tRNA Gene Cluster Research Articles

Factors targeting MED12 to drive tumorigenesis?

Mediator Subcomplex 12 (MED12) is part of the transcriptional preinitiation machinery. Mutations of its gene predominantly occur in two types of highly frequent benign tumors, uterine leiomyomas and fibroadenomas of the breast, where they apparently act as driver mutations. Nevertheless, their presence is not restricted to benign tumors having been found at considerable frequencies in uterine leiomyosarcomas, malignant phyllodes tumors, and chronic lymphocytic leukemia also. Most of the mutations are located within exon 2 of the gene but in rare cases the intron 1/exon 2 boundary or exon 1 are affected. As to their type, predominantly single nucleotide exchanges with a hotspot in one codon are found, but small deletions clustering around that hotspot also are not uncommon. According to their presumed classification as gain-of-function mutations, these latter deletions are leaving the open reading frame intact. As to the types of mutations, so far no apparent differences between the tumor entities affected have emerged. Interestingly, this pattern with small deletions clustered around the hotspot of single nucleotide exchanges resembles that seen as a result of targeted gene editing. In contrast to other driver mutations the percentage of MED12-mutation positive tumors of independent clonal origin increases with the number of tumors per patient suggesting unknown etiological factors supporting site specific mutagenesis. These factors may act by inducing simultaneous site-specific double strand breaks the erroneous repair of which may lead to corresponding mutations. As inducers of DNA damage and its repair such as foreign nucleic acids of the microbiome displaying sequence homology to the putative target site might play a role. Interestingly, a 16 base pair homology of the hotspot to a putative terminator base-paired hairpin sequence of a Staphylococcus aureus tRNA gene cluster has been noted which might form R-loop like structures with its target sequence thus inducing said changes.

Next-generation sequencing data used to determine the mitochondrial genomes and a preliminary phylogeny of Verophasmatodea insects

We used Illumina next-generation sequencing data to generate the nearly complete mitochondrial genomes (mitogenomes) of three phasmatodeans (Megalophasma granulata, Calvisia medogensis, and Phyllium tibetense) without the aid of additional techniques such as long-range PCR or cloning. The most surprising finding was the presence of a novel gene arrangement “trnR-trnA-trnN-trnSAGN-trnE-trnF” (genes underlined are encoded by the minority strand) that was detected in M. granulata. The ancestral order of this tRNA gene cluster is typically “trnA-trnR-trnN-trnSAGN-trnE-trnF”. However, trnA was inserted between trnR and trnN in M. granulata, and this represents a rare case of gene rearrangement in Phasmatodea. Phylogenetic analyses were conducted on the concatenated nucleotide sequences of all protein-coding genes and two ribosomal RNA genes. Both maximum likelihood and Bayesian analyses failed to support the monophyly of Areolatae, Anareolatae, Diapheromeridae, Phasmatidae, Heteropterygidae, Necrosciinae, and Necrosciini. However, the monophyly of several lower taxonomic groups were confirmed in our analysis. For instance, two Ramulus species and Entoria okinawaensis were recovered in the monophyletic Clitumnini, and Phraortes, Phyllium, and Bacillus species formed individual monophyletic clades. Our results support the hypothesis that wing-loss independently occurred several times in Verophasmatodea.

A long terminal repeat retrotransposon of Schizosaccharomyces japonicus integrates upstream of RNA pol III transcribed genes.

BackgroundTransposable elements (TEs) are common constituents of centromeres. However, it is not known what causes this relationship. Schizosaccharomyces japonicus contains 10 families of Long Terminal Repeat (LTR)-retrotransposons and these elements cluster in centromeres and telomeres. In the related yeast, Schizosaccharomyces pombe LTR-retrotransposons Tf1 and Tf2 are distributed in the promoter regions of RNA pol II transcribed genes. Sequence analysis of TEs indicates that Tj1 of S. japonicus is related to Tf1 and Tf2, and uses the same mechanism of self-primed reverse transcription. Thus, we wondered why these related retrotransposons localized in different regions of the genome.ResultsTo characterize the integration behavior of Tj1 we expressed it in S. pombe. We found Tj1 was active and capable of generating de novo integration in the chromosomes of S. pombe. The expression of Tj1 is similar to Type C retroviruses in that a stop codon at the end of Gag must be present for efficient integration. 17 inserts were sequenced, 13 occurred within 12 bp upstream of tRNA genes and 3 occurred at other RNA pol III transcribed genes. The link between Tj1 integration and RNA pol III transcription is reminiscent of Ty3, an LTR-retrotransposon of Saccharomyces cerevisiae that interacts with TFIIIB and integrates upstream of tRNA genes.ConclusionThe integration of Tj1 upstream of tRNA genes and the centromeric clustering of tRNA genes in S. japonicus demonstrate that the clustering of this TE in centromere sequences is due to a unique pattern of integration.

