Functional characterization of the Mycobacterium abscessus genome coupled with condition specific transcriptomics reveals conserved molecular strategies for host adaptation and persistence.

BACKGROUND: Mycobacterium abscessus subsp. abscessus (MAB) is a highly drug resistant mycobacterium and the most common respiratory pathogen among the rapidly growing non-tuberculous mycobacteria. MAB is also one of the most deadly of the emerging cystic fibrosis (CF) pathogens requiring prolonged treatment with multiple antibiotics. In addition to its "mycobacterial" virulence genes, the genome of MAB harbours a large accessory genome, presumably acquired via lateral gene transfer including homologs shared with the CF pathogens Pseudomonas aeruginosa and Burkholderia cepacia. While multiple genome sequences are available there is little functional genomics data available for this important pathogen. RESULTS: We report here the first multi-omics approach to characterize the primary transcriptome, coding potential and potential regulatory regions of the MAB genome utilizing differential RNA sequencing (dRNA-seq), RNA-seq, Ribosome profiling and LC-MS proteomics. In addition we attempt to address the genomes contribution to the molecular systems that underlie MAB's adaptation and persistence in the human host through an examination of MABs transcriptional response to a number of clinically relevant conditions. These include hypoxia, exposure to sub-inhibitory concentrations of antibiotics and growth in an artificial sputum designed to mimic the conditions within the cystic fibrosis lung. CONCLUSIONS: Our integrated map provides the first comprehensive view of the primary transcriptome of MAB and evidence for the translation of over one hundred new short open reading frames (sORFs). Our map will act as a resource for ongoing functional genomics characterization of MAB and our transcriptome data from clinically relevant stresses informs our understanding of MAB's adaptation to life in the CF lung. MAB's adaptation to growth in artificial CF sputum highlights shared metabolic strategies with other CF pathogens including P. aeruginosa and mirrors the transcriptional responses that lead to persistence in mycobacteria. These strategies include an increased reliance on amino acid metabolism, and fatty acid catabolism and highlights the relevance of the glyoxylate shunt to growth in the CF lung. Our data suggests that, similar to what is seen in chronically persisting P. aeruginosa, progression towards a biofilm mode of growth would play a more prominent role in a longer-term MAB infection. Finally, MAB's transcriptional response to antibiotics highlights the role of antibiotic modifications enzymes, active transport and the evolutionarily conserved WhiB7 driven antibiotic resistance regulon.

Genome sequencing of the extinct Eurasian wild aurochs, Bos primigenius, illuminates the phylogeography and evolution of cattle.

BACKGROUND: Domestication of the now-extinct wild aurochs, Bos primigenius, gave rise to the two major domestic extant cattle taxa, B. taurus and B. indicus. While previous genetic studies have shed some light on the evolutionary relationships between European aurochs and modern cattle, important questions remain unanswered, including the phylogenetic status of aurochs, whether gene flow from aurochs into early domestic populations occurred, and which genomic regions were subject to selection processes during and after domestication. Here, we address these questions using whole-genome sequencing data generated from an approximately 6,750-year-old British aurochs bone and genome sequence data from 81 additional cattle plus genome-wide single nucleotide polymorphism data from a diverse panel of 1,225 modern animals. RESULTS: Phylogenomic analyses place the aurochs as a distinct outgroup to the domestic B. taurus lineage, supporting the predominant Near Eastern origin of European cattle. Conversely, traditional British and Irish breeds share more genetic variants with this aurochs specimen than other European populations, supporting localized gene flow from aurochs into the ancestors of modern British and Irish cattle, perhaps through purposeful restocking by early herders in Britain. Finally, the functions of genes showing evidence for positive selection in B. taurus are enriched for neurobiology, growth, metabolism and immunobiology, suggesting that these biological processes have been important in the domestication of cattle. CONCLUSIONS: This work provides important new information regarding the origins and functional evolution of modern cattle, revealing that the interface between early European domestic populations and wild aurochs was significantly more complex than previously thought.

