Structure of Tetrahymena telomerase-bound CST with polymerase α-primase.

Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single-strand 3' overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN21,2. TPP1 and POT1 associate with the 3' overhang, with POT1 binding the G-strand3 and TPP1 (in complex with TIN24) recruiting telomerase via interaction with telomerase reverse transcriptase5 (TERT). The telomere DNA ends are replicated and maintained by telomerase6, for the G-strand, and subsequently DNA polymerase α-primase7,8 (PolαPrim), for the C-strand9. PolαPrim activity is stimulated by the heterotrimeric complex CTC1-STN1-TEN110-12 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex-telomerase-core ribonucleoprotein, p50, CST and PolαPrim-that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis.

A Hexameric Ribozyme Nanostructure Formed by Double-Decker Assembly of a Pair of Triangular Ribozyme Trimers.

The modular architecture of naturally occurring ribozymes makes them a promising class of structural platform for the design and assembly of three-dimensional (3D) RNA nanostructures, into which the catalytic ability of the platform ribozyme can be installed. We have constructed and analyzed RNA nanostructures with polygonal-shaped (closed) ribozyme oligomers by assembling unit RNAs derived from the Tetrahymena group I intron with a typical modular architecture. In this study, we dimerized ribozyme trimers with a triangular shape by introducing three pillar units. The resulting double-decker nanostructures containing six ribozyme units were characterized biochemically and their structures were observed by atomic force microscopy. The double-decker hexamers exhibited higher catalytic activity than the parent ribozyme trimers.

Hidden genomic evolution in a morphospecies-The landscape of rapidly evolving genes in Tetrahymena.

A morphospecies is defined as a taxonomic species based wholly on morphology, but often morphospecies consist of clusters of cryptic species that can be identified genetically or molecularly. The nature of the evolutionary novelty that accompanies speciation in a morphospecies is an intriguing question. Morphospecies are particularly common among ciliates, a group of unicellular eukaryotes that separates 2 kinds of nuclei-the silenced germline nucleus (micronucleus [MIC]) and the actively expressed somatic nucleus (macronucleus [MAC])-within a common cytoplasm. Because of their very similar morphologies, members of the Tetrahymena genus are considered a morphospecies. We explored the hidden genomic evolution within this genus by performing a comprehensive comparative analysis of the somatic genomes of 10 species and the germline genomes of 2 species of Tetrahymena. These species show high genetic divergence; phylogenomic analysis suggests that the genus originated about 300 million years ago (Mya). Seven universal protein domains are preferentially included among the species-specific (i.e., the youngest) Tetrahymena genes. In particular, leucine-rich repeat (LRR) genes make the largest contribution to the high level of genome divergence of the 10 species. LRR genes can be sorted into 3 different age groups. Parallel evolutionary trajectories have independently occurred among LRR genes in the different Tetrahymena species. Thousands of young LRR genes contain tandem arrays of exactly 90-bp exons. The introns separating these exons show a unique, extreme phase 2 bias, suggesting a clonal origin and successive expansions of 90-bp-exon LRR genes. Identifying LRR gene age groups allowed us to document a Tetrahymena intron length cycle. The youngest 90-bp exon LRR genes in T. thermophila are concentrated in pericentromeric and subtelomeric regions of the 5 micronuclear chromosomes, suggesting that these regions act as genome innovation centers. Copies of a Tetrahymena Long interspersed element (LINE)-like retrotransposon are very frequently found physically adjacent to 90-bp exon/intron repeat units of the youngest LRR genes. We propose that Tetrahymena species have used a massive exon-shuffling mechanism, involving unequal crossing over possibly in concert with retrotransposition, to create the unique 90-bp exon array LRR genes.

LF4/MOK and a CDK-related kinase regulate the number and length of cilia in Tetrahymena.

