Plasmodium falciparum translational machinery condones polyadenosine repeats.

Plasmodium falciparum is a causative agent of human malaria. Sixty percent of mRNAs from its extremely AT-rich (81%) genome harbor long polyadenosine (polyA) runs within their ORFs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate polyA runs cause ribosome stalling and frameshifting, triggering mRNA surveillance pathways and attenuating protein synthesis. Here, we show that P. falciparum is an exception to this rule. We demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately translated in P. falciparum cells. We show that polyA runs do not elicit any response from No Go Decay (NGD) or result in the production of frameshifted proteins. This is in stark contrast to what we observe in human cells or T. thermophila, an organism with similar AT-content. Finally, using stalling reporters we show that Plasmodium cells evolved not to have a fully functional NGD pathway.

Boundaries of eliminated heterochromatin of Tetrahymena are positioned by the DNA-binding protein Ltl1.

During differentiation of the Tetrahymena thermophila somatic nucleus, its germline-derived DNA undergoes extensive reorganization including the removal of ∼50 Mb from thousands of loci called internal eliminated sequences (IESs). IES-associated chromatin is methylated on lysines 9 and 27 of histone H3, marking newly formed heterochromatin for elimination. To ensure that this reorganized genome maintains essential coding and regulatory sequences, the boundaries of IESs must be accurately defined. In this study, we show that the developmentally expressed protein encoded by Lia3-Like 1 (LTL1) (Ttherm_00499370) is necessary to direct the excision boundaries of particular IESs. In ΔLTL1 cells, boundaries of eliminated loci are aberrant and heterogeneous. The IESs regulated by Ltl1 are distinct from those regulated by the guanine-quadruplex binding Lia3 protein. Ltl1 has a general affinity for double stranded DNA (Kd ∼ 350 nM) and binds specifically to a 50 bp A+T rich sequence flanking each side of the D IES (Kd ∼ 43 nM). Together these data reveal that Ltl1 and Lia3 control different subsets of IESs and that their mechanisms for flanking sequence recognition are distinct.

Transgenerational Inheritance: Parental Guidance Suggested.

RNAs have been attractive candidates to transmit epigenetic information over multiple generations. In Tetrahymena, a new study demonstrates that the selective degradation of small RNAs that occurs by interaction with the parental genome can communicate altered patterns of heterochromatin formation and DNA elimination in offspring.

Diversification of HP1-like Chromo Domain Proteins in Tetrahymena thermophila.

Proteins that possess a chromo domain are well-known for their roles in heterochromatin assembly and maintenance. The Heterochromatin Protein 1 (HP1) family, with a chromo domain and carboxy-terminal chromo shadow domain, targets heterochromatin through interaction with histone H3 methylated on lysine 9 (H3K9me2/3). The structural and functional diversity of these proteins observed in both fission yeast and metazoans correlate with chromatin specialization. To expand these studies, we examined chromo domain proteins in the ciliate Tetrahymena thermophila, which has functionally diverse and developmentally regulated heterochromatin domains. We identified thirteen proteins similar to HP1. Together they possess only a fraction of the possible chromo domain subtypes and most lack a recognizable chromo shadow domain. Using fluorescence microscopy to track chromatin localization of tagged proteins through the life cycle, we show evidence that in T. thermophila this family has diversified with biological roles in RNAi-directed DNA elimination, germline genome structure, and somatic heterochromatin. Those proteins with H3K27me3 binding sequence characteristics localize to chromatin in mature nuclei, whereas those with H3K9me2/3 binding characteristics localize to developing nuclei undergoing DNA elimination. Findings point to an expanded and diversified family of chromo domain proteins that parallels heterochromatin diversity in ciliates.

Rapid generation of hypomorphic mutations.

Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.

One genome's junk is another's garbage.

Experiments on a single-celled ciliate reveal how mobile genetic elements can shape a genome, even one which is not transcriptionally active.

DRH1, a p68-related RNA helicase gene, is required for chromosome breakage in Tetrahymena.