Delineating transcriptional networks of prognostic gene signatures refines treatment recommendations for lymph node-negative breast cancer patients.

The majority of women diagnosed with lymph node-negative breast cancer are unnecessarily treated with damaging chemotherapeutics after surgical resection. This highlights the importance of understanding and more accurately predicting patient prognosis. In the present study, we define the transcriptional networks regulating well-established prognostic gene expression signatures. We find that the same set of transcriptional regulators consistently lie upstream of both 'prognosis' and 'proliferation' gene signatures, suggesting that a central transcriptional network underpins a shared phenotype within these signatures. Strikingly, the master transcriptional regulators within this network predict recurrence risk for lymph node-negative breast cancer better than currently used multigene prognostic assays, particularly in estrogen receptor-positive patients. Simultaneous examination of p16(INK4A) expression, which predicts tumours that have bypassed cellular senescence, revealed that intermediate levels of p16(INK4A) correlate with an intact pRB pathway and improved survival. A combination of these master transcriptional regulators and p16(INK4A), termed the OncoMasTR score, stratifies tumours based on their proliferative and senescence capacity, facilitating a clearer delineation of lymph node-negative breast cancer patients at high risk of recurrence, and thus requiring chemotherapy. Furthermore, OncoMasTR accurately classifies over 60% of patients as 'low risk', an improvement on existing prognostic assays, which has the potential to reduce overtreatment in early-stage patients. Taken together, the present study provides new insights into the transcriptional regulation of cellular proliferation in breast cancer and provides an opportunity to enhance and streamline methods of predicting breast cancer prognosis.

Relaxed selection drives a noisy noncoding transcriptome in members of the Mycobacterium tuberculosis complex.

Related species are often used to understand the molecular underpinning of virulence through examination of a shared set of biological features attributable to a core genome of orthologous genes. An important but insufficiently studied issue, however, is the extent to which the regulatory architectures are similarly conserved. A small number of studies have compared the primary transcriptomes of different bacterial species, but few have compared closely related species with clearly divergent evolutionary histories. We addressed the impact of differing modes of evolution within the genus Mycobacterium through comparison of the primary transcriptome of M. marinum with that of a closely related lineage, M. bovis. Both are thought to have evolved from an ancestral generalist species, with M. bovis and other members of the M. tuberculosis complex having subsequently undergone downsizing of their genomes during the transition to obligate pathogenicity. M. marinum, in contrast, has retained a large genome, appropriate for an environmental organism, and is a broad-host-range pathogen. We also examined changes over a shorter evolutionary time period through comparison of the primary transcriptome of M. bovis with that of another member of the M. tuberculosis complex (M. tuberculosis) which possesses an almost identical genome but maintains a distinct host preference. Importance: Our comparison of the transcriptional start site (TSS) maps of M. marinum and M. bovis uncovers a pillar of conserved promoters, noncoding RNA (NCRNA), and a genome-wide signal in the -35 promoter regions of both species. We identify evolutionarily conserved transcriptional attenuation and highlight its potential contribution to multidrug resistance mediated through the transcriptional regulator whiB7. We show that a species population history is reflected in its transcriptome and posit relaxed selection as the main driver of an abundance of canonical -10 promoter sites in M. bovis relative to M. marinum. It appears that transcriptome composition in mycobacteria is driven primarily by the availability of such sites and that their frequencies diverge significantly across the mycobacterial clade. Finally, through comparison of M. bovis and M. tuberculosis, we illustrate that single nucleotide polymorphism (SNP)-driven promoter differences likely underpin many of the transcriptional differences between M. tuberculosis complex lineages.

Control of reproductive floral organ identity specification in Arabidopsis by the C function regulator AGAMOUS.