Characterization of the mitochondrial genome of Amolops tuberodepressus (Anura: Ranidae)

Amolops tuberodepressus is a vulnerable torrent frog, and only know distributed in the Wuliang Mountain in southwestern China. In the present study, the mitochondrial DNA (mtDNA) sequence of A. tuberodepressus was determined. The genome was 18 348 bp in length, and it contained 37 genes (13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs), one partial control region and one light strand replication origin. The gene rearrangement was observed within the WANCY tRNA gene cluster region, which similar to other Amolops species. In this paper, we utilized 13 protein-coding genes of A. tuberodepressus and other 10 closely ranid species to construct the species phylogenetic tree to verify the A. tuberodepressus was accuracy.

Phylogenetic relationships among four new complete mitogenome sequences of Pelophylax (Amphibia: Anura) from the Balkans and Cyprus

We present the full-length mitogenome sequences of four European water frog species: Pelophylax cypriensis, P. epeiroticus, P. kurtmuelleri and P. shqipericus. The mtDNA size varied from 17,363 to 17,895 bp, and its organization with the LPTF tRNA gene cluster preceding the 12 S rRNA gene displayed the typical Neobatrachian arrangement. Maximum likelihood and Bayesian inference revealed a well-resolved mtDNA phylogeny of seven European Pelophylax species. The uncorrected p-distance for among Pelophylax mitogenomes was 9.6 (range 0.01–0.13). Most divergent was the P. shqipericus mitogenome, clustering with the “P. lessonae” group, in contrast to the other three new Pelophylax mitogenomes related to the “P. bedriagae/ridibundus” lineage. The new mitogenomes resolve ambiguities of the phylogenetic placement of P. cretensis and P. epeiroticus.

Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of Occidozyga martensii.

In this study, the complete nucleotide sequence (18,321 bp) of the mitochondrial (mt) genome of the round-tongued floating frog, Occidozyga martensii was determined. Although, the base composition and codon usage of O. martensii conformed to the typical vertebrate patterns, this mt genome contained 23 tRNAs (a tandem duplication of tRNA-Met gene). The LTPF tRNA-gene cluster, and the derived position of the ND5 gene downstream of the control region, were present in this mitogenome. Moreover, we found that in the WANCY tRNA-gene cluster, the tRNA-Asn gene was located between the tRNATyr and COI genes instead of between the tRNA-Ala and tRNA-Cys genes, which is a novel mtDNA gene rearrangement in vertebrates. Based on the concatenated nucleotide sequences of the 13 protein-coding genes, phylogenetic analysis (BI, ML, MP) was performed to further clarify the phylogenetic relations of this species within anurans.

Complete mitochondrial genome of Amolops mantzorum (Anura: Ranidae)

Sichuan torrent frog, Amolops mantzorum (family Ranidae, suborder Neobatrachia), possesses heteromorphic sex chromosomes unusual characteristics among amphibians. We determined the complete nucleotide sequence of the A. mantzorum mitogenome. This genome is 17,744 bp in length and contains 37 genes, 1 control region, and 1 light strand replication origin typically found in vertebrate mtDNAs. In the A. mantzorum mitogenome, a novel gene arrangement is observed within the WANCY tRNA gene cluster region. This mt gene arrangement seems to be usable as a molecular maker to distinguish to this species from other species in the genus Amolops.

