Beta class amino methyltransferases from bacteria to humans: evolution and structural consequences.

S-adenosyl-l-methionine dependent methyltransferases catalyze methyl transfers onto a wide variety of target molecules, including DNA and RNA. We discuss a family of methyltransferases, those that act on the amino groups of adenine or cytosine in DNA, have conserved motifs in a particular order in their amino acid sequence, and are referred to as class beta MTases. Members of this class include M.EcoGII and M.EcoP15I from Escherichia coli, Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM), the MTA1-MTA9 complex from the ciliate Oxytricha, and the mammalian MettL3-MettL14 complex. These methyltransferases all generate N6-methyladenine in DNA, with some members having activity on single-stranded DNA as well as RNA. The beta class of methyltransferases has a unique multimeric feature, forming either homo- or hetero-dimers, allowing the enzyme to use division of labor between two subunits in terms of substrate recognition and methylation. We suggest that M.EcoGII may represent an ancestral form of these enzymes, as its activity is independent of the nucleic acid type (RNA or DNA), its strandedness (single or double), and its sequence (aside from the target adenine).

Identification of a DNA N6-Adenine Methyltransferase Complex and Its Impact on Chromatin Organization.

DNA N6-adenine methylation (6mA) has recently been described in diverse eukaryotes, spanning unicellular organisms to metazoa. Here, we report a DNA 6mA methyltransferase complex in ciliates, termed MTA1c. It consists of two MT-A70 proteins and two homeobox-like DNA-binding proteins and specifically methylates dsDNA. Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression. We investigate the impact of 6mA on nucleosome occupancy in vitro by reconstructing complete, full-length Oxytricha chromosomes harboring 6mA in native or ectopic positions. We show that 6mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a diverged DNA N6-adenine methyltransferase and defines the role of 6mA in chromatin organization.

Transcription-independent functions of an RNA polymerase II subunit, Rpb2, during genome rearrangement in the ciliate, Oxytricha trifallax.

The RNA polymerase II (Pol-II) holoenzyme, responsible for messenger RNA production, typically consists of 10-12 subunits. Our laboratory previously demonstrated that maternally deposited, long, noncoding, template RNAs are essential for programmed genome rearrangements in the ciliate Oxytricha trifallax. Here we show that such RNAs are bidirectionally transcribed and transported to the zygotic nucleus. The gene encoding the second-largest subunit of Pol-II, Rpb2, has undergone gene duplication, and the two paralogs, Rpb2-a and -b, display different expression patterns. Immunoprecipitation of double-stranded RNAs identified an association with Rpb2-a. Through immunoprecipitation and mass spectrometry, we show that Rpb2-a in early zygotes appears surprisingly unassociated with other Pol II subunits. A partial loss of function of Rpb2-a leads to an increase in expression of transposons and other germline-limited satellite repeats. We propose that evolutionary divergence of the Rpb2 paralogs has led to acquisition of transcription-independent functions during sexual reproduction that may contribute to the negative regulation of germline gene expression.

A functional role for transposases in a large eukaryotic genome.

Despite comprising much of the eukaryotic genome, few transposons are active, and they usually confer no benefit to the host. Through an exaggerated process of genome rearrangement, Oxytricha trifallax destroys 95% of its germline genome during development. This includes the elimination of all transposon DNA. We show that germline-limited transposase genes play key roles in this process of genome-wide DNA excision, which suggests that transposases function in large eukaryotic genomes containing thousands of active transposons. We show that transposase gene expression occurs during germline-soma differentiation and that silencing of transposase by RNA interference leads to abnormal DNA rearrangement in the offspring. This study suggests a new important role in Oxytricha for this large portion of genomic DNA that was previously thought of as junk.

Proposed function of the accumulation of plasma membrane-type Ca2+-ATPase mRNA in resting cysts of the ciliate Sterkiella histriomuscorum.

From an mRNA differential-display analysis of the encystment-excystment cycle of the ciliate Sterkiella histriomuscorum, we have isolated an expressed sequence tag encoding a plasma membrane-type Ca2+-ATPase (PMCA). PMCAs are located either in the plasma membranes or in the membranes of intracellular organelles, and their function is to pump calcium either out of the cell or into the intracellular calcium stores, respectively. The S. histriomuscorum macronuclear PMCA gene (ShPMCA) and its corresponding cDNA were cloned; it is the first member of the Ca2+-ATPase family identified in Sterkiella. The predicted protein of 1,065 amino acids exhibits 37% identity with PMCAs of diverse organisms. A phylogenetic analysis showed its relatedness to homologs of two alveolates: the ciliate Paramecium tetraurelia and the apicomplexan Toxoplasma gondii. Overexpression of the protein ShPMCA failed to rescue the wild-type phenotype of three Ca2+-ATPase-defective mutant strains of Saccharomyces cerevisiae; this failure contrasts with the reported ability of the PMCAs of parasites to complement defects in yeast. ShPMCA mRNA is markedly accumulated during encystment and in resting cysts, suggesting a function during excystment. To address the possibility of a signaling role for calcium at excystment, the capacity of calcium to induce excystment was examined.