The floral organ identity factor AGAMOUS (AG) is a key regulator of Arabidopsis thaliana flower development, where it is involved in the formation of the reproductive floral organs as well as in the control of meristem determinacy. To obtain insights into how AG specifies organ fate, we determined the genes and processes acting downstream of this C function regulator during early flower development and distinguished between direct and indirect effects. To this end, we combined genome-wide localization studies, gene perturbation experiments, and computational analyses. Our results demonstrate that AG controls flower development to a large extent by controlling the expression of other genes with regulatory functions, which are involved in mediating a plethora of different developmental processes. One aspect of this function is the suppression of the leaf development program in emerging floral primordia. Using trichome initiation as an example, we demonstrate that AG inhibits an important aspect of leaf development through the direct control of key regulatory genes. A comparison of the gene expression programs controlled by AG and the B function regulators APETALA3 and PISTILLATA, respectively, showed that while they control many developmental processes in conjunction, they also have marked antagonistic, as well as independent activities.

Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro.

BACKGROUND: Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA. RESULTS: A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology. CONCLUSIONS: This study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA.

Mycobacterial lineages causing pulmonary and extrapulmonary tuberculosis, Ethiopia.

Molecular typing of 964 specimens from patients in Ethiopia with lymph node or pulmonary tuberculosis showed a similar distribution of Mycobacterium tuberculosis strains between the 2 disease manifestations and a minimal role for M. bovis. We report a novel phylogenetic lineage of M. tuberculosis strongly associated with the Horn of Africa.

Genome of Acanthamoeba castellanii highlights extensive lateral gene transfer and early evolution of tyrosine kinase signaling.

BACKGROUND: The Amoebozoa constitute one of the primary divisions of eukaryotes, encompassing taxa of both biomedical and evolutionary importance, yet its genomic diversity remains largely unsampled. Here we present an analysis of a whole genome assembly of Acanthamoeba castellanii (Ac) the first representative from a solitary free-living amoebozoan. RESULTS: Ac encodes 15,455 compact intron-rich genes, a significant number of which are predicted to have arisen through inter-kingdom lateral gene transfer (LGT). A majority of the LGT candidates have undergone a substantial degree of intronization and Ac appears to have incorporated them into established transcriptional programs. Ac manifests a complex signaling and cell communication repertoire, including a complete tyrosine kinase signaling toolkit and a comparable diversity of predicted extracellular receptors to that found in the facultatively multicellular dictyostelids. An important environmental host of a diverse range of bacteria and viruses, Ac utilizes a diverse repertoire of predicted pattern recognition receptors, many with predicted orthologous functions in the innate immune systems of higher organisms. CONCLUSIONS: Our analysis highlights the important role of LGT in the biology of Ac and in the diversification of microbial eukaryotes. The early evolution of a key signaling facility implicated in the evolution of metazoan multicellularity strongly argues for its emergence early in the Unikont lineage. Overall, the availability of an Ac genome should aid in deciphering the biology of the Amoebozoa and facilitate functional genomic studies in this important model organism and environmental host.

Polycomb PHF19 binds H3K36me3 and recruits PRC2 and demethylase NO66 to embryonic stem cell genes during differentiation.

Polycomb group proteins are repressive chromatin modifiers with essential roles in metazoan development, cellular differentiation and cell fate maintenance. How Polycomb proteins access active chromatin to confer transcriptional silencing during lineage transitions remains unclear. Here we show that the Polycomb repressive complex 2 (PRC2) component PHF19 binds trimethylated histone H3 Lys36 (H3K36me3), a mark of active chromatin, via its Tudor domain. PHF19 associates with the H3K36me3 demethylase NO66, and it is required to recruit the PRC2 complex and NO66 to stem cell genes during differentiation, leading to PRC2-mediated trimethylation of histone H3 Lys27 (H3K27), loss of H3K36me3 and transcriptional silencing. We propose a model whereby PHF19 functions during mouse embryonic stem cell differentiation to transiently bind the H3K36me3 mark via its Tudor domain, forming essential contact points that allow recruitment of PRC2 and H3K36me3 demethylase activity to active gene loci during their transition to a Polycomb-repressed state.

Molecular basis for the specification of floral organs by APETALA3 and PISTILLATA.