The p68 DEAD box helicases comprise a widely conserved protein family involved in a large range of biological processes including transcription, splicing and translation. The genome of the ciliate Tetrahymena thermophile encodes two p68-like helicases, Drh1p and Lia2p. We show that DRH1 is essential for growth and completion of development. In growing cells, Drh1p is excluded from the nucleus and accumulates near cortical basal bodies. In contrast, during sexual reproduction, this protein localizes to meiotic micronuclei, initially in punctate foci in regions where centromeres and telomeres are known to reside and later in post-zygotic differentiating somatic macronuclei. Differentiation of the macronuclear genome involves extensive DNA rearrangements including fragmentation of the five pairs of germline-derived chromosomes into 180 chromosomal sub-fragments that are stabilized by de novo telomere deletion. In addition, thousands of internal eliminated sequences (IESs) are excised from loci dispersed throughout the genome. Strains with DRH1 deleted from the germline nuclei, which do not express the protein during post-zygotic development, fail to fragment the developing macronuclear chromosomes. IES excision still occurs in the absence of DRH1 zygotic expression; thus, Drh1p is the first protein found to be specifically required for chromosome breakage but not DNA elimination.

A Parallel G Quadruplex-Binding Protein Regulates the Boundaries of DNA Elimination Events of Tetrahymena thermophila.

Guanine (G)-rich DNA readily forms four-stranded quadruplexes in vitro, but evidence for their participation in genome regulation is limited. We have identified a quadruplex-binding protein, Lia3, that controls the boundaries of germline-limited, internal eliminated sequences (IESs) of Tetrahymena thermophila. Differentiation of this ciliate's somatic genome requires excision of thousands of IESs, targeted for removal by small-RNA-directed heterochromatin formation. In cells lacking LIA3 (ΔLIA3), the excision of IESs bounded by specific G-rich polypurine tracts was impaired and imprecise, whereas the removal of IESs without such controlling sequences was unaffected. We found that oligonucleotides containing these polypurine tracts formed parallel G-quadruplex structures that are specifically bound by Lia3. The discovery that Lia3 binds G-quadruplex DNA and controls the accuracy of DNA elimination at loci with specific G-tracts uncovers an unrecognized potential of quadruplex structures to regulate chromosome organization.

SUMOylation is developmentally regulated and required for cell pairing during conjugation in Tetrahymena thermophila.

The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins regulates numerous nuclear events in eukaryotes, including transcription, mitosis and meiosis, and DNA repair. Despite extensive interest in nuclear pathways within the field of ciliate molecular biology, there have been no investigations of the SUMO pathway in Tetrahymena. The developmental program of sexual reproduction of this organism includes cell pairing, micronuclear meiosis, and the formation of a new somatic macronucleus. We identified the Tetrahymena thermophila SMT3 (SUMO) and UBA2 (SUMO-activating enzyme) genes and demonstrated that the corresponding green fluorescent protein (GFP) tagged gene products are found predominantly in the somatic macronucleus during vegetative growth. Use of an anti-Smt3p antibody to perform immunoblot assays with whole-cell lysates during conjugation revealed a large increase in SUMOylation that peaked during formation of the new macronucleus. Immunofluorescence using the same antibody showed that the increase was localized primarily within the new macronucleus. To initiate functional analysis of the SUMO pathway, we created germ line knockout cell lines for both the SMT3 and UBA2 genes and found both are essential for cell viability. Conditional Smt3p and Uba2p cell lines were constructed by incorporation of the cadmium-inducible metallothionein promoter. Withdrawal of cadmium resulted in reduced cell growth and increased sensitivity to DNA-damaging agents. Interestingly, Smt3p and Uba2p conditional cell lines were unable to pair during sexual reproduction in the absence of cadmium, consistent with a function early in conjugation. Our studies are consistent with multiple roles for SUMOylation in Tetrahymena, including a dynamic regulation associated with the sexual life cycle.

LIA4 encodes a chromoshadow domain protein required for genomewide DNA rearrangements in Tetrahymena thermophila.

Extensive DNA elimination occurs as part of macronuclear differentiation during Tetrahymena sexual reproduction. The identification of sequences to excise is guided by a specialized RNA interference (RNAi) machinery that targets the methylation of histone H3 lysine 9 (K9) and K27 on chromatin associated with these internal eliminated sequences (IESs). This modified chromatin is reorganized into heterochromatic subnuclear foci, which is a hallmark of their subsequent elimination. Here, we demonstrate that Lia4, a chromoshadow domain-containing protein, is an essential component in this DNA elimination pathway. LIA4 knockout (ΔLIA4) lines fail to excise IESs from their developing somatic genome and arrest at a late stage of conjugation. Lia4 acts after RNAi-guided heterochromatin formation, as both H3K9 and H3K27 methylation are established. Nevertheless, without LIA4, these cells fail to form the heterochromatic foci associated with DNA rearrangement, and Lia4 accumulates in the foci, indicating that Lia4 plays a key role in their structure. These data indicate a critical role for Lia4 in organizing the nucleus during Tetrahymena macronuclear differentiation.

