Genomic Heterogeneity in a Natural Archaeal Population Suggests a Model of tRNA Gene Disruption

Understanding the mechanistic basis of the disruption of tRNA genes, as manifested in the intron-containing and split tRNAs found in Archaea, will provide considerable insight into the evolution of the tRNA molecule. However, the evolutionary processes underlying these disruptions have not yet been identified. Previously, a composite genome of the deep-branching archaeon Caldiarchaeum subterraneum was reconstructed from a community genomic library prepared from a C. subterraneum–dominated microbial mat. Here, exploration of tRNA genes from the library reveals that there are at least three types of heterogeneity at the tRNAThr(GGU) gene locus in the Caldiarchaeum population. All three involve intronic gain and splitting of the tRNA gene. Of two fosmid clones found that encode tRNAThr(GGU), one (tRNAThr-I) contains a single intron, whereas another (tRNAThr-II) contains two introns. Notably, in the clone possessing tRNAThr-II, a 5′ fragment of the tRNAThr-I (tRNAThr-F) gene was observed 1.8-kb upstream of tRNAThr-II. The composite genome contains both tRNAThr-II and tRNAThr-F, although the loci are >500 kb apart. Given that the 1.8-kb sequence flanked by tRNAThr-F and tRNAThr-II is predicted to encode a DNA recombinase and occurs in six regions of the composite genome, it may be a transposable element. Furthermore, its dinucleotide composition is most similar to that of the pNOB8-type plasmid, which is known to integrate into archaeal tRNA genes. Based on these results, we propose that the gain of the tRNA intron and the scattering of the tRNA fragment occurred within a short time frame via the integration and recombination of a mobile genetic element

Discovery of permuted and recently split transfer RNAs in Archaea

Background: As in eukaryotes, precursor transfer RNAs in Archaea often contain introns that are removed in tRNA maturation. Two unrelated archaeal species display unique pre-tRNA processing complexity in the form of split tRNA genes, in which two to three segments of tRNAs are transcribed from different loci, then trans-spliced to form a mature tRNA. Another rare type of pre-tRNA, found only in eukaryotic algae, is permuted, where the 3 ’ half is encoded upstream of the 5 ’ half, and must be processed to be functional. Results: Using an improved version of the gene-finding program tRNAscan-SE, comparative analyses and experimental verifications, we have now identified four novel trans-spliced tRNA genes, each in a different species of the Desulfurococcales branch of the Archaea: tRNA Asp(GUC) in Aeropyrum pernix and Thermosphaera aggregans, and tRNA Lys(CUU) in Staphylothermus hellenicus and Staphylothermus marinus. Each of these includes features surprisingly similar to previously studied split tRNAs, yet comparative genomic context analysis and phylogenetic distribution suggest several independent, relatively recent splitting events. Additionally, we identified the first examples of permuted tRNA genes in Archaea: tRNA iMet(CAU) and tRNA Tyr(GUA) in Thermofilum pendens, which appear to be permuted in the same arrangement seen previously in red alga. Conclusions: Our findings illustrate that split tRNAs are sporadically spread across a major branch of the Archaea

In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems of type I use a Cas ribonucleoprotein complex for antiviral defense (Cascade) to mediate the targeting and degradation of foreign DNA. To address molecular features of the archaeal type I-A Cascade interference mechanism, we established the in vitro assembly of the Thermoproteus tenax Cascade from six recombinant Cas proteins, synthetic CRISPR RNAs (crRNAs) and target DNA fragments. RNA-Seq analyses revealed the processing pattern of crRNAs from seven T. tenax CRISPR arrays. Synthetic crRNA transcripts were matured by hammerhead ribozyme cleavage. The assembly of type I-A Cascade indicates that Cas3′ and Cas3′′ are an integral part of the complex, and the interference activity was shown to be dependent on the crRNA and the matching target DNA. The reconstituted Cascade was used to identify sequence motifs that are required for efficient DNA degradation and to investigate the role of the subunits Cas7 and Cas3′′ in the interplay with other Cascade subunits

Change of tRNA identity leads to a divergent orthogonal histidyl-tRNA synthetase/tRNAHis pair

Mature tRNAHis has at its 5′-terminus an extra guanylate, designated as G−1. This is the major recognition element for histidyl-tRNA synthetase (HisRS) to permit acylation of tRNAHis with histidine. However, it was reported that tRNAHis of a subgroup of α-proteobacteria, including Caulobacter crescentus, lacks the critical G−1 residue. Here we show that recombinant C. crescentus HisRS allowed complete histidylation of a C. crescentus tRNAHis transcript (lacking G−1). The addition of G−1 did not improve aminoacylation by C. crescentus HisRS. However, mutations in the tRNAHis anticodon caused a drastic loss of in vitro histidylation, and mutations of bases A73 and U72 also reduced charging. Thus, the major recognition elements in C. crescentus tRNAHis are the anticodon, the discriminator base and U72, which are recognized by the divergent (based on sequence similarity) C. crescentus HisRS. Transplantation of these recognition elements into an Escherichia coli tRNAHis template, together with addition of base U20a, created a competent substrate for C. crescentus HisRS. These results illustrate how a conserved tRNA recognition pattern changed during evolution. The data also uncovered a divergent orthogonal HisRS/tRNAHis pair

Crystal Structure of the RNA Recognition Motif of Yeast Translation Initiation Factor eIF3b Reveals Differences to Human eIF3b