How different organs are formed from small sets of undifferentiated precursor cells is a key question in developmental biology. To understand the molecular mechanisms underlying organ specification in plants, we studied the function of the homeotic selector genes APETALA3 (AP3) and PISTILLATA (PI), which control the formation of petals and stamens during Arabidopsis flower development. To this end, we characterized the activities of the transcription factors that AP3 and PI encode throughout flower development by using perturbation assays as well as transcript profiling and genomewide localization studies, in combination with a floral induction system that allows a stage-specific analysis of flower development by genomic technologies. We discovered considerable spatial and temporal differences in the requirement for AP3/PI activity during flower formation and show that they control different sets of genes at distinct phases of flower development. The genomewide identification of target genes revealed that AP3/PI act as bifunctional transcription factors: they activate genes involved in the control of numerous developmental processes required for organogenesis and repress key regulators of carpel formation. Our results imply considerable changes in the composition and topology of the gene network controlled by AP3/PI during the course of flower development. We discuss our results in light of a model for the mechanism underlying sex-determination in seed plants, in which AP3/PI orthologues might act as a switch between the activation of male and the repression of female development.

The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium.

More than 50 y of research have provided great insight into the physiology, metabolism, and molecular biology of Salmonella enterica serovar Typhimurium (S. Typhimurium), but important gaps in our knowledge remain. It is clear that a precise choreography of gene expression is required for Salmonella infection, but basic genetic information such as the global locations of transcription start sites (TSSs) has been lacking. We combined three RNA-sequencing techniques and two sequencing platforms to generate a robust picture of transcription in S. Typhimurium. Differential RNA sequencing identified 1,873 TSSs on the chromosome of S. Typhimurium SL1344 and 13% of these TSSs initiated antisense transcripts. Unique findings include the TSSs of the virulence regulators phoP, slyA, and invF. Chromatin immunoprecipitation revealed that RNA polymerase was bound to 70% of the TSSs, and two-thirds of these TSSs were associated with σ(70) (including phoP, slyA, and invF) from which we identified the -10 and -35 motifs of σ(70)-dependent S. Typhimurium gene promoters. Overall, we corrected the location of important genes and discovered 18 times more promoters than identified previously. S. Typhimurium expresses 140 small regulatory RNAs (sRNAs) at early stationary phase, including 60 newly identified sRNAs. Almost half of the experimentally verified sRNAs were found to be unique to the Salmonella genus, and <20% were found throughout the Enterobacteriaceae. This description of the transcriptional map of SL1344 advances our understanding of S. Typhimurium, arguably the most important bacterial infection model.

Methicillin resistance alters the biofilm phenotype and attenuates virulence in Staphylococcus aureus device-associated infections.

Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to ß-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R602H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to ß-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are frequently responsible for device-related infections in immuno-compromised patients.

European 2--a clonal complex of Mycobacterium bovis dominant in the Iberian Peninsula.

Mycobacterium bovis isolates from the Iberian Peninsula are dominated by strains with spoligotype patterns deleted for spacer 21. Whole-genome sequencing of three Spanish strains with spacer 21 missing in their spoligotype pattern revealed a series of SNPs and subsequent screening of a selection of these SNPs identified one in gene guaA that is specific to these strains. This group of strains from the Iberian Peninsula missing spoligotype spacer 21 represents a new clonal complex of M. bovis, defined by the SNP profile with a distinct spoligotype signature. We have named this clonal complex European 2 (Eu2) and found that it was present at low frequency in both France and Italy and absent from the British Isles.

Sequencing illustrates the transcriptional response of Legionella pneumophila during infection and identifies seventy novel small non-coding RNAs.

