RNomics and Modomics in the halophilic archaea Haloferax volcanii: Identification of RNA modification genes

Department of Microbiology, University of Florida, Gainsville, FL 32611, Florida, USA.
BMC Genomics (Impact Factor: 3.99). 11/2008; 9(1):470. DOI: 10.1186/1471-2164-9-470
Source: PubMed


Naturally occurring RNAs contain numerous enzymatically altered nucleosides. Differences in RNA populations (RNomics) and pattern of RNA modifications (Modomics) depends on the organism analyzed and are two of the criteria that distinguish the three kingdoms of life. If the genomic sequences of the RNA molecules can be derived from whole genome sequence information, the modification profile cannot and requires or direct sequencing of the RNAs or predictive methods base on the presence or absence of the modifications genes.
By employing a comparative genomics approach, we predicted almost all of the genes coding for the t+rRNA modification enzymes in the mesophilic moderate halophile Haloferax volcanii. These encode both guide RNAs and enzymes. Some are orthologous to previously identified genes in Archaea, Bacteria or in Saccharomyces cerevisiae, but several are original predictions.
The number of modifications in t+rRNAs in the halophilic archaeon is surprisingly low when compared with other Archaea or Bacteria, particularly the hyperthermophilic organisms. This may result from the specific lifestyle of halophiles that require high intracellular salt concentration for survival. This salt content could allow RNA to maintain its functional structural integrity with fewer modifications. We predict that the few modifications present must be particularly important for decoding, accuracy of translation or are modifications that cannot be functionally replaced by the electrostatic interactions provided by the surrounding salt-ions. This analysis also guides future experimental validation work aiming to complete the understanding of the function of RNA modifications in Archaeal translation.

