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 furoviruses—fungus-transmitted rod-shaped positive-strand plant viruses—has suggested the presence of tRNA-like structures (TLSs) at the 3′ 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-1α · GTP (Kd= 2 to 11 nM), whereas valylated PMTV, PCV, and IPCV RNAs bound EF-1α · GTP weakly (Kd≥ 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

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

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

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 5′–3′ 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 3′-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 3′ TLS but is stimulated by the presence of a 5′-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 3′-end that can be valylated and then form a stable complex with translation elongation factor eEF1A·GTP. Transcription of this RNA by TYMV RNA-dependent RNA polymerase (RdRp) to yield minus strands has previously been shown to initiate within the 3′-CCA sequence. We have now demonstrated that minus strand synthesis is strongly repressed upon the binding of eEF1A·GTP to the valylated viral RNA. eEF1A·GTP had no effect on RNA synthesis templated by non-aminoacylated RNA. Higher eEF1A·GTP levels were needed to repress minus strand synthesis templated by valyl-EMV TLS RNA, which binds eEF1A·GTP with lower affinity than does valyl-TYMV RNA. Repression by eEF1A·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 eEF1A·GTP binding occurs early during infection to help coordinate the competing translation and replication functions of the genomic RNA

Genome sequences of bacteria closely associated with the dominant Nostocales cyanobacteria of recent HABs in the US Pacific Northwest

We have assembled over a dozen genomes of bacteria that remained attached after filtration and/or washing to cyanobacterial material derived from recent blooms in freshwater lakes in the US Pacific NW. PacBio and Illumina metagenomes were generated from samples, and when sufficient sequencing reads from heterotrophic bacteria were present to allow genome assembly, those reads typically represented one or at most three distinct bacteria, whose reads were present at levels lower than those of the cyanobacterium. We interpret these genomes as representing phycosphere bacteria. The taxonomic groups represented were diverse, including alphaproteobacteria, betaproteobacteria, Bacteroidetes, Verrucomicrobia, Planctomycetes, Gemmatimonadetes and Vampirovibrionales. These were associated with a range of Nostocales ADA clade cyanobacteria. Typically, there were no close relatives of the new genomes in the genome or 16S rRNA database. In addition to these, a dominant cyanobacterium genome, Dolichospermum sp. DET69, was assembled from 5 samples from Detroit Reservoir, Oregon, taken across 4 years. From each sample, a near-identical Opitutus sp. genome was assembled. This suggests a recurring and specific interaction, perhaps one that survives the boom/bust cycle of over-wintering, after which an emerging bloom arises from a few surviving filaments or from newly germinated akinetes