BACKGROUND: The multi-subunit eukaryotic initiation factor3 (eIF3) plays a central role in the initiation step of protein synthesis in eukaryotes. One of its large subunits, eIF3b, serves as a scaffold within eIF3 as it interacts with several other subunits. It harbors an RNA Recognition Motif (RRM), which is shown to be a non-canonical RRM in human as it is not capable to interact with oligonucleotides, but rather interacts with eIF3j, a sub-stoichiometric subunit of eIF3. PRINCIPAL FINDING: We have analyzed the high-resolution crystal structure of the eIF3b RRM domain from yeast. It exhibits the same fold as its human ortholog, with similar charge distribution on the surface interacting with the eIF3j in human. Thermodynamic analysis of the interaction between yeast eIF3b-RRM and eIF3j revealed the same range of enthalpy change and dissociation constant as for the human proteins, providing another line of evidence for the same mode of interaction between eIF3b and eIF3j in both organisms. However, analysis of the surface charge distribution of the putative RNA-binding β-sheet suggested that in contrast to its human ortholog, it potentially could bind oligonucleotides. Three-dimensional positioning of the so called "RNP1" motif in this domain is similar to other canonical RRMs, suggesting that this domain might indeed be a canonical RRM, conferring oligonucleotide binding capability to eIF3 in yeast. Interaction studies with yeast total RNA extract confirmed the proposed RNA binding activity of yeast eIF3b-RRM. CONCLUSION: We showed that yeast eIF3b-RRM interacts with eIF3j in a manner similar to its human ortholog. However, it shows similarities in the oligonucleotide binding surface to canonical RRMs and interacts with yeast total RNA. The proposed RNA binding activity of eIF3b-RRM may help eIF3 to either bind to the ribosome or recruit the mRNA to the 43S pre-initiation complex

A Model of Proto-Anti-Codon RNA Enzymes Requiring l-Amino Acid Homochirality

All living organisms encode the 20 natural amino acid units of polypeptides using a universal scheme of triplet nucleotide “codons”. Disparate features of this codon scheme are potentially informative of early molecular evolution: (i) the absence of any codons for d-amino acids; (ii) the odd combination of alternate codon patterns for some amino acids; (iii) the confinement of synonymous positions to a codon’s third nucleotide; (iv) the use of 20 specific amino acids rather than a number closer to the full coding potential of 64; and (v) the evolutionary relationship of patterns in stop codons to amino acid codons. Here I propose a model for an ancestral proto-anti-codon RNA (pacRNA) auto-aminoacylation system and show that pacRNAs would naturally manifest features of the codon table. I show that pacRNAs could implement all the steps for auto-aminoacylation: amino acid coordination, intermediate activation of the amino acid by the 5′-end of the pacRNA, and 3′-aminoacylation of the pacRNA. The anti-codon cradles of pacRNAs would have been able to recognize and coordinate only a small number of l-amino acids via hydrogen bonding. A need for proper spatial coordination would have limited the number of chargeable amino acids for all anti-codon sequences, in addition to making some anti-codon sequences unsuitable. Thus, the pacRNA model implies that the idiosyncrasies of the anti-codon table and l-amino acid homochirality co-evolved during a single evolutionary period. These results further imply that early life consisted of an aminoacylated RNA world with a richer enzymatic potential than ribonucleotides alone

A genomic analysis of the archaeal system Ignicoccus hospitalis-Nanoarchaeum equitans

Sequencing of the complete genome of Ignicoccus hospitalis gives insight into its association with another species of Archaea, Nanoarchaeum equitans

Proteomic Characterization of Cellular and Molecular Processes that Enable the Nanoarchaeum equitans-Ignicoccus hospitalis Relationship

Nanoarchaeum equitans, the only cultured representative of the Nanoarchaeota, is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. The molecular mechanisms that enable this relationship are unknown. Using whole-cell proteomics, differences in the relative abundance of >75% of predicted protein-coding genes from both Archaea were measured to identify the specific response of I. hospitalis to the presence of N. equitans on its surface. A purified N. equitans sample was also analyzed for evidence of interspecies protein transfer. The depth of cellular proteome coverage achieved here is amongst the highest reported for any organism. Based on changes in the proteome under the specific conditions of this study, I. hospitalis reacts to N. equitans by curtailing genetic information processing (replication, transcription) in lieu of intensifying its energetic, protein processing and cellular membrane functions. We found no evidence of significant Ignicoccus biosynthetic enzymes being transported to N. equitans. These results suggest that, under laboratory conditions, N. equitans diverts some of its host's metabolism and cell cycle control to compensate for its own metabolic shortcomings, thus appearing to be entirely dependent on small, transferable metabolites and energetic precursors from I. hospitalis

Structure-Function Analysis of Human TYW2 Enzyme Required for the Biosynthesis of a Highly Modified Wybutosine (yW) Base in Phenylalanine-tRNA

Posttranscriptional modifications are critical for structure and function of tRNAs. Wybutosine (yW) and its derivatives are hyper-modified guanosines found at the position 37 of eukaryotic and archaeal tRNAPhe. TYW2 is an enzyme that catalyzes α-amino-α-carboxypropyl transfer activity at the third step of yW biogenesis. Using complementation of a ΔTYW2 strain, we demonstrate here that human TYW2 (hTYW2) is active in yeast and can synthesize the yW of yeast tRNAPhe. Structure-guided analysis identified several conserved residues in hTYW2 that interact with S-adenosyl-methionine (AdoMet), and mutation studies revealed that K225 and E265 are critical residues for the enzymatic activity. We previously reported that the human TYW2 is overexpressed in breast cancer. However, no difference in the tRNAPhe modification status was observed in either normal mouse tissue or a mouse tumor model that overexpresses Tyw2, indicating that hTYW2 may have a role in tumorigenesis unrelated to yW biogenesis