Second generation sequencing has prompted a number of groups to re-interrogate the transcriptomes of several bacterial and archaeal species. One of the central findings has been the identification of complex networks of small non-coding RNAs that play central roles in transcriptional regulation in all growth conditions and for the pathogen's interaction with and survival within host cells. Legionella pneumophila is a gram-negative facultative intracellular human pathogen with a distinct biphasic lifestyle. One of its primary environmental hosts in the free-living amoeba Acanthamoeba castellanii and its infection by L. pneumophila mimics that seen in human macrophages. Here we present analysis of strand specific sequencing of the transcriptional response of L. pneumophila during exponential and post-exponential broth growth and during the replicative and transmissive phase of infection inside A. castellanii. We extend previous microarray based studies as well as uncovering evidence of a complex regulatory architecture underpinned by numerous non-coding RNAs. Over seventy new non-coding RNAs could be identified; many of them appear to be strain specific and in configurations not previously reported. We discover a family of non-coding RNAs preferentially expressed during infection conditions and identify a second copy of 6S RNA in L. pneumophila. We show that the newly discovered putative 6S RNA as well as a number of other non-coding RNAs show evidence for antisense transcription. The nature and extent of the non-coding RNAs and their expression patterns suggests that these may well play central roles in the regulation of Legionella spp. specific traits and offer clues as to how L. pneumophila adapts to its intracellular niche. The expression profiles outlined in the study have been deposited into Genbank's Gene Expression Omnibus (GEO) database under the series accession GSE27232.

Sequencing and analysis of an Irish human genome.

BACKGROUND: Recent studies generating complete human sequences from Asian, African and European subgroups have revealed population-specific variation and disease susceptibility loci. Here, choosing a DNA sample from a population of interest due to its relative geographical isolation and genetic impact on further populations, we extend the above studies through the generation of 11-fold coverage of the first Irish human genome sequence. RESULTS: Using sequence data from a branch of the European ancestral tree as yet unsequenced, we identify variants that may be specific to this population. Through comparisons with HapMap and previous genetic association studies, we identified novel disease-associated variants, including a novel nonsense variant putatively associated with inflammatory bowel disease. We describe a novel method for improving SNP calling accuracy at low genome coverage using haplotype information. This analysis has implications for future re-sequencing studies and validates the imputation of Irish haplotypes using data from the current Human Genome Diversity Cell Line Panel (HGDP-CEPH). Finally, we identify gene duplication events as constituting significant targets of recent positive selection in the human lineage. CONCLUSIONS: Our findings show that there remains utility in generating whole genome sequences to illustrate both general principles and reveal specific instances of human biology. With increasing access to low cost sequencing we would predict that even armed with the resources of a small research group a number of similar initiatives geared towards answering specific biological questions will emerge.

A complete mitochondrial genome sequence from a mesolithic wild aurochs (Bos primigenius).

BACKGROUND: The derivation of domestic cattle from the extinct wild aurochs (Bos primigenius) has been well-documented by archaeological and genetic studies. Genetic studies point towards the Neolithic Near East as the centre of origin for Bos taurus, with some lines of evidence suggesting possible, albeit rare, genetic contributions from locally domesticated wild aurochsen across Eurasia. Inferences from these investigations have been based largely on the analysis of partial mitochondrial DNA sequences generated from modern animals, with limited sequence data from ancient aurochsen samples. Recent developments in DNA sequencing technologies, however, are affording new opportunities for the examination of genetic material retrieved from extinct species, providing new insight into their evolutionary history. Here we present DNA sequence analysis of the first complete mitochondrial genome (16,338 base pairs) from an archaeologically-verified and exceptionally-well preserved aurochs bone sample. METHODOLOGY: DNA extracts were generated from an aurochs humerus bone sample recovered from a cave site located in Derbyshire, England and radiocarbon-dated to 6,738+/-68 calibrated years before present. These extracts were prepared for both Sanger and next generation DNA sequencing technologies (Illumina Genome Analyzer). In total, 289.9 megabases (22.48%) of the post-filtered DNA sequences generated using the Illumina Genome Analyzer from this sample mapped with confidence to the bovine genome. A consensus B. primigenius mitochondrial genome sequence was constructed and was analysed alongside all available complete bovine mitochondrial genome sequences. CONCLUSIONS: For all nucleotide positions where both Sanger and Illumina Genome Analyzer sequencing methods gave high-confidence calls, no discrepancies were observed. Sequence analysis reveals evidence of heteroplasmy in this sample and places this mitochondrial genome sequence securely within a previously identified aurochsen haplogroup (haplogroup P), thus providing novel insights into pre-domestic patterns of variation. The high proportion of authentic, endogenous aurochs DNA preserved in this sample bodes well for future efforts to determine the complete genome sequence of a wild ancestor of domestic cattle.

