del monte) groups with Australian marsupials, suggesting a marsupial colonization of Australia on two occasions or, alternatively, a migration of an Australian marsupial lineage to South America. Molecular estimates suggest that the deepest marsupial divergences took place 64-62 million years before present (MYBP), implying that the radiation of recent marsupials took place after the K/T (Cretaceous/Tertiary) boundary. The South American marsupial lineages are all characterized by a putatively non-functional tRNA for lysine, a potential RNA editing of the tRNA for asparagine, and a rearrangement of tRNA genes at the origin of light strand replication.

Animals↗

PubMed2004

Activation induced deaminase: the importance of being specific.

Activation-induced deaminase (AID) is required for class switch recombination and somatic hypermutation in immunoglobulin genes. Although the preponderance of evidence suggests that AID functions by deaminating deoxycytidine in DNA, the question remains whether it can also deaminate cytidine in mRNA, as originally proposed based on its homology to RNA-editing enzymes. Recently, the biological relevance of assaying mammalian enzymes for DNA deaminase activity using Escherichia coli DNA as a reporter has been questioned, representing another round in the ongoing debate.

APOBEC-1 Deaminase↗

PubMed2004

Multiple domains control the subcellular localization and activity of ETR-3, a regulator of nuclear and cytoplasmic RNA processing events.

Embryonic lethal abnormal vision (ELAV) type RNA binding protein 3 (ETR-3; also called NAPOR, CUGBP2, or BRUNOL3) has been implicated in the regulation of nuclear and cytoplasmic RNA processing events, including alternative splicing, RNA editing, stability and translation. Here, we report that the ETR-3 protein contains multiple regions that control its subcellular localization and are important for its activity as a splicing regulator. We cloned ETR-3 from chicken heart and fused it to the C terminus of green fluorescent protein (GFPcETR3vL). GFPcETR3vL is found predominantly in the nucleus and is an active regulator of alternative splicing in cotransfection assays with a cardiac troponin T minigene. ETR-3 contains two N-terminal RNA recognition motifs (RRMs), a 210-amino acid divergent domain, and a C-terminal RRM. We demonstrate that the C terminus contains a strong nuclear localization signal overlapping the third RRM, which can confer nuclear localization on a normally cytoplasmic pyruvate kinase chimera. Additional deletions revealed nuclear localization and export activities in the divergent domain of ETR-3, as well as regions within the first two RRMs that are important for cytoplasmic localization. The nuclear export activity of the divergent domain is sensitive to leptomycin B, indicating that export to the cytoplasm is mediated via a CRM1-dependent pathway. The C terminus and a region within the divergent domain were also shown to be important for splicing activity of ETR-3. This is the first characterization of protein domains involved in mediating the subcellular localization and splicing activity of a member of the CELF family of RNA processing regulators.

Alternative Splicing↗

PubMed2006

Kdr allelic variation in pyrethroid resistant mosquitoes, Culex quinquefasciatus (S.).

The loss of target site sensitivity to insecticides resulting from a substitution of leucine to phenylalanine, termed the kdr mutation, in the voltage-gated sodium channel of the insect nervous system is known to be important in insecticide resistance. However, little is known about the molecular basis of the genotype and kdr-mediated resistance phenotype relationship. This study investigated whether the functional polymorphism of the L-to-F kdr mutation that determines resistance phenotype undergoes DNA variation or goes through transcriptional regulatory variation. We detected no correlation for the kdr allele at the genomic DNA level with levels of susceptibility and resistance to insecticide. However, we find a strong correlation between the kdr allelic expression and levels of insecticide resistance and susceptibility through RNA allelic variation and RNA editing. These findings shed new light on the role of transcriptional regulation in the kdr-mediated resistance in mosquitoes and its connection with the genotype-resistance phenotype relationship.

Alleles↗

PubMed1990

A set of tRNAs that lack either the T psi C arm or the dihydrouridine arm: towards a minimal tRNA adaptor.

The mitochondrial DNA (mtDNA) molecules of the nematode worms, Caenorhabditis elegans and Ascaris suum contain 22 putative genes for non-standard forms of tRNAs. The inferred transcripts can be folded into 20 separate structures each resembling a tRNA whose T psi C arm and variable loop are replaced with a simple loop of 6-12 nucleotides. In two further structures [that resemble tRNAs for ser(UCN) and ser(AGN)], the dihydrouridine arm is replaced by a loop of 5-8 nucleotides. By hybridizing mt-tRNA gene-specific oligonucleotide probes to nematode RNAs, we have obtained evidence for transcription of at least nine C.elegans and three A.suum mt-tRNA genes. Each transcript (tRNA) is the exact size predicted from the respective DNA sequence, to which three nucleotides, presumably CCA, have been added following transcription. An exception was C.elegans mt-tRNAasn, most molecules of which had one nucleotide (plus CCA) more than predicted from the gene. The data presented strongly support the conclusion that the functional mt-tRNAs of nematode worms are direct transcripts (with only CCA addition) of the structurally unusual mt-tRNA genes. There is no evidence of trans-splicing or RNA editing to add the sequences missing from these nonstandard tRNAs. We presume, therefore, that the non-standard forms are active in mitochondrial protein synthesis.

