Faculty of Science

Abstract The Oxytricha trifallax mitochondrial genome contains the largest sequenced ciliate mitochondrial chromosome (��� 70 kb) plus a��� 5-kb linear plasmid bearing mitochondrial telomeres. We identify two new ciliate split genes (rps3 and nad2) as well as four new mitochondrial genes (ribosomal small subunit protein genes: rps-2, 7, 8, 10), previously undetected in ciliates due to their extreme divergence.

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 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.

RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa. Maternal RNA templates provide both an organizing guide for DNA rearrangements and a template that can transport somatic mutations to the next generation. This opportunity for RNAmediated genome rearrangement and DNA repair is profound in the ciliate Oxytricha, which deletes 95% of its germline genome during development in a process that severely fragments its chromosomes and then sorts and reorders the hundreds of thousands of pieces remaining. Oxytricha's somatic nuclear genome is therefore an epigenome formed through RNA templates and signals arising from the previous generation. Furthermore, this mechanism of RNA-mediated epigenetic inheritance can function across multiple generations, and the discovery of maternal template RNA molecules has revealed new biological roles for RNA and has hinted at the power of RNA molecules to sculpt genomic information in cells.

RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa. Maternal RNA templates provide both an organizing guide for DNA rearrangements and a template that can transport somatic mutations to the next generation. This opportunity for RNAmediated genome rearrangement and DNA repair is profound in the ciliate Oxytricha, which deletes 95% of its germline genome during development in a process that severely fragments its chromosomes and then sorts and reorders the hundreds of thousands of pieces remaining. Oxytricha's somatic nuclear genome is therefore an epigenome formed through RNA templates and signals arising from the previous generation. Furthermore, this mechanism of RNA-mediated epigenetic inheritance can function across multiple generations, and the discovery of maternal template RNA molecules has revealed new biological roles for RNA and has hinted at the power of RNA molecules to sculpt genomic information in cells.

Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs 1 . Splicing is guided locally by short conserved sequences, but genes typically contain many potential splice sites, and the mechanisms specifying the correct sites remain poorly understood. In most organisms, short introns recognized by the intron definition mechanism 2 cannot be efficiently predicted solely on the basis of sequence motifs 3 . In multicellular eukaryotes, long introns are recognized through exon definition 2 and most genes produce multiple mRNA variants through alternative splicing 4 . The nonsense-mediated mRNA decay 5,6 (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals 7,8 . Here we show that the tiny introns of the ciliate Paramecium tetraurelia are under strong selective pressure to cause premature termination of mRNA translation in the event of intron retention, and that the same bias is observed among the short introns of plants, fungi and animals. By knocking down the two P. tetraurelia genes encoding UPF1, a protein that is crucial in NMD, we show that the intrinsic efficiency of splicing varies widely among introns and that NMD activity can significantly reduce the fraction of unspliced mRNAs. The results suggest that, independently of alternative splicing, species with large intron numbers universally rely on NMD to compensate for suboptimal splicing efficiency and accuracy.

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.

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

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.

2009 marks not only the 200 th anniversary of Darwin's birth but also publication of the first scientific evolutionary theory, Lamarck's