The length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. The multiciliated protist model, Tetrahymena, carries two types of cilia (oral and locomotory) and the length of the locomotory cilia is dependent on their position with the cell. In Tetrahymena, loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their number. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. We suggest that the distribution of LF4/MOK along the cilium delivers a uniform dose of inhibition to IFT trains that travel from the base to the tip. In a longer cilium, the IFT machinery may experience a higher cumulative dose of inhibition by LF4/MOK. Thus, LF4/MOK activity could be a readout of cilium length that helps to balance the rate of IFT-driven assembly with the rate of disassembly at steady state. We used a forward genetic screen to identify a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. Loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the cyclin-binding motif and acting upstream of RCKs. The new genetic tools we developed here for Tetrahymena have potential for further dissection of the principles of cilia length regulation in multiciliated cells.

How to Kinetically Dissect an RNA Machine.

RNA-based machines are ubiquitous in Nature and increasingly important for medicines. They fold into complex, dynamic structures that process information and catalyze reactions, including reactions that generate new RNAs and proteins across biology. What are the experimental strategies and steps that are necessary to understand how these complex machines work? Two 1990 papers from Herschlag and Cech on "Catalysis of RNA Cleavage by the Tetrahymena thermophila Ribozyme" provide a master class in dissecting an RNA machine through kinetics approaches. By showing how to propose a kinetic framework, fill in the numbers, do cross-checks, and make comparisons across mutants and different RNA systems, the papers illustrate how to take a mechanistic approach and distill the results into general insights that are difficult to attain through other means.

Ultra low doses and biological amplification: Approaching Avogadro's number.

This paper describes evidence establishing that ultra-low doses of diverse chemical agents at concentrations from 10-18 to 10-24 M (e.g., approaching and/or less than 1 atom or molecule of a substance/cell based on Avogadro's constant - 6.022×1023/mole) are capable of engaging receptor and intracellular signaling systems to elicit reproducible effects in a variety of species, from unicellular organisms to humans. Multiple experimental studies have shown that only one or very few molecules are needed to activate a cell and/or entire organism via cascade(s) of amplification mechanisms and processes. For example, ultra-low dose ligand exposure was able to activate both an individual cell, and ~3000 to 25,000 neighboring cells on average, by about 50%. Such activation of cells and whole organisms typically displayed hormetic-biphasic dose responses. These findings indicate that numerous, diverse phylogenetic systems have evolved highly sensitive detection and signaling mechanisms to enhance survival functions, such as defense against infectious agents, responses to diverse types of pheromone communications (e.g., alarm, sexual attraction), and development of several types of cellular protection/resilience processes. This suggests that ultra-low dose effects may be far more common than have been recognized to date. We posit that such findings have important implications for evolutionary theory, ecological and systems biology, and clinical medicine.

Pyridine Nucleotide-Linked Lactate Dehydrogenase of Tetrahymena: Evidence for D- and L-Enzymes in the Mitochondria.

An NAD-linked lactate dehydrogenase (LDH) in a crude mitochondrial fraction obtained from Tetrahymena homogenates was previously reported by this laboratory. This fraction contains the NADH and succinate oxidase system as well as the mitochondrial cytochromes and carries out oxidative phosphorylation. The preparation catalyzes the oxidation of D- and L-lactate linked only to certain analogs of NAD; it has not been possible to demonstrate NAD-dependent D- or L-lactate oxidation nor is there any evidence that either of these enzymes is a flavoprotein as indicated by their inability to reduce directly certain artificial electron acceptors. A lactate racemase is not present.

Development of an expression system in Tetrahymena inner arm dyneins and motile properties of the single-headed subspecies (Dyh8p and Dyh12p).

Diverse inner arm dyneins cooperate with outer arm dyneins to produce ciliary beating. This study demonstrates an expression system for inner arm dyneins in Tetrahymena. The motor domain of inner arm dynein (Dyh8p or Dyh12p) was fused with the tail of outer arm dynein (Dyh3p) and expressed in viable DYH3-knockout (vKO-DYH3) cells. The chimeric dyneins were observed in the oral apparatus and cilia on the cell bodies, and did not change the swimming speed of vKO-DYH3 cells. In a gliding assay, the motor domains of Dyh8p and Dyh12p moved toward the minus ends of microtubules at 0.8 and 0.3 µm/s, respectively. The gliding velocities of Dyh8p and Dyh12p were decreased in 5 mM ATP but not increased in 0.1 or 0.5 mM ADP. This expression system will be useful for molecular studies on diverse inner arm dyneins.