Animals↗

PubMed2005

Differential proteome analysis of aging in rat skeletal muscle.

To identify the mechanisms underlying muscle aging, we have undertaken a high-resolution differential proteomic analysis of gastrocnemius muscle in young adults, mature adults, and old LOU/c/jall rats. Two-dimensional gel electrophoresis and subsequent MALDI-ToF mass spectrometry analyses led to the identification of 40 differentially expressed proteins. Strikingly, most differences characterized old (30-month) animals, whereas young (7-month) and mature (18-month) adults exhibited similar patterns of expression. Important modifications in contractile (actin, myosin light-chains, troponins-T) and cytoskeletal (desmin, tubulin) proteins, and in essential regulatory proteins (gelsolin, myosin binding proteins, CapZ-beta, P23), likely account for dysfunctions in old muscle force generation and speed of contraction. Other features support decreases in cytosolic (triose-phosphate isomerase, enolase, glycerol-3-P dehydrogenase, creatine kinase) and mitochondrial (isocitrate dehydrogenase, cytochrome-c oxidase) energy metabolisms. Muscle aging is often associated with increased oxidative stress. Accordingly, we observed differential regulation of molecular chaperones (hsp20, hsp27, reticuloplasmin ER60) and of proteins implicated in reactive aldehyde detoxification (aldehyde dehydrogenase, glutathione transferase, glyoxalase). We further noticed up-regulation of proteins involved in transcriptional elongation (RNA capping protein) and RNA-editing (Apobec2). Most of these proteins were previously unrecognized as differentially expressed in old muscles, and they represent novel starting points for elucidating the mechanisms of muscle aging.

Aging↗

PubMed2006

Sodium channel gene expression associated with pyrethroid resistant house flies and German cockroaches.

The voltage-gated sodium channels of the insect nervous system are the primary target of DDT and pyrethroid insecticides. The loss of target site sensitivity to insecticides resulting from a substitution of leucine to phenylalanine, termed the L-to-F kdr mutation, in the sodium channel of the insect nervous system is known to be important in insecticide resistance. Yet, little is known about the molecular basis underlying the genotype and kdr-mediated resistance phenotype relationship. Here we report a systematic study of resistance-associated kdr allelic expression within and among resistant and susceptible house fly and German cockroach populations. We compared genomic DNA and RNA sequences within the same individuals from different insect strains, finding no correlation for the kdr allele at the genomic DNA level with levels of susceptibility or resistance to insecticide. However, there was a strong correlation between kdr allele expression and the levels of insecticide resistance. This correlation is probably regulated through RNA variation and RNA editing. These results suggest a role for posttranscriptional regulation in the connection of the sodium channel genotype and its mutation-mediated resistance phenotype.

Alleles↗

PubMed1990

cis rescue of a mutated reverse transcriptase gene of human hepatitis B virus by creation of an internal ATG.

Using mutational analysis, we have investigated the translation strategy of the reverse transcriptase gene (pol) of human hepatitis B virus. It has been proposed that this pol gene product is synthesized as a core-pol fusion protein from a polycistronic mRNA template via ribosomal frameshifting, a mechanism often seen in retroelements. Our data indicate that creation of a novel ATG initiation codon near the original ATG can compensate for a lethal missense mutation at the first ATG position of the pol open reading frame. Genetic analysis has rigorously ruled out the possibilities of frameshifting, non-ATG initiation, or RNA editing. These results are discussed in the context of a 5'-end entry model versus a novel model of direct internal entry of ribosomes.

Base Sequence↗

PubMed1989

Regulation of gene expression during adipocyte differentiation: a review.