The complete mitochondrial genome of Feirana taihangnica (Anura: Dicroglossidae)

The complete mitochondrial genome sequence of the Feirana taihangnica (Anura: Dicroglossidae) was determined. It is a circular molecule of 17,412 bp in length, containing 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a control region. The tRNAThr gene located in the LTPF tRNA gene cluster typically found in other anurans is absent from the F. taihangnica mtDNA and a tandem duplication of tRNAMet gene is observed. The A+T content of the overall base compositon of H-strand is 57.2% and the length of control region is 1972 bp with 61.8% A+T content.

Gene rearrangements in gekkonid mitochondrial genomes with shuffling, loss, and reassignment of tRNA genes.

BackgroundVertebrate mitochondrial genomes (mitogenomes) are 16–18 kbp double-stranded circular DNAs that encode a set of 37 genes. The arrangement of these genes and the major noncoding region is relatively conserved through evolution although gene rearrangements have been described for diverse lineages. The tandem duplication-random loss model has been invoked to explain the mechanisms of most mitochondrial gene rearrangements. Previously reported mitogenomic sequences for geckos rarely included gene rearrangements, which we explore in the present study.ResultsWe determined seven new mitogenomic sequences from Gekkonidae using a high-throughput sequencing method. The Tropiocolotes tripolitanus mitogenome involves a tandem duplication of the gene block: tRNAArg, NADH dehydrogenase subunit 4L, and NADH dehydrogenase subunit 4. One of the duplicate copies for each protein-coding gene may be pseudogenized. A duplicate copy of the tRNAArg gene appears to have been converted to a tRNAGln gene by a C to T base substitution at the second anticodon position, although this gene may not be fully functional in protein synthesis. The Stenodactylus petrii mitogenome includes several tandem duplications of tRNALeu genes, as well as a translocation of the tRNAAla gene and a putative origin of light-strand replication within a tRNA gene cluster. Finally, the Uroplatus fimbriatus and U. ebenaui mitogenomes feature the apparent loss of the tRNAGlu gene from its original position. Uroplatus fimbriatus appears to retain a translocated tRNAGlu gene adjacent to the 5’ end of the major noncoding region.ConclusionsThe present study describes several new mitochondrial gene rearrangements from Gekkonidae. The loss and reassignment of tRNA genes is not very common in vertebrate mitogenomes and our findings raise new questions as to how missing tRNAs are supplied and if the reassigned tRNA gene is fully functional. These new examples of mitochondrial gene rearrangements in geckos should broaden our understanding of the evolution of mitochondrial gene arrangements.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-930) contains supplementary material, which is available to authorized users.

TRNA gene copy number variation in humans

The human tRNAome consists of more than 500 interspersed tRNA genes comprising 51 anticodon families of largely unequal copy number. We examined tRNA gene copy number variation (tgCNV) in six individuals; two kindreds of two parents and a child, using high coverage whole genome sequence data. Such differences may be important because translation of some mRNAs is sensitive to the relative amounts of tRNAs and because tRNA competition determines translational efficiency vs. fidelity and production of native vs. misfolded proteins. We identified several tRNA gene clusters with CNV, which in some cases were part of larger iterations. In addition there was an isolated tRNALysCUU gene that was absent as a homozygous deletion in one of the parents. When assessed by semiquantitative PCR in 98 DNA samples representing a wide variety of ethnicities, this allele was found deleted in hetero- or homozygosity in all groups at ~50% frequency. This is the first report of copy number variation of human tRNA genes. We conclude that tgCNV exists at significant levels among individual humans and discuss the results in terms of genetic diversity and prior genome wide association studies (GWAS) that suggest the importance of the ratio of tRNALys isoacceptors in Type-2 diabetes.

Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation

Despite numerous studies on specific sumoylated transcriptional regulators, the global role of SUMO on chromatin in relation to transcription regulation remains largely unknown. Here, we determined the genome-wide localization of SUMO1 and SUMO2/3, as well as of UBC9 (encoded by UBE2I) and PIASY (encoded by PIAS4), two markers for active sumoylation, along with Pol II and histone marks in proliferating versus senescent human fibroblasts together with gene expression profiling. We found that, whereas SUMO alone is widely distributed over the genome with strong association at active promoters, active sumoylation occurs most prominently at promoters of histone and protein biogenesis genes, as well as Pol I rRNAs and Pol III tRNAs. Remarkably, these four classes of genes are up-regulated by inhibition of sumoylation, indicating that SUMO normally acts to restrain their expression. In line with this finding, sumoylation-deficient cells show an increase in both cell size and global protein levels. Strikingly, we found that in senescent cells, the SUMO machinery is selectively retained at histone and tRNA gene clusters, whereas it is massively released from all other unique chromatin regions. These data, which reveal the highly dynamic nature of the SUMO landscape, suggest that maintenance of a repressive environment at histone and tRNA loci is a hallmark of the senescent state. The approach taken in our study thus permitted the identification of a common biological output and uncovered hitherto unknown functions for active sumoylation at chromatin as a key mechanism that, in dynamically marking chromatin by a simple modifier, orchestrates concerted transcriptional regulation of a network of genes essential for cell growth and proliferation.

The complete mitochondrial genome of Arctic Calanus hyperboreus (Copepoda, Calanoida) reveals characteristic patterns in calanoid mitochondrial genome

Copepoda is the most diverse and abundant group of crustaceans, but its phylogenetic relationships are ambiguous. Mitochondrial (mt) genomes are useful for studying evolutionary history, but only six complete Copepoda mt genomes have been made available and these have extremely rearranged genome structures. This study determined the mt genome of Calanus hyperboreus, making it the first reported Arctic copepod mt genome and the first complete mt genome of a calanoid copepod. The mt genome of C. hyperboreus is 17,910bp in length and it contains the entire set of 37 mt genes, including 13 protein-coding genes, 2 rRNAs, and 22 tRNAs. It has a very unusual gene structure, including the longest control region reported for a crustacean, a large tRNA gene cluster, and reversed GC skews in 11 out of 13 protein-coding genes (84.6%). Despite the unusual features, comparing this genome to published copepod genomes revealed retained pan-crustacean features, as well as a conserved calanoid-specific pattern. Our data provide a foundation for exploring the calanoid pattern and the mechanisms of mt gene rearrangement in the evolutionary history of the copepod mt genome.

Expression and processing of an unusual tRNA gene cluster in the cyanobacteriumAnabaenasp. PCC 7120

Anabaena sp. PCC 7120 is a filamentous cyanobacterium that bears a cluster of 26 tRNA genes and pseudogenes in the delta plasmid. The sequences of these tRNAs suggest that they have been acquired by horizontal gene transfer from another organism. The cluster is transcribed as a single transcript that is quickly processed to individual tRNAs. RNase P and RNase Z, in vitro, are able to process precursors containing some of these tRNAs. Deletion of the cluster causes no obvious phenotype or effect on growth under diverse culture conditions, indicating that the tRNAs encoded in the cluster are not required for growth under laboratory conditions, although they are aminoacylated in vivo. We have studied a possible tRNA(Ser) [tRNA(Ser) GCU(2)] present in the cluster with a sequence that deviates from consensus. This tRNA is processed in vitro by RNaseP at the expected position. In addition, this tRNA(Ser) GCU is specifically aminoacylated with serine by an Anabaena sp. PCC 7120 crude extract. These data indicate that tRNA(Ser) GCU(2) is fully functional, despite its unusual structure. Similar clusters are found in other three cyanobacteria whose genomes have been sequenced.

The complete mitochondrial genome of Hoplobatrachus rugulosus (Anura: Dicroglossidae)

The mitochondrial genome of Hoplobatrachus rugulosus (Anura: Dicroglossidae) is a circular molecule of 20,309 bp in length, containing 39 genes (including the extra copy of ND5 and tRNAMet genes). The following four distinctive features are observed: the cluster of rearranged tRNA genes (TPF tRNA gene cluster), the translocation of tRNALeu(CUN) and ND5 genes, the tandem duplication of tRNAMet genes (Met1 and Met2), and the duplicated d-loop–ND5 regions.