Analysis of 5'- or 3'-terminal tRNA editing: mitochondrial 5' tRNA editing in Acanthamoeba castellanii as the exemplar.

Editing processes that result in the structural retailoring of the aminoacyl acceptor stems of mitochondrial tRNAs are the focus of this chapter. This type of tRNA editing is the most frequently observed and widely distributed and involves nucleotide replacement within the 5' or 3' half of the aminoacyl acceptor stem in either a template-directed or a template-independent fashion. We provide a detailed protocol that allows demarcation of 5'-terminal tRNA editing events from those occurring on the 3' side of the acceptor stem. We present the mitochondrial 5' tRNA editing system in Acanthamoeba castellanii as the exemplar of terminal tRNA editing. The methodology involves RNA ligase-mediated circularization of tRNAs, cDNA synthesis primed by tRNA-specific oligonucleotides, amplification of cDNA via polymerase chain reaction, and cloning and sequencing of multiple products. This approach permits (1) simultaneous determination of 5' and 3' acceptor stem sequences, (2) delineation of 5' or 3' editing, (3) identification of mature tRNAs (characterized by having a 3'-CCA(OH) motif), (4) identification of processing/editing intermediates, and (5) mechanistic insights.

Methods for analysis of mitochondrial tRNA editing in Acanthamoeba castellanii.

We present here methodology for assaying 5'-terminal editing of mitochondrial tRNAs in the amoeboid protist Acanthamoeba castellanii. This type of editing involves replacement of one or more nucleotides within the first three positions at the 5' end of a tRNA substrate. The assay procedure involves RNA ligase-mediated joining of the 5' and 3' ends of a tRNA, use of the resulting circularized tRNA as template for cDNA synthesis primed by tRNA-specific primers over a region that encompasses the ligated 5' and 3' halves of the acceptor stem, amplification of cDNA via polymerase chain reaction, and cloning and sequencing of double-stranded cDNA product. This approach has the advantage that it simultaneously reveals potential editing events on the 5' and 3' side of an acceptor stem, as well as serves to identify mature tRNAs (characterized by having a 3'-CCAOH motif) and partially processed intermediates.

tRNAHis maturation: an essential yeast protein catalyzes addition of a guanine nucleotide to the 5' end of tRNAHis.

All tRNAHis molecules are unusual in having an extra 5' GMP residue (G(-1)) that, in eukaryotes, is added after transcription and RNase P cleavage. Incorporation of this G(-1) residue is a rare example of nucleotide addition occurring at an RNA 5' end in a normal phosphodiester linkage. We show here that the essential Saccharomyces cerevisiae ORF YGR024c (THG1) is responsible for this guanylyltransferase reaction. Thg1p was identified by survey of a genomic collection of yeast GST-ORF fusion proteins for addition of [alpha-32P]GTP to tRNAHis. End analysis confirms the presence of G(-1). Thg1p is required for tRNAHis guanylylation in vivo, because cells depleted of Thg1p lack G(-1) in their tRNAHis. His6-Thg1p purified from Escherichia coli catalyzes the guanylyltransferase step of G(-1) addition using a ppp-tRNAHis substrate, and appears to catalyze the activation step using p-tRNAHis and ATP. Thg1p is highlye conserved in eukaryotes, where G(-1) addition is necessary, and is not found in eubacteria, where G(-1) is genome-encoded. Thus, Thg1p is the first member of a new family of enzymes that can catalyze phosphodiester bond formation at the 5' end of RNAs, formally in a 3'-5' direction. Surprisingly, despite its varied activities, Thg1p contains no recognizable catalytic or functional domains.