Morphogenesis: a Mob rules from the rear.

The giant ciliated protozoan Stentor coeruleus is re-emerging as a model organism for morphogenesis and patterning in unicellular organisms. A new study provides evidence that cytokinesis and morphogenesis are mechanistically linked through the Mob1 protein.

Mutations in Pdd1 reveal distinct requirements for its chromodomain and chromoshadow domain in directing histone methylation and heterochromatin elimination.

Pdd1, a specialized HP1-like protein, is required for genome-wide DNA rearrangements that restructure a previously silent germ line genome into an active somatic genome during macronuclear differentiation of Tetrahymena thermophila. We deleted or otherwise mutated conserved regions of the protein to investigate how its different domains promote the excision of thousands of internal eliminated sequences (IESs). Previous studies revealed that Pdd1 contributes to recognition of IES loci after they are targeted by small-RNA-guided methylation of histone H3 on lysine 27 (H3K27), subsequently aids the establishment of H3K9 methylation, and recruits proteins that lead to excision. The phenotypes we observed for different Pdd1 alleles showed that each of the two chromodomains and the chromoshadow domain (CSD) have distinct contributions during somatic genome differentiation. Chromodomain 1 (CD1) is essential for conjugation as either its deletion or the substitution of two key aromatic amino acid residues (the W97A W100A mutant) is lethal. These mutations caused mislocalization of a cyan fluorescent protein (CFP)-tagged protein, prevented the establishment of histone H3 dimethylated on K9 (H3K9me2), and abolished IES excision. Nevertheless, the requirement for CD1 could be bypassed by recruiting Pdd1 directly to an IES by addition of a specific DNA binding domain. Chromodomain 2 (CD2) was necessary for producing viable progeny, but low levels of H3K9me2 and IES excision still occurred. A mutation in the chromoshadow domain (CSD) prevented Pdd1 focus formation but still permitted ∼17% of conjugants to produce viable progeny. However, this mutant was unable to stimulate excision when recruited to an ectopic IES, indicating that this domain is important for recruitment of excision factors.

Epigenetics of ciliates.

Research using ciliates revealed early examples of epigenetic phenomena and continues to provide novel findings. These protozoans maintain separate germline and somatic nuclei that carry transcriptionally silent and active genomes, respectively. Examining the differences in chromatin within distinct nuclei of Tetrahymena identified histone variants and established that transcriptional regulators act by modifying histones. Formation of somatic nuclei requires both transcriptional activation of silent chromatin and large-scale DNA elimination. This somatic genome remodeling is directed by homologous RNAs, acting with an RNA interference (RNAi)-related machinery. Furthermore, the content of the parental somatic genome provides a homologous template to guide this genome restructuring. The mechanisms regulating ciliate DNA rearrangements reveal the surprising power of homologous RNAs to remodel the genome and transmit information transgenerationally.

LIA5 is required for nuclear reorganization and programmed DNA rearrangements occurring during tetrahymena macronuclear differentiation.

During macronuclear differentiation of the ciliate Tetrahymena thermophila, genome-wide DNA rearrangements eliminate nearly 50 Mbp of germline derived DNA, creating a streamlined somatic genome. The transposon-like and other repetitive sequences to be eliminated are identified using a piRNA pathway and packaged as heterochromatin prior to their removal. In this study, we show that LIA5, which encodes a zinc-finger protein likely of transposon origin, is required for both chromosome fragmentation and DNA elimination events. Lia5p acts after the establishment of RNAi-directed heterochromatin modifications, but prior to the excision of the modified sequences. In ∆LIA5 cells, DNA elimination foci, large nuclear sub-structures containing the sequences to be eliminated and the essential chromodomain protein Pdd1p, do not form. Lia5p, unlike Pdd1p, is not stably associated with these structures, but is required for their formation. In the absence of Lia5p, we could recover foci formation by ectopically inducing DNA damage by UV treatment. Foci in both wild-type or UV-treated ∆LIA5 cells contain dephosphorylated Pdd1p. These studies of LIA5 reveal that DNA elimination foci form after the excision of germ-line limited sequences occurs and indicate that Pdd1p reorganization is likely mediated through a DNA damage response.