TRNAomics: tRNA gene copy number variation and codon use provide bioinformatic evidence of a new anticodon:codon wobble pair in a eukaryote

tRNA genes are interspersed throughout eukaryotic DNA, contributing to genome architecture and evolution in addition to translation of the transcriptome. Codon use correlates with tRNA gene copy number in noncomplex organisms including yeasts. Synonymous codons impact translation with various outcomes, dependent on relative tRNA abundances. Availability of whole-genome sequences allowed us to examine tRNA gene copy number variation (tgCNV) and codon use in four Schizosaccharomyces species and Saccharomyces cerevisiae. tRNA gene numbers vary from 171 to 322 in the four Schizosaccharomyces despite very high similarity in other features of their genomes. In addition, we performed whole-genome sequencing of several related laboratory strains of Schizosaccharomyces pombe and found tgCNV at a cluster of tRNA genes. We examined for the first time effects of wobble rules on correlation of tRNA gene number and codon use and showed improvement for S. cerevisiae and three of the Schizosaccharomyces species. In contrast, correlation in Schizosaccharomyces japonicus is poor due to markedly divergent tRNA gene content, and much worsened by the wobble rules. In japonicus, some tRNA iso-acceptor genes are absent and others are greatly reduced relative to the other yeasts, while genes for synonymous wobble iso-acceptors are amplified, indicating wobble use not apparent in any other eukaryote. We identified a subset of japonicus-specific wobbles that improves correlation of codon use and tRNA gene content in japonicus. We conclude that tgCNV is high among Schizo species and occurs in related laboratory strains of S. pombe (and expectedly other species), and tRNAome-codon analyses can provide insight into species-specific wobble decoding.

The complete mitochondrial genome of Pseudochauhanea macrorchis (Monogenea: Chauhaneidae) revealed a highly repetitive region and a gene rearrangement hot spot in Polyopisthocotylea

The complete mitochondrial genome of Pseudochauhanea macrorchis was determined and compared with other monogenean mitochondrial genomes from GenBank. The circular genome was 15,031 bp in length and encoded 36 genes (12 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs) typically found in flatworms. Structures of the mitochondrial genome were mostly concordant with that known for Microcotyle sebastis and Polylabris halichoeres, but also contained two noted features-a gene rearrangement hot spot and the highly repetitive region (HRR) in major non-coding region (NCR). The gene rearrangement hot spot located between the cox3 and nad5 genes, including a cluster of tRNA genes, nad6 gene and one major NCR. The HRR seemed to be a unique feature of the polyopisthocotylean mitochondrial genomes. In conclusion, the present study provided new molecular data for future studies of the comparative mitochondrial genomics and also served as a resource of markers for the studies of species populations and monogenean phylogenetics.

Complete mitochondrial genome of Nanorana pleskei (Amphibia: Anura: Dicroglossidae) and evolutionary characteristics

Abstract The complete mitochondrial genome of Nanorana pleskei from the Qinghai-Tibet Plateau was sequenced. It includes 17,660 base pairs, containing 13 protein-coding genes, two rRNAs and 23 tRNAs. A tandem duplication of tRNAMet gene was found in this mitochondrial genome, and the similarity between the two tRNAMet genes is 85.8%, being the highest in amphibian mitochondrial genomes sequenced thus far. Based on gene organization, 24 types were found from 145 amphibian mitochondrial genomes. Type 1 was present in 108 species, type 11 in 11 species, types 5, 16, 17, and 20 each in two species, and the others each present in one species. Fifteen types were found in Anura, being the most diversity in three orders of the Lissamphibia. Our phylogenetic results using 11 protein-coding gene sequences of 145 amphibian mitochondrial genomes strongly support the monophyly of the Lissamphibia, as well as its three orders, the Gymnophiona, Caudata, and Anura, among which the relationships were ((Gymnophiona (Caudata, Anura)). Based on the phylogenetic trees, type 1 was recognized as the ancestral type for amphibians, and type 11 was the synapomorphic type for the Neobatrachia. Gene rearrangements among lineages provide meaningful phylogenetic information. The rearrangement of the LTPF tRNA gene cluster and the translocation of the ND5 gene only found in the Neobatrachia support the monophyly of this group; similarly, the tandem duplication of the tRNAMet genes only found in the Dicroglossidae support the monophyly of this family.