A substrate for telomerase.

Recent data indicates that controlled in vivo synthesis of telomeric DNA is a 'ménage à trois', in which sister chromatid termini paired by telomere end-binding protein constitute a core substrate for a telomerase dimer. Such an arrangement could serve to fine tune synthesis of telomeric DNA and might ensure similar telomerase processivity for paired sister chromatid termini, possibly to secure the maintenance of individual telomere lengths in daughter cells. It also suggests that the bona fide end-capping protein, which is likely to regulate telomerase by restricting access to chromosome termini, has escaped detection in man.

Evolution and assembly of an extremely scrambled gene.

The process of gene unscrambling in hypotrichous ciliates represents one of nature's ingenious solutions to the problem of gene assembly. With some essential genes scrambled in as many as 51 pieces, these ciliates rely on sequence and structural cues to rebuild their fragmented genes and genomes. Here we report the complex pattern of scrambling in the DNA polymerase alpha gene of Stylonychia lemnae. The germline (micronuclear) copy of this gene is broken into 48 pieces with 47 dispersed over two loci, with no asymmetry in the placement of coding segments on either strand. Direct repeats present at the boundaries between coding and noncoding sequences provide pointers to help guide assembly of the functional (macronuclear) gene. We investigate the evolution of this complex gene in three hypotrichous species.

Telomerase reverse transcriptase genes identified in Tetrahymena thermophila and Oxytricha trifallax.

Telomerase reverse transcriptase (TERT) has been identified as the catalytic subunit of the chromosome end-replicating enzyme in Euplotes, yeasts, and mammals. However, it was not reported among the protein components of purified Tetrahymena telomerase, the first telomerase identified and the most thoroughly studied. It therefore seemed possible that Tetrahymena used an alternative telomerase that lacked a TERT protein. We now report the cloning and sequencing of a Tetrahymena thermophila gene whose encoded protein has the properties expected for a TERT, including large size (133 kDa), basicity (calculated pI = 10.0), and reverse transcriptase sequence motifs with telomerase-specific features. The expression of mRNA from the Tetrahymena TERT gene increases dramatically at 2-5 h after conjugation, preceding de novo addition of telomeres to macronuclear DNA molecules. We also report the cloning and sequencing of the ortholog from Oxytricha trifallax. The Oxytricha macronuclear TERT gene has no introns, whereas that of Tetrahymena has 18 introns. Sequence comparisons reveal a new amino acid sequence motif (CP), conserved among the ciliated protozoan TERTs, and allow refinement of previously identified motifs. A phylogenetic tree of the known TERTs follows the phylogeny of the organisms in which they are found, consistent with an ancient origin rather than recent transposition. The conservation of TERTs among eukaryotes supports the model that telomerase has a conserved core (TERT plus the RNA subunit), with other subunits of the holoenzyme being more variable among species.

Evolution of internal eliminated segments and scrambling in the micronuclear gene encoding DNA polymerase alpha in two Oxytricha species.

To learn about the evolution of internal eliminated segments (IESs) and gene scrambling in hypotrichous ciliates we determined the structure of the micronuclear (germline) gene encoding DNA polymerasealpha(DNA polalpha) in Oxytricha trifallax and compared it to the previously published structure of the germline DNA polalphagene in Oxytricha nova . The DNA polalphagene of O.trifallax contains 51 macronuclear-destined segments (MDSs) separated by 50 IESs, compared to 45 MDSs and 44 IESs in the O.nova gene. This means that IESs and MDSs have been gained and/or lost during evolutionary divergence of the two species. Most of the MDSs are highly scrambled in a similar non-random pattern in the two species. We present a model to explain how IESs, non-scrambled MDSs and scrambled MDSs may be added and/or eliminated during evolution. Corresponding IESs in the two species differ totally in sequence, and junctions between MDSs and IESs are shifted by 1-18 bp in O.trifallax compared to the O.nova gene. In both species a short region of the gene is distantly separated from the main part of the gene. Comparison of the gene in the two species shows that IESs and scrambling are highly malleable over evolutionary time.

The germline gene encoding DNA polymerase alpha in the hypotrichous ciliate Oxytricha nova is extremely scrambled.

We report the structure of the micronuclear (germline) gene encoding the large catalytic subunit of DNA polymerase alpha (DNA pol alpha) in the ciliate Oxytricha nova. It contains 44 internal eliminated segments (IESs) that divide the gene into 45 macronuclear-destined segments (MDSs) that are in a non-randomly scrambled order with an inversion near the gene center. Odd numbered MDSs 29-43, containing 230 bp out of a total of 4938 bp of macronuclear sequence, are missing from the 14 kb cloned gene. The missing MDSs have not been located but are at least several kilobases from the main body of the gene. The remarkably scrambled DNA pol alpha gene must be extensively cut, re-ordered and spliced and an inversion must occur to produce an unscrambled, functional version of the gene during development of a new macronucleus. Unscrambling is hypothesized to occur by a homologous recombination mechanism guided by repeat sequences at MDS ends.