A Tetrahymena Piwi bound to mature tRNA 3' fragments activates the exonuclease Xrn2 for RNA processing in the nucleus.

Emerging evidence suggests that Argonaute (Ago)/Piwi proteins have diverse functions in the nucleus and cytoplasm, but the molecular mechanisms employed in the nucleus remain poorly defined. The Tetrahymena thermophila Ago/Piwi protein Twi12 is essential for growth and functions in the nucleus. Twi12-bound small RNAs (sRNAs) are 3' tRNA fragments that contain modified bases and thus are attenuated for base pairing to targets. We show that Twi12 assembles an unexpected complex with the nuclear exonuclease Xrn2. Twi12 functions to stabilize and localize Xrn2, as well as to stimulate its exonuclease activity. Twi12 function depends on sRNA binding, which is required for its nuclear import. Depletion of Twi12 or Xrn2 induces a cellular ribosomal RNA processing defect known to result from limiting Xrn2 activity in other organisms. Our findings suggest a role for an Ago/Piwi protein and 3' tRNA fragments in nuclear RNA metabolism.

Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena.

The ciliated protozoan Tetrahymena undergoes extensive programmed DNA elimination when the germline micronucleus produces the new macronucleus during sexual reproduction. DNA elimination is epigenetically controlled by DNA sequences of the parental macronuclear genome, and this epigenetic regulation is mediated by small RNAs (scan RNAs [scnRNAs]) of ∼28-30 nucleotides that are produced and function by an RNAi-related mechanism. Here, we examine scnRNA production and turnover by deep sequencing. scnRNAs are produced exclusively from the micronucleus and nonhomogeneously from a variety of chromosomal locations. scnRNAs are preferentially derived from the eliminated sequences, and this preference is mainly determined at the level of transcription. Despite this bias, a significant fraction of scnRNAs is also derived from the macronuclear-destined sequences, and these scnRNAs are degraded during the course of sexual reproduction. These results indicate that the pattern of DNA elimination in the new macronucleus is shaped by the biased transcription in the micronucleus and the selective degradation of scnRNAs in the parental macronucleus.

Intercepting noncoding messages between germline and soma.

There is growing evidence to support the notion that small RNAs derived from noncoding RNAs (ncRNAs) are mobile carriers of epigenetic information in diverse eukaryotic systems. However, challenges remain in defining what messages are being sent and how. In the August 1, 2012, issue of Genes & Development, Schoeberl and colleagues (pp. 1729-1742) reported a detailed analysis of the turnover of small RNAs during the sexual reproduction of the ciliated protozoan Tetrahymena. The results revealed surprisingly complicated roles played by small RNAs in shaping the communication between the germline and the soma.

A Tetrahymena Hsp90 co-chaperone promotes siRNA loading by ATP-dependent and ATP-independent mechanisms.

The loading of small interfering RNAs (siRNAs) and microRNAs into Argonaute proteins is enhanced by Hsp90 and ATP in diverse eukaryotes. However, whether this loading also occurs independently of Hsp90 and ATP remains unclear. We show that the Tetrahymena Hsp90 co-chaperone Coi12p promotes siRNA loading into the Argonaute protein Twi1p in both ATP-dependent and ATP-independent manners in vitro. The ATP-dependent activity requires Hsp90 and the tetratricopeptide repeat (TPR) domain of Coi12p, whereas these factors are dispensable for the ATP-independent activity. Both activities facilitate siRNA loading by counteracting the Twi1p-binding protein Giw1p, which is important to specifically sort the 26- to 32-nt siRNAs to Twi1p. Although Coi12p lacking its TPR domain does not bind to Hsp90, it can partially restore the siRNA loading and DNA elimination defects of COI12 knockout cells, suggesting that Hsp90- and ATP-independent loading of siRNA occurs in vivo and plays a physiological role in Tetrahymena.

Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci.