Origin of most primitive mRNAs and genetic codes via interactions between primitive tRNA ribozymes.

The origin and early evolution of genetic codon system and early mRNAs were analyzed from a viewpoint of primordial gene theory and the poly-tRNA theory. A hypothetical 25-amino acid (aa)-primordial peptide was deduced from internal aa-sequence homology of adenylate kinases. Theoretical models were made which can reasonably explain how primitive tRNA(s) could have had converted to be earliest mRNAs via interactions between presumptive anticodons and (poly-)tRNA ribozyme. Transfer-RNA gene clusters in the trrnD- and rrnB-operons of Bacillus subtilis seemed to be relics of early peptide-synthesizing RNA machine. Detailed analyses revealed that the poly-tRNA regions in these operons are true relics of RNA-machine for making a 16-aa trrnD-peptide and a 21-aa rrnB-peptide, whose aa sequences are in the order of aa specificities of tRNAs in the tRNA gene clusters of the trrnD-operon and rrnB-operon, respectively. The primordial gene-encoded peptide deduced from adenylate kinases were found to be a genuine homologue of the rrnB-peptide. Various protein superfamilies were found to have evolved from either of these two types of primitive peptides. Earliest mRNAs were concluded to have evolved from tRNAGly (trrnD-mRNA) or tRNAHis (rrnB-mRNA), where trrnD- and rrnB-mRNAs are hypothetical primitive mRNAs complementary to the tandem arrangement of 16 or 21 anticodons of tRNAs in the trrnD-operon and rrnB-operon, respectively. The poly-tRNA model is considered to be an excellent theory, because it can reasonably explain origins of both genetic codes and earliest mRNAs, and because the hypothesis can be statistically evaluated by base-identity levels in proper alignments. The genetic codon system is a typical mature semeiotic system within a cell, and the genesis of the genetic codon system was discussed from an aspect of de Saussure's semeiology. Arbitrary correspondence between (anti)codon and aa would be most plausibly a result of semeiotic culture system of intracellular tRNA-riboorganismic society.

Cohesinopathy mutations disrupt the subnuclear organization of chromatin

In Saccharomyces cerevisiae, chromatin is spatially organized within the nucleus with centromeres clustering near the spindle pole body, telomeres clustering into foci at the nuclear periphery, ribosomal DNA repeats localizing within a single nucleolus, and transfer RNA (tRNA) genes present in an adjacent cluster. [corrected] Furthermore, certain genes relocalize from the nuclear interior to the periphery upon transcriptional activation. The molecular mechanisms responsible for the organization of the genome are not well understood. We find that evolutionarily conserved proteins in the cohesin network play an important role in the subnuclear organization of chromatin. Mutations that cause human cohesinopathies had little effect on chromosome cohesion, centromere clustering, or viability when expressed in yeast. However, two mutations in particular lead to defects in (a) GAL2 transcription and recruitment to the nuclear periphery, (b) condensation of mitotic chromosomes, (c) nucleolar morphology, and (d) tRNA gene-mediated silencing and clustering of tRNA genes. We propose that the cohesin network affects gene regulation by facilitating the subnuclear organization of chromatin.

Complete nucleotide sequence and gene organization of the mitochondrial genome of Paa spinosa (Anura: Ranoidae)

The mt genome of Paa spinosa (Anura: Ranoidae) is a circular molecule of 18,012 bp in length, containing 38 genes (including an extra copy of tRNA-Met gene). This mt genome is characterized by three distinctive features: a cluster of rearranged tRNA genes (LTPF tRNA gene cluster), a tandem duplication of tRNA-Met gene (Met1 and Met2), and distinct repeat regions at both 5′ and 3′-sides in the control region. Comparing the locations and the sequences of all tRNA-Met genes among Ranoidae, and constructing NJ tree of the nucleotide of those tRNA-Met genes, we suggested a tandem duplication of tRNA-Met gene can be regarded as a synapomorphy of Dicroglossinae. To further investigate the phylogenetic relationships of anurans, phylogenetic analyses (BI, ML and MP) based on the nucleotide dataset and the corresponding amino acid dataset of 11 protein-coding genes (except ND5 and ATP8) arrived at the similar topology.