The duplication of entire genomes has long been recognized as having great potential for evolutionary novelties, but the mechanisms underlying their resolution through gene loss are poorly understood. Here we show that in the unicellular eukaryote Paramecium tetraurelia, a ciliate, most of the nearly 40,000 genes arose through at least three successive whole-genome duplications. Phylogenetic analysis indicates that the most recent duplication coincides with an explosion of speciation events that gave rise to the P. aurelia complex of 15 sibling species. We observed that gene loss occurs over a long timescale, not as an initial massive event. Genes from the same metabolic pathway or protein complex have common patterns of gene loss, and highly expressed genes are over-retained after all duplications. The conclusion of this analysis is that many genes are maintained after whole-genome duplication not because of functional innovation but because of gene dosage constraints.Ciliates are unique among unicellular organisms in that they separate germline and somatic functions 1 . Each cell harbours two kinds of nucleus, namely silent diploid micronuclei and highly polyploid macronuclei. The latter are unusual in that they contain an extensively rearranged genome streamlined for expression and divide by a non-mitotic process. Only micronuclei undergo meiosis to perpetuate genetic information; the macronuclei are lost at each sexual generation and develop anew from the micronuclear lineage.In Paramecium the exact number of micronuclear chromosomes (more than 50) and the structures of their centromeres and telomeres remain unknown. During macronuclear development, these chromosomes are amplified to about 800 copies and undergo two types of DNA elimination event. Tens of thousand of short, unique copy elements (internal eliminated sequences) are removed by a precise mechanism that leads to the reconstitution of functional genes 2 .Transposable elements and other repeated sequences are removed by an imprecise mechanism leading either to chromosome fragmentation and de novo telomere addition or to variable internal deletions 3 . These rearrangements occur after a few rounds of endoreplication, leading to some heterogeneity in the sequences abutting the imprecisely eliminated regions 3 . The sizes of the resulting, acentric macronuclear chromosomes range from 50-1,000 kilobases (kb) as measured by pulsed-field gel electrophoresis. Because the sexual process of autogamy results in an entirely homozygous genotype 4 , the macronuclear DNA that was sequenced was genetically homogeneous.The Paramecium genome sequence The Paramecium macronuclear genome sequence was established with the use of a whole-genome shotgun and assembly strategy. Paired-end sequencing of plasmid and bacterial artificial chromosome (BAC) clones provided a coverage of 13 genome equivalents (Supplementary Table S1). We assembled the sequence reads with Arachne 5 in 1,907 contigs connected in 697 scaffolds of size greater than 2 kb, giving a total coverage of 72...

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of ∼45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a ∼10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated.

With more chromosomes than any other sequenced genome, the macronuclear genome of Oxytricha trifallax has a unique and complex architecture, including alternative fragmentation and predominantly single-gene chromosomes.

Genome-wide DNA rearrangements occur in many eukaryotes but are most exaggerated in ciliates, making them ideal model systems for epigenetic phenomena. During development of the somatic macronucleus, Oxytricha trifallax destroys 95% of its germ line, severely fragmenting its chromosomes, and then unscrambles hundreds of thousands of remaining fragments by permutation or inversion. Here we demonstrate that DNA or RNA templates can orchestrate these genome rearrangements in Oxytricha, supporting an epigenetic model for sequence-dependent comparison between germline and somatic genomes. A complete RNA cache of the maternal somatic genome may be available at a specific stage during development to provide a template for correct and precise DNA rearrangement. We show the existence of maternal RNA templates that could guide DNA assembly, and that disruption of specific RNA molecules disables rearrangement of the corresponding gene. Injection of artificial templates reprogrammes the DNA rearrangement pathway, suggesting that RNA molecules guide genome rearrangement.Parental RNA transcripts and microRNAs are critical for programming development in metazoa 1-4 , raising the possibility that altered RNA molecules can reprogramme patterning on a developmental or evolutionary timescale 5 . Despite the suggestion of template-directed events involving "an ancestral RNA-sequence cache" 6 there has been limited evidence for a direct role of RNA as a template of information across generations 7,8 . Information transfer from RNA to DNA usually involves polymerization 9 . Here we show that RNA molecules can also organize DNA rearrangements, expanding the epigenetic influence of RNA beyond gene expression and priming or directing DNA and RNA synthesis, editing, modification or repair 9-11 .O. trifallax is a unicellular eukaryote harbouring two kinds of nuclei: germline micronuclei and somatic macronuclei. Diploid micronuclei are transcriptionally inert during vegetative growth but they transmit the germline genome through subsequent generations. Effectively polyploid macronuclei provide all vegetative gene expression, but degrade after fertilization, when new micronuclei and macronuclei develop. DNA differentiation in ciliates such as Oxytricha (also called Sterkiella) involves massive chromosome fragmentation and deletion of transposons and internally eliminated sequences (IESs), accomplishing 95% genome Author Information TEBPα and TEBPβ macronucleus and micronucleus sequences have been submitted to GenBank under accession numbers EU047938-EU047941. Reprints and permissions information is available at www.nature.com/reprints. Correspondence and requests for materials should be addressed to L. RNAi against putative templates disrupts rearrangementTo test the hypothesis that putative maternal RNA templates influence rearrangement, we induced RNA interference (RNAi) to target homologous RNA degradation. Oxytricha cells, before and during conjugation, were fed Escherichia coli producing double stranded RNA fragments of two m...