The differentiation of adipose precursor cells is accompanied by the acquisition of adipocyte-specific messenger (m) RNAs allowing characteristic changes in protein composition. The development of methods for cloning and characterizing individual genes has provided the opportunity to study selective gene expression by adipocytes at the molecular level. In this review, the information obtained to date regarding transcriptional and post-transcriptional regulatory mechanisms utilized by adipocytes is summarized. Included are descriptions of conserved DNA sequences found in noncoding regions of adipose genes and of how protein-DNA interactions at these regions are thought to regulate the initiation of transcription. Among the transcription factors implemented in regulation of adipocyte-specific gene expression are the protein product of the proto-oncogene c-fos, the triiodothyronine receptor and steroid hormone receptors. Data also are discussed that indicate that RNA editing and determinants of mRNA stability contribute to the adipocyte phenotype. It is hoped that a review of the regulatory aspects of gene expression during adipocyte differentiation will identify areas deserving further study by researchers interested in understanding adipose development.

Adipose Tissue↗

PubMed1992

Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA. A primitive form of plant mitochondrial genome.

Analysis of the mitochondrial DNA of a liverwort Marchantia polymorpha by electron microscopy and restriction endonuclease mapping indicated that the liverwort mitochondrial genome was a single circular molecule of about 184,400 base-pairs. We have determined the complete sequence of the liverwort mitochondrial DNA and detected 94 possible genes in the sequence of 186,608 base-pairs. These included genes for three species of ribosomal RNA, 29 genes for 27 species of transfer RNA and 30 open reading frames (ORFs) for functionally known proteins (16 ribosomal proteins, 3 subunits of H(+)-ATPase, 3 subunits of cytochrome c oxidase, apocytochrome b protein and 7 subunits of NADH ubiquinone oxidoreductase). Three ORFs showed similarity to ORFs of unknown function in the mitochondrial genomes of other organisms. Furthermore, 29 ORFs were predicted as possible genes by using the index of G + C content in first, second and third letters of codons (42.0 +/- 10.9%, 37.0 +/- 13.2% and 26.4 +/- 9.4%, respectively) obtained from the codon usages of identified liverwort genes. To date, 32 introns belonging to either group I or group II intron have been found in the coding regions of 17 genes including ribosomal RNA genes (rrn18 and rrn26), a transfer RNA gene (trnS) and a pseudogene (psi nad7). RNA editing was apparently lacking in liverwort mitochondria since the nucleotide sequences of the liverwort mitochondrial DNA were well-conserved at the DNA level.

Base Sequence↗

PubMed2006

Dual targeting of a single tRNA(Trp) requires two different tryptophanyl-tRNA synthetases in Trypanosoma brucei.

The mitochondrion of Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by the import of a small fraction of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single-copy genes, suggesting the use of the same enzyme in the cytosol and mitochondrion. However, the T. brucei genome contains two distinct genes for eukaryotic tryptophanyl-tRNA synthetase (TrpRS). RNA interference analysis established that both TrpRS1 and TrpRS2 are essential for growth and required for cytosolic and mitochondrial tryptophanyl-tRNA formation, respectively. Decoding the mitochondrial tryptophan codon UGA requires mitochondria-specific C-->U RNA editing in the anticodon of the imported tRNA(Trp). In vitro charging assays with recombinant TrpRS enzymes demonstrated that the edited anticodon and the mitochondria-specific thiolation of U33 in the imported tRNA(Trp) act as antideterminants for the cytosolic TrpRS1. The existence of two TrpRS enzymes, therefore, can be explained by the need for a mitochondrial synthetase with extended substrate specificity to achieve aminoacylation of the imported thiolated and edited tRNA(Trp). Thus, the notion that, in an organism, all nuclear-encoded tRNAs assigned to a given amino acid are charged by a single aminoacyl-tRNA synthetase, is not universally valid.

Amino Acid Sequence↗

PubMed1997

Functionally distinct double-stranded RNA-binding domains associated with alternative splice site variants of the interferon-inducible double-stranded RNA-specific adenosine deaminase.

The double-stranded RNA-specific adenosine deaminase (ADAR) is an interferon-inducible RNA-editing enzyme implicated in the site-selective deamination of adenosine to inosine in viral RNAs and cellular pre-mRNAs. We have isolated and characterized human genomic clones of the ADAR gene and cDNA clones encoding splice site variants of the ADAR protein. Southern blot and sequence analyses revealed that the gene spans about 30 kilobase pairs and consists of 15 exons. The codon phasing of the splice site junctions of exons 3, 5, and 7 that encode the three copies of the highly conserved RNA-binding R-motif (RI, RII, and RIII) was exactly conserved and identical to those R-motif exons of the interferon-inducible RNA-dependent protein kinase. Alternative splice site variants of the 1226-amino acid ADAR-a protein, designated b and c, were identified that differed in exons 6 and 7. ADAR-b was a 5'-splice site variant that possessed a 26-amino acid deletion within exon 7; ADAR-c was a 3'-splice site variant that possessed an additional 19-amino acid deletion within exon 6. The wild-type ADAR-a, -b, and -c proteins all possessed comparable double-stranded RNA-specific adenosine deaminase activity. However, mutational analysis of the R-motifs revealed that the exon 6 and 7 deletions of ADAR-b and -c variants altered the functional importance of each of the three R-motifs.