Transgenerational transmission of genome-regulatory epigenetic information can determine phenotypes in the progeny of sexual reproduction. Sequence specificity of transgenerational regulation derives from small RNAs assembled into Piwi-protein complexes. Known targets of transgenerational regulation are primarily transposons and transposon-derived sequences. Here, we extend the scope of Piwi-mediated transgenerational regulation to include unique noncoding RNA loci. Ciliates such as Tetrahymena have a phenotypically silent germline micronucleus and an expressed somatic macronucleus, which is differentiated anew from a germline genome copy in sexual reproduction. We show that the nuclear-localized Tetrahymena Piwi protein Twi8p shuttles from parental to zygotic macronuclei. Genetic elimination of Twi8p has no phenotype for cells in asexual growth. On the other hand, cells lacking Twi8p arrest in sexual reproduction with zygotic nuclei that retain the germline genome structure, without the DNA elimination and fragmentation required to generate a functional macronucleus. Twi8p-bound small RNAs originate from long-noncoding RNAs with a terminal hairpin, which become detectable in the absence of Twi8p. Curiously, the loci that generate Twi8p-bound small RNAs are essential for asexual cell growth, even though Twi8 RNPs are essential only in sexual reproduction. Our findings suggest the model that Twi8 RNPs act on silent germline chromosomes to permit their conversion to expressed macronuclear chromosomes. Overall this work reveals that a Piwi protein carrying small RNAs from long-noncoding RNA loci has transgenerational function in establishing zygotic nucleus competence for gene expression.

Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating.

Cilia beating is powered by the inner and outer dynein arms (IDAs and ODAs). These multi-subunit macrocomplexes are arranged in two rows on each outer doublet along the entire cilium length, except its distal end. To generate cilia beating, the activity of ODAs and IDAs must be strictly regulated locally by interactions with the dynein arm-associated structures within each ciliary unit and coordinated globally in time and space between doublets and along the axoneme. Here, we provide evidence of a novel ciliary complex composed of two conserved WD-repeat proteins, Fap43p and Fap44p. This complex is adjacent to another WD-repeat protein, Fap57p, and most likely the two-headed inner dynein arm, IDA I1. Loss of either protein results in altered waveform, beat stroke and reduced swimming speed. The ciliary localization of Fap43p and Fap44p is interdependent in the ciliate Tetrahymena thermophila.

Effects of molecular crowding on a bimolecular group I ribozyme and its derivative that self-assembles to form ribozyme oligomers.

In the RNA world, enrichment of self-replicating RNAs would have been beneficial to their survival, amplification, and evolution. Self-assembly of RNAs may be a strategy by which they enrich themselves. We examined the effects of molecular crowding on the activity of a bimolecular group I ribozyme and its derivative that self-assembles to form ribozyme oligomers. In a comparative activity assay using PEG as a molecular crowder, PEG rescued mutations in the parent bimolecular ribozyme more effectively than those in the oligomeric form.

Biogenic triamine and tetraamine activate core catalytic ability of Tetrahymena group I ribozyme in the absence of its large activator module.

Group I intron ribozymes share common core elements that form a three-dimensional structure responsible for their catalytic activity. This core structure is unstable without assistance from additional factors that stabilize its tertiary structure. We examined biogenic triamine and tetraamine and also their fragments for their abilities to stabilize a structurally unstable group I ribozyme, ΔP5 ribozyme, derived from the Tetrahymena group I intron ribozyme by deleting its large activator module. Biogenic triamine (spermidine) and tetraamine (spermine) efficiently activated the ΔP5 ribozyme under conditions where the ribozyme was virtually inactive. These observations suggested that polyamines are promising small molecule modulators to activate and possibly inhibit the core catalytic ability of group I ribozymes.

Mutual detoxification of mercury and selenium in unicellular Tetrahymena.