In eukaryotes, small RNAs (sRNAs) have key roles in development, gene expression regulation, and genome integrity maintenance. In ciliates, such as Paramecium, sRNAs form the heart of an epigenetic system that has evolved from core eukaryotic gene silencing components to selectively target DNA for deletion. In Paramecium, somatic genome development from the germline genome accurately eliminates the bulk of typically gene-interrupting, noncoding DNA. We have discovered an sRNA class (internal eliminated sequence [IES] sRNAs [iesRNAs]), arising later during Paramecium development, which originates from and precisely delineates germline DNA (IESs) and complements the initial sRNAs ("scan" RNAs [scnRNAs]) in targeting DNA for elimination. We show that whole-genome duplications have facilitated successive differentiations of Paramecium Dicer-like proteins, leading to cooperation between Dcl2 and Dcl3 to produce scnRNAs and to the production of iesRNAs by Dcl5. These innovations highlight the ability of sRNA systems to acquire capabilities, including those in genome development and integrity.

Summary Genome duality in ciliated protozoa offers a unique system to showcase their epigenome as a model of inheritance. In Oxytricha, the somatic genome is responsible for vegetative growth, while the germline contributes DNA to the next sexual generation. Somatic nuclear development removes all transposons and other so-called “junk DNA”, which comprise ~95% of the germline. We demonstrate that Piwi-interacting small RNAs (piRNAs) from the maternal nucleus can specify genomic regions for retention in this process. Oxytricha piRNAs map primarily to the somatic genome, representing the ~5% of the germline that is retained. Furthermore, injection of synthetic piRNAs corresponding to normally-deleted regions leads to their retention in later generations. Our findings highlight small RNAs (sRNAs) as powerful transgenerational carriers of epigenetic information for genome programming.

Distinct small RNA pathways are involved in the two types of homology-dependent effects described in Paramecium tetraurelia, as shown by a functional analysis of Dicer and Dicer-like genes and by the sequencing of small RNAs. The siRNAs that mediate post-transcriptional gene silencing when cells are fed with double-stranded RNA (dsRNA) were found to comprise two subclasses. DCR1-dependent cleavage of the inducing dsRNA generates ∼23-nt primary siRNAs from both strands, while a different subclass of ∼24-nt RNAs, characterized by a short untemplated poly-A tail, is strictly antisense to the targeted mRNA, suggestive of secondary siRNAs that depend on an RNA-dependent RNA polymerase. An entirely distinct pathway is responsible for homology-dependent regulation of developmental genome rearrangements after sexual reproduction. During early meiosis, the DCL2 and DCL3 genes are required for the production of a highly complex population of ∼25-nt scnRNAs from all types of germline sequences, including both strands of exons, introns, intergenic regions, transposons and Internal Eliminated Sequences. A prominent 5′-UNG signature, and a minor fraction showing the complementary signature at positions 21–23, indicate that scnRNAs are cleaved from dsRNA precursors as duplexes with 2-nt 3′ overhangs at both ends, followed by preferential stabilization of the 5′-UNG strand.

SummaryThe prevailing view of the nuclear genetic code is that it is largely frozen and unambiguous. Flexibility in the nuclear genetic code has been demonstrated in ciliates that reassign standard stop codons to amino acids, resulting in seven variant genetic codes, including three previously undescribed ones reported here. Surprisingly, in two of these species, we find efficient translation of all 64 codons as standard amino acids and recognition of either one or all three stop codons. How, therefore, does the translation machinery interpret a “stop” codon? We provide evidence, based on ribosomal profiling and “stop” codon depletion shortly before coding sequence ends, that mRNA 3′ ends may contribute to distinguishing stop from sense in a context-dependent manner. We further propose that such context-dependent termination/readthrough suppression near transcript ends enables genetic code evolution.