Genome biology: the sleek and oh-so chic Oxytricha nanochromosomes.

The somatic nucleus of Oxytricha trifallax contains over 15,000 different chromosomes, most containing a single gene. Analysis of this 50 Mb genome uncovers novel regulatory strategies and adaptive potential when gene copy number and allelic frequency are no longer constrained by genetic linkage.

Transformation and strain engineering of Tetrahymena.

Transformation of Tetrahymena by microinjection of DNA was established 25 years ago. This rather labor-intensive technique has since been shelved, replaced by less time consuming and more efficient methods, electroporation and biolistics. Conjugative electroporation is the method of choice for introducing autonomously replicating, rDNA-based vectors into Tetrahymena. These are maintained as high-copy linear mini-chromosomes. Versatile expression cassettes in these vectors facilitate expression of most genes. Transformation efficiencies are sufficiently high to permit screens using expression libraries. Biolistic transformation is primarily used to introduce DNA for integration into the genome by homologous recombination. This technique has greatly enhanced strain engineering of Tetrahymena through facilitating the disruption of genes (creating targeted knockout cell lines) or epitope-tagging coding regions, allowing researchers to take full advantage of the sequenced genome. The presence of both germline and somatic nuclei in these cells requires different strategies to target DNA to the desired compartment. This presents challenges, including the need to engineer the polygenic macronuclear genome, which has nearly 50 copies of each gene. However, separate manipulation of functionally distinct genomes provides experimental opportunities, especially for the analysis of essential genes, by modifying the silent micronucleus then subsequently examining phenotypes in the next sexual generation. The flexibility to engineer strains as needed makes Tetrahymena a facile system with which to answer many biological questions.

Zygotic expression of the double-stranded RNA binding motif protein Drb2p is required for DNA elimination in the ciliate Tetrahymena thermophila.

Double-stranded RNA binding motif (DSRM)-containing proteins play many roles in the regulation of gene transcription and translation, including some with tandem DSRMs that act in small RNA biogenesis. We report the characterization of the genes for double-stranded RNA binding proteins 1 and 2 (DRB1 and DRB2), two genes encoding nuclear proteins with tandem DSRMs in the ciliate Tetrahymena thermophila. Both proteins are expressed throughout growth and development but exhibit distinct peaks of expression, suggesting different biological roles. In support of this, we show that expression of DRB2 is essential for vegetative growth while DRB1 expression is not. During conjugation, Drb1p and Drb2p localize to distinct nuclear foci. Cells lacking all DRB1 copies are able to produce viable progeny, although at a reduced rate relative to wild-type cells. In contrast, cells lacking germ line DRB2 copies, which thus cannot express Drb2p zygotically, fail to produce progeny, arresting late into conjugation. This arrest phenotype is accompanied by a failure to organize the essential DNA rearrangement protein Pdd1p into DNA elimination bodies and execute DNA elimination and chromosome breakage. These results implicate zygotically expressed Drb2p in the maturation of these nuclear structures, which are necessary for reorganization of the somatic genome.

DNA elimination in ciliates: transposon domestication and genome surveillance.

Ciliated protozoa extensively remodel their somatic genomes during nuclear development, fragmenting their chromosomes and removing large numbers of internal eliminated sequences (IESs). The sequences eliminated are unique and repetitive DNAs, including transposons. Recent studies have identified transposase proteins that appear to have been domesticated and are used by these cells to eliminate DNA not wanted in the somatic macronucleus. This DNA elimination process is guided by meiotically produced small RNAs, generated in the germline nucleus, that recognize homologous sequences leading to their removal. These scan RNAs are found in complexes with PIWI proteins. Before they search the developing genome for IESs to eliminate, they scan the parental somatic nucleus and are removed from the pool if they match homologous sequences in that previously reorganized genome. In Tetrahymena, the scan RNAs target heterochromatin modifications to mark IESs for elimination. This DNA elimination pathway in ciliates shares extensive similarity with piRNA-mediated silencing of metazoans and highlights the remarkable ability of homologous RNAs to shape developing genomes.