Adenosine Deaminase↗

PubMed1996

Genetic stability of a porcine rotavirus RNA segment during repeated plaque isolation.

The porcine group A rotavirus CC86 was characterized to explore its utility as a tool for mutation analysis. It has a semiduplication of the gene 11 RNA segment. Nucleotide sequence determination of cDNA confirmed that the NSP5 coding sequence and the conserved nontranslated termini of the RNA segment were retained. A comparison of the NSP5 genes of CC86 and CN86 that were isolated from the same fecal specimen showed eight base pair changes, suggesting that CN86 was not the immediate progenitor of CC86. Synthesis of NSP5 in monkey MA104 cells infected with CC86, CN86, or simian rotavirus SA11 was compared by one- and two-dimensional polyacrylamide gel electrophoresis. NSP5 from all three viruses had similar posttranslational modifications, and no difference in the expression levels was observed. To experimentally address the genetic stability of CC86 segment 11, the virus was passaged by serial plaque to plaque transfer. The repeated genetic bottlenecking led to a gradual loss of fitness. This effect is not observed when virus is passaged by the standard method of moderate dilution. Nucleotide sequence analysis of cDNA clones isolated from viral segment 11 RNA of virus from plaque-to-plaque passage numbers 0, 1, 4, and 8 showed occasional base substitutions, mostly in the NSP5 coding sequence. Two mutations, leading to His-to-Arg and Lys-to-Arg replacements, respectively, in NSP5 were established in the virus population. Forward and reverse base pair changes (A-U<-->G-C) at the two sites appeared to be concerted and take place at a very high frequency, suggesting that a mechanism equivalent to RNA editing might operate. The overall mutation rate of segment 11 was much lower, having a calculated maximal value of 5 x 10(-5) per replicated base.

Animals↗

PubMed1992

Silent mitochondrial and active nuclear genes for subunit 2 of cytochrome c oxidase (cox2) in soybean: evidence for RNA-mediated gene transfer.

In most plants and other eukaryotes investigated, the mitochondrial genome carries the gene encoding subunit 2 of cytochrome c oxidase (cox2). In this paper, we show that the previously reported mitochondrial cox2 of soybean is actually silent, and that there is an expressed, single-copy, nucleus-encoded cox2. Molecular cloning and sequence analysis of cox2 cDNA and genomic clones show that the soybean nuclear gene encodes an N-terminal extension that resembles a signal sequence for mitochondrial import and whose coding sequence is separated by an intron from that corresponding to mtDNA-encoded cox2. Comparison of soybean mitochondrial and nuclear cox2 sequences clearly indicates that in an ancestor of soybean, cox2 was transferred from the mitochondrion to the nucleus via a C-to-U edited RNA intermediate.

Amino Acid Sequence↗

PubMed2005

Nuclear localization of the Nipah virus W protein allows for inhibition of both virus- and toll-like receptor 3-triggered signaling pathways.

The Nipah virus V and W proteins, which are encoded by the P gene via RNA editing, have a common N-terminal domain but unique C-terminal domains. They localize to the cytoplasm and nucleus, respectively, and have both been shown to function as inhibitors of JAK/STAT signaling. Here we report that V and W proteins also block virus activation of the beta interferon (IFN-beta) promoter and the IFN regulatory factor 3 (IRF3)-responsive IFN-stimulated gene 54 promoter. Surprisingly, only W protein shows strong inhibition of promoter activation in response to stimulation of Toll-like receptor 3 (TLR3) by extracellular double-stranded RNA. This activity is dependent on the nuclear localization of W protein. Within the unique C-terminal domain of W protein, we have identified a nuclear localization signal (NLS) that requires basic residues at positions 439, 440, and 442. This NLS is responsible for mediating the preferential interaction of W protein with karyopherin-alpha 3 and karyopherin-alpha 4. Nuclear localization of W protein therefore enables it to target both virus and TLR3 pathways, whereas the cytoplasmic V protein is restricted to inhibiting the virus pathway. We propose that this discrepancy is in part due to the V protein being less able to block signaling in response to the kinase, TBK-1, whereas both V and W can prevent promoter activation in response to IKKepsilon. We demonstrate that, when the TLR3 pathway is stimulated, the levels of phosphorylated IRF3 are reduced in the presence of W protein but not V protein, confirming the differential effects of these proteins and illustrating that W protein-mediated inhibition is due to a loss of active IRF3.

Amino Acid Sequence↗