41 research outputs found
Transfer RNA Mimicry in a New Group of Positive-Strand RNA Plant Viruses, the Furoviruses: Differential Aminoacylation between the RNA Components of One Genome
AbstractRecent sequencing of the genomes of several furovirusesāfungus-transmitted rod-shaped positive-strand plant virusesāhas suggested the presence of tRNA-like structures (TLSs) at the 3ā² ends of the genomic RNAs. We show here that the genomic RNAs of soil-borne wheat mosaic virus (SBWMV), beet soil-borne virus (BSBV), potato mop-top virus (PMTV), peanut clump virus (PCV), and Indian peanut clump virus (IPCV) all possess functional TLSs that are capable of high-efficiency valylation. While the SBWMV, BSBV, and PMTV TLSs are similar to those found in tymoviruses, the PCV and IPCV TLSs harbor an insertion of about 40 nucleotides between the two halves of the TLS. The valylated SBWMV and BSBV RNAs formed tight complexes with wheat germ EF-1Ī± Ā· GTP (Kd= 2 to 11 nM), whereas valylated PMTV, PCV, and IPCV RNAs bound EF-1Ī± Ā· GTP weakly (Kdā„ 50 nM). The TLS of PCV RNA2 differs from PCV RNA1 in lacking the major valine identity nucleotide in the anticodon and consequently is capable of only very inefficient valylation. This is the first case of differential aminoacylation between the RNA components of one genome
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Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria
With the expansion of the RNA world, antisense strategies have become widespread to manipulate nuclear gene expression but organelle genetic systems have remained aside. The present work opens the field to mitochondria. We demonstrate that customized RNAs expressed from a nuclear transgene and driven by a transfer RNA-like (tRNA-like) moiety are taken up by mitochondria in plant cells. The process appears to follow the natural tRNA import specificity, suggesting that translocation indeed occurs through the regular tRNA uptake pathway. Upon validation of the strategy with a reporter sequence, we developed a chimeric catalytic RNA composed of a specially designed trans-cleaving hammerhead ribozyme and a tRNA mimic. Organelle import of the chimeric ribozyme and specific target cleavage within mitochondria were demonstrated in transgenic tobacco cell cultures and Arabidopsis thaliana plants, providing the first directed knockdown of a mitochondrial RNA in a multicellular eukaryote. Further observations point to mitochondrial messenger RNA control mechanisms related to the plant developmental stage and culture conditions. Transformation of mitochondria is only accessible in yeast and in the unicellular alga Chlamydomonas. Based on the widespread tRNA import pathway, our data thus make a breakthrough for direct investigation and manipulation of mitochondrial genetics
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Structural and functional analysis of the finished genome of the recently isolated toxic Anabaena sp WA102
Background:
Very few closed genomes of the cyanobacteria that commonly produce toxic blooms in lakes and reservoirs are available, limiting our understanding of the properties of these organisms. A new anatoxin-a-producing member of the Nostocaceae, Anabaena sp. WA102, was isolated from a freshwater lake in Washington State, USA, in 2013 and maintained in non-axenic culture.
Results:
The Anabaena sp. WA102 5.7 Mbp genome assembly has been closed with long-read, single-molecule sequencing and separately a draft genome assembly has been produced with short-read sequencing technology. The closed and draft genome assemblies are compared, showing a correlation between long repeats in the genome and the many gaps in the short-read assembly. Anabaena sp. WA102 encodes anatoxin-a biosynthetic genes, as does its close relative Anabaena sp. AL93 (also introduced in this study). These strains are distinguished by differences in the genes for light-harvesting phycobilins, with Anabaena sp. AL93 possessing a phycoerythrocyanin operon. Biologically relevant structural variants in the Anabaena sp. WA102 genome were detected only by long-read sequencing: a tandem triplication of the anaBCD promoter region in the anatoxin-a synthase gene cluster (not triplicated in Anabaena sp. AL93) and a 5-kbp deletion variant present in two-thirds of the population. The genome has a large number of mobile elements (160). Strikingly, there was no synteny with the genome of its nearest fully assembled relative, Anabaena sp. 90.