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    • "This analysis identified one of the methyltransferases belonging to COG1901, encompassing an a/b knot fold (also named SPOUT) superfamily of methyltransferases as a valid candidate (Tkaczuk et al. 2007). This prediction fits with the observation that genes of this family usually cluster with pus10 in several archaeal genomes (Grosjean et al. 2008a). Lastly, the crystal structure of a COG1901 family member, Mj1640 from M. jannaschii, was solved in complex with AdoMet at 1.4 A ˚ resolution (Chen and Yuan 2010). "
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    ABSTRACT: The methylation of pseudouridine (Ψ) at position 54 of tRNA, producing m(1)Ψ, is a hallmark of many archaeal species, but the specific methylase involved in the formation of this modification had yet to be characterized. A comparative genomics analysis had previously identified COG1901 (DUF358), part of the SPOUT superfamily, as a candidate for this missing methylase family. To test this prediction, the COG1901 encoding gene, HVO_1989, was deleted from the Haloferax volcanii genome. Analyses of modified base contents indicated that while m(1)Ψ was present in tRNA extracted from the wild-type strain, it was absent from tRNA extracted from the mutant strain. Expression of the gene encoding COG1901 from Halobacterium sp. NRC-1, VNG1980C, complemented the m(1)Ψ minus phenotype of the ΔHVO_1989 strain. This in vivo validation was extended with in vitro tests. Using the COG1901 recombinant enzyme from Methanocaldococcus jannaschii (Mj1640), purified enzyme Pus10 from M. jannaschii and full-size tRNA transcripts or TΨ-arm (17-mer) fragments as substrates, the sequential pathway of m(1)Ψ54 formation in Archaea was reconstituted. The methylation reaction is AdoMet dependent. The efficiency of the methylase reaction depended on the identity of the residue at position 55 of the TΨ-loop. The presence of Ψ55 allowed the efficient conversion of Ψ54 to m(1)Ψ54, whereas in the presence of C55, the reaction was rather inefficient and no methylation reaction occurred if a purine was present at this position. These results led to renaming the Archaeal COG1901 members as TrmY proteins.
    RNA 03/2012; 18(3):421-33. DOI:10.1261/rna.030841.111 · 4.94 Impact Factor
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    • "The genome sequence of Hfx. volcanii was also used for a bioinformatic prediction of the modification sites of stable RNAs, the guide RNAs involved in RNA modification, and RNA-modifying enzymes [28] [29]. The number of predicted modifications was astonishingly low, and this was also interpreted as an adaptation to the high salt cytoplasm because RNA stability is higher under high salt conditions. "
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    ABSTRACT: The genome sequence of Haloferax volcanii is available and several comparative genomic in silico studies were performed that yielded novel insight for example into protein export, RNA modifications, small non-coding RNAs, and ubiquitin-like Small Archaeal Modifier Proteins. The full range of functional genomic methods has been established and results from transcriptomic, proteomic and metabolomic studies are discussed. Notably, Hfx. volcanii is together with Halobacterium salinarum the only prokaryotic species for which a translatome analysis has been performed. The results revealed that the fraction of translationally-regulated genes in haloarchaea is as high as in eukaryotes. A highly efficient genetic system has been established that enables the application of libraries as well as the parallel generation of genomic deletion mutants. Facile mutant generation is complemented by the possibility to culture Hfx. volcanii in microtiter plates, allowing the phenotyping of mutant collections. Genetic approaches are currently used to study diverse biological questions-from replication to posttranslational modification-and selected results are discussed. Taken together, the wealth of functional genomic and genetic tools make Hfx. volcanii a bona fide archaeal model species, which has enabled the generation of important results in recent years and will most likely generate further breakthroughs in the future.
    Archaea 11/2011; 2011(3):602408. DOI:10.1155/2011/602408 · 2.71 Impact Factor
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    • "Both strains show the presence of C13 and C22 in tRNA Met (Fig. 5A,B), suggesting that neither the formation of C13 nor C22 are Cbf5-mediated. Probably C13 is produced by TruD/Pus7 because a homolog is present in H. volcanii (Grosjean et al. 2008), and P. abyssi Pus7 can produce C13 in vitro (Muller et al. 2009). Similar analysis using CMCT reactions suggests that C39 in elongator tRNA Met is also not Cbf5-mediated (Fig. 5C,D) thus is not guide RNA dependent, whereas 29-O-methylation of U at the same position 39 in tRNA Trp of H. volcanii is guide RNA dependent (Clouet d'Orval et al. 2001; Singh et al. 2004). "
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    ABSTRACT: Pseudouridine (Ψ), the isomer of uridine, is commonly found at various positions of noncoding RNAs of all organisms. Ψ residues are formed by a number of single- or multisite specific Ψ synthases, which generally act as stand-alone proteins. In addition, in Eukarya and Archaea, specific ribonucleoprotein complexes, each containing a distinct box H/ACA guide RNA and four core proteins, can produce Ψ at many sites of different cellular RNAs. Cbf5 is the core Ψ synthase in these complexes. Using Haloferax volcanii as an archaeal model organism, we show that, contrary to eukaryotes, the Cbf5 homolog (HVO_2493) is not essential in this archaeon. The Cbf5-deleted strain of H. volcanii completely lacks Ψ at positions 1940, 1942, 2605, and 2591 (Escherichia coli positions 1915, 1917, 2572, and 2586) of its 23S rRNA, and contains reduced steady-state levels of some box H/ACA RNAs. Archaeal Cbf5 is known to have tRNA Ψ55 synthase activity in vitro but we could not confirm this activity in vivo in H. volcanii. Conversely, the Pus10 (previously PsuX) homolog (HVO_1979), which can produce tRNA Ψ55, as well as Ψ54 in vitro, is shown here to be essential in H. volcanii, whereas the corresponding tRNA Ψ55 synthases, Pus4 and TruB, are not essential in yeast and E. coli, respectively. Finally, we demonstrate that HVO_1852, the TruA/Pus3 homolog, is responsible for the pseudouridylation of position 39 in H. volcanii tRNAs and that the corresponding gene is not essential.
    RNA 07/2011; 17(7):1367-80. DOI:10.1261/rna.2712811 · 4.94 Impact Factor
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