Selenium (Se) is commonly recognized as a protective element with an antagonistic effect against mercury (Hg) toxicity. However, the mechanisms of this Hg-Se antagonism are complex and remain controversial. To gain insight into the Hg-Se antagonism, a type of unicellular eukaryotic protozoa (Tetrahymena malaccensis, T. malaccensis) was selected and individually or jointly exposed to two Hg and three Se species. We found that Se species showed different toxic effects on the proliferation of T. malaccensis with the toxicity following the order: selenite (Se(IV))>selenomethionine (SeMeth)>selenate (Se(VI)). The Hg-Se antagonism in Tetrahymena was observed because the joint toxicity significantly decreased under co-exposure to highly toxic dosages of Hg and Se versus individual toxicity. Unlike Se(IV) and Se(VI), non-toxic dosage of SeMeth significantly decreased the Hg toxicity, revealing the influence of the Se species and dosages on the Hg-Se antagonism. Unexpectedly, inorganic divalent Hg (Hg2+) and monomethylmercury (MeHg) also displayed detoxification towards extremely highly toxic dosages of Se, although their detoxifying efficiency was discrepant. These results suggested mutual Hg-Se detoxification in T. malaccensis, which was highly dependent on the dosages and species of both elements. As compared to other species, SeMeth and MeHg promoted the Hg-Se joint effects to a higher degree. Additionally, the Hg contents decreased for all the Hg-Se co-exposed groups, revealing a sequestering effect of Se towards Hg in T. malaccensis.

The DEAD-Box Protein CYT-19 Uses Arginine Residues in Its C-Tail To Tether RNA Substrates.

DEAD-box proteins are nonprocessive RNA helicases that play diverse roles in cellular processes. The Neurospora crassa DEAD-box protein CYT-19 promotes mitochondrial group I intron splicing and functions as a general RNA chaperone. CYT-19 includes a disordered, arginine-rich "C-tail" that binds RNA, positioning the helicase core to capture and unwind nearby RNA helices. Here we probed the C-tail further by varying the number and positions of arginines within it. We found that removing sets of as few as four of the 11 arginines reduced RNA unwinding activity (kcat/KM) to a degree equivalent to that seen upon removal of the C-tail, suggesting that a minimum or "threshold" number of arginines is required. In addition, a mutant with 16 arginines displayed RNA unwinding activity greater than that of wild-type CYT-19. The C-tail modifications impacted unwinding only of RNA helices within constructs that included an adjacent helix or structured RNA element that would allow C-tail binding, indicating that the helicase core remained active in the mutants. In addition, changes in RNA unwinding efficiency of the mutants were mirrored by changes in functional RNA affinity, as determined from the RNA concentration dependence of ATPase activity, suggesting that the C-tail functions primarily to increase RNA affinity. Interestingly, the salt concentration dependence of RNA unwinding activity is unaffected by C-tail composition, suggesting that the C-tail uses primarily hydrogen bonding, not electrostatic interactions, to bind double-stranded RNA. Our results provide insights into how an unstructured C-tail contributes to DEAD-box protein activity and suggest parallels with other families of RNA- and DNA-binding proteins.

Biased assembly of the nuclear pore complex is required for somatic and germline nuclear differentiation in Tetrahymena.

Ciliates have two functionally distinct nuclei, a somatic macronucleus (MAC) and a germline micronucleus (MIC) that develop from daughter nuclei of the last postzygotic division (PZD) during the sexual process of conjugation. Understanding this nuclear dimorphism is a central issue in ciliate biology. We show, by live-cell imaging of Tetrahymena, that biased assembly of the nuclear pore complex (NPC) occurs immediately after the last PZD, which generates anterior-posterior polarized nuclei: MAC-specific NPCs assemble in anterior presumptive MACs but not in posterior presumptive MICs. MAC-specific NPC assembly in the anterior nuclei occurs much earlier than transport of Twi1p, which is required for MAC genome rearrangement. Correlative light-electron microscopy shows that addition of new nuclear envelope (NE) precursors occurs through the formation of domains of redundant NE, where the outer double membrane contains the newly assembled NPCs. Nocodazole inhibition of the second PZD results in assembly of MAC-specific NPCs in the division-failed zygotic nuclei, leading to failure of MIC differentiation. Our findings demonstrate that NPC type switching has a crucial role in the establishment of nuclear differentiation in ciliates.