Telomerase RNA localized in the replication band and spherical subnuclear organelles in hypotrichous ciliates.

The intranuclear distribution of telomere DNA-binding protein and telomerase RNA in hypotrichous ciliates was revealed by indirect fluorescent antibody staining and in situ hybridization. The Oxytricha telomere protein colocalized with DNA, both being dispersed throughout the macronucleus except for numerous spherical foci that contained neither DNA nor the protein. Surprisingly, the telomerase RNA was concentrated in these foci; therefore, much of telomerase does not colocalize with telomeres. These foci persist through the cell cycle. They may represent sites of assembly, transport or stockpiling of telomerase and other ribonucleoproteins. During S phase, the macronuclear DNA replication machinery is organized into a disc-shaped structure called the replication band. Telomerase RNA is enriched in the replication band as judged by fluorescence intensity. We conclude that the localization of a subfraction of telomerase is coordinated with semiconservative DNA replication.

Oligonucleotides complementary to the Oxytricha nova telomerase RNA delineate the template domain and uncover a novel mode of primer utilization.

The telomerase reverse transcriptase uses an essential RNA subunit as a template to direct telomeric DNA synthesis. The 190-nucleotide Oxytricha nova telomerase RNA was identified by using an oligonucleotide probe complementary to the predicted CCCCAAAA template. This RNA displays extensive sequence similarity to the Euplotes crassus telomerase RNA and carries the same 5' CAAAACCCCAAAACC 3' telomeric domain. Antisense oligonucleotides were used to map the boundaries of the functional template and to investigate the mechanism of primer recognition and elongation. On the basis of their ability to inhibit or to prime telomerase, oligonucleotides were classified into three categories. Category 1 oligonucleotides, which extended 5' of residue 42 in the RNA, abolished elongation of (T4G4)3 and (G4T4)3 primers in vitro. In contrast, oligonucleotides terminating between residues 42 and 50 (categories 2 and 3), served as efficient telomerase primers. We conclude that the O. nova template comprises residues 42 to 50 in the 190-nucleotide RNA, a different set of nucleotides than are used by the E. crassus enzyme. Category 2 primer reactions amassed short products, and their abundance could be decreased by altering the 5' sequence of the primer, consistent with the two-primer-binding-site model for telomerase. Category 3 primers generated a bimodal distribution of short and long products, each having a unique elongation profile. The long-product profile is inconsistent with sequence-specific primer alignment. Rather, each primer was extended by the same register of TTTTGGGG repeats, suggesting shuttling to a default position within the template. The parallels between telomerase and RNA polymerase elongation mechanisms are discussed.

A gene-sized DNA molecule encoding the catalytic subunit of DNA polymerase alpha in the macronucleus of Oxytricha nova.

We have isolated a gene-sized molecule encoding the catalytic subunit of DNA polymerase alpha from a macronuclear genomic library of Oxytricha nova, by using a 0.7-kb fragment of the corresponding human gene as a hybridization probe. Two different versions of the gene are present in the macronucleus, one with an EcoRI site (RI+) and one without an EcoRI site (RI-). The cloned RI- version has been characterized. It is 4938 bp in length, excluding telomeres. It consists of a 329-bp 5' leader, a 4479-bp coding region and a 130-bp 3' trailer. The deduced amino-acid sequence shares conserved regions with the yeast and human polypeptides. We also demonstrate by Southern analysis that gene-sized molecules of similar size, homologous to the isolated O. nova gene are present in the mac genome of closely and distantly related hypotrichs.

A proposed superfamily of transposase genes: transposon-like elements in ciliated protozoa and a common "D35E" motif.

The transposon-like elements TBE1, Tec1, and Tec2 of hypotrichous ciliated protozoa appear to encode a protein that belongs to the IS630-Tc1 family of transposases. The Anabaena IS895 transposase also is placed in this family. We note that most family members transpose into the dinucleotide target, TA, and that members with eukaryotic hosts have a tendency for somatic excision that is carried to an extreme by the ciliate elements. Alignments including the additional members, and also mariner elements, show that transposases of this family share strongly conserved residues in a large C-terminal portion, including a fully conserved dipeptide, Asp-Glu (DE), and a block consisting of a fully conserved Asp and highly conserved Glu, separated by 34 or 35 residues (D35E). This D35E motif likely is homologous to the previously characterized D35E motif of the family of retroviral-retrotransposon integrases and IS3-like transposases. Because it is known that the IS3-retroposon D35E region is a critical portion of a domain capable of various in vitro transposition-related reactions, the results suggest that the two families share homologous catalytic transposase domains and that members of both families may share a common transposition mechanism.