Conclusion:
Structural and functional genome analyses indicate that Anabaena sp. WA102 has a flexible genome. Genome closure, which can be readily achieved with long-read sequencing, reveals large scale (e.g., gene order) and local structural features that should be considered in understanding genome evolution and functioKeywords: PacBio, Illumina, Mobilome, Cyanobacteria, Anabaena, Long-read sequencing, Structural variation, Anatoxin-a, Synteny, Tandem repeatKeywords: PacBio, Illumina, Mobilome, Cyanobacteria, Anabaena, Long-read sequencing, Structural variation, Anatoxin-a, Synteny, Tandem repea
Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria
With the expansion of the RNA world, antisense strategies have become widespread to manipulate nuclear gene expression but organelle genetic systems have remained aside. The present work opens the field to mitochondria. We demonstrate that customized RNAs expressed from a nuclear transgene and driven by a transfer RNA-like (tRNA-like) moiety are taken up by mitochondria in plant cells. The process appears to follow the natural tRNA import specificity, suggesting that translocation indeed occurs through the regular tRNA uptake pathway. Upon validation of the strategy with a reporter sequence, we developed a chimeric catalytic RNA composed of a specially designed trans-cleaving hammerhead ribozyme and a tRNA mimic. Organelle import of the chimeric ribozyme and specific target cleavage within mitochondria were demonstrated in transgenic tobacco cell cultures and Arabidopsis thaliana plants, providing the first directed knockdown of a mitochondrial RNA in a multicellular eukaryote. Further observations point to mitochondrial messenger RNA control mechanisms related to the plant developmental stage and culture conditions. Transformation of mitochondria is only accessible in yeast and in the unicellular alga Chlamydomonas. Based on the widespread tRNA import pathway, our data thus make a breakthrough for direct investigation and manipulation of mitochondrial genetics
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A closely-related clade of globally distributed bloom-forming cyanobacteria within the Nostocales
In order to better understand the relationships among current Nostocales cyanobacterial blooms, eight genomes were sequenced from cultured isolates or from environmental metagenomes of recent planktonic Nostocales blooms. Phylogenomic analysis of publicly available sequences placed the new genomes among a group of 15 genomes from four continents in a distinct ADA Glade (Anabaena/Dolichospenruun/Aphanizomenon) within the Nostocales. This Glade contains four species-level groups, two of which include members with both Anabaena-like and Aphanizomenon-flos-aquae-like morphology. The genomes contain many repetitive genetic elements and a sizable pangenome, in which ABC-type transporters are highly represented. Alongside common core genes for photosynthesis, the differentiation of N-2-fixing heterocysts, and the uptake and incorporation of the major nutrients P, N and S, we identified several gene pathways in the pangenome that may contribute to niche partitioning. Genes for problematic secondary metabolites-cyanotoxins and taste-and-odor compounds-were sporadically present, as were other polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene clusters. By contrast, genes predicted to encode the ribosomally generated bacteriocin peptides were found in all genomes
Close spacing of AUG initiation codons confers dicistronic character on a eukaryotic mRNA
TYMV RNA supports the translation of two proteins, p69 and p206, from AUG initiation codons 7 nucleotides apart. We have studied the translation of this overlapping dicistronic mRNA with luciferase reporter RNAs electroporated into cowpea protoplasts and in toe-printing studies that map ribosomes stalled during initiation in wheat germ extracts. Agreement between these two assays indicates that the observed effects reflect ribosome initiation events. The robust expression from the downstream AUG(206) codon was dependent on its closeness to the upstream AUG(69) codon. Stepwise separation of these codons resulted in a gradual increase in upstream initiation and decrease in downstream initiation, and expression was converted from dicistronic to monocistronic. Selection by ribosomes for initiation between the nearby AUG codons was responsive to the sequence contexts that govern leaky scanning, but the normally strong position effect favoring upstream initiation was greatly diminished. Similar dicistronic expression was supported for RNAs with altered initiation sequences and for RNAs devoid of flanking viral sequences. Closely spaced AUG codons may thus represent an under-recognized strategy for bicistronic expression from eukaryotic mRNAs. The initiation behavior observed in these studies suggests that 5ā²ā3ā² ribosome scanning involves backward excursions averaging about 15 nucleotides
Cap- and initiator tRNA-dependent initiation of TYMV polyprotein synthesis by ribosomes: Evaluation of the Trojan horse model for TYMV RNA translation
Turnip yellow mosaic virus (TYMV) RNA directs the translation of two overlapping open reading frames. Competing models have been previously published to explain ribosome access to the downstream polyprotein cistron. The Trojan horse model, based on cell-free experiments, proposes noncanonical cap-independent initiation in which the 3ā²-terminal tRNA-like structure (TLS) functionally replaces initiator tRNA, and the valine bound to the TLS becomes cis-incorporated into viral protein. The initiation coupling model, based on in vivo expression and ribosome toe-printing studies, proposes a variation of canonical leaky scanning. Here, we have re-examined the wheat germ extract experiments that led to the Trojan horse model, incorporating a variety of controls. We report that (1) translation in vitro from the polyprotein AUG of TYMV RNA is unchanged after removal of the 3ā² TLS but is stimulated by the presence of a 5ā²-cap; (2) the presence of free cap analog or edeine (which interferes with initiation at the ribosomal P site and its tRNA(i) (Met) involvement) inhibits translation from the polyprotein AUG; (3) the toe-prints of immediately post-initiation ribosomes on TYMV RNA are similar with and without an intact TLS; and (4) significant deacylation of valyl-TYMV RNA in wheat germ extract can complicate the detection of cis-incorporation. These results favor the initiation coupling model
eEF1A binding to aminoacylated viral RNA represses minus strand synthesis by TYMV RNA-dependent RNA polymerase
AbstractThe genomic RNA of Turnip yellow mosaic virus (TYMV) has an 82-nucleotide-long tRNA-like structure at its 3ā²-end that can be valylated and then form a stable complex with translation elongation factor eEF1AĀ·GTP. Transcription of this RNA by TYMV RNA-dependent RNA polymerase (RdRp) to yield minus strands has previously been shown to initiate within the 3ā²-CCA sequence. We have now demonstrated that minus strand synthesis is strongly repressed upon the binding of eEF1AĀ·GTP to the valylated viral RNA. eEF1AĀ·GTP had no effect on RNA synthesis templated by non-aminoacylated RNA. Higher eEF1AĀ·GTP levels were needed to repress minus strand synthesis templated by valyl-EMV TLS RNA, which binds eEF1AĀ·GTP with lower affinity than does valyl-TYMV RNA. Repression by eEF1AĀ·GTP was also observed with a methionylated variant of TYMV RNA and with aminoacylated tRNAHis, tRNAAla, and tRNAPhe transcripts. It is proposed that minus strand repression by eEF1AĀ·GTP binding occurs early during infection to help coordinate the competing translation and replication functions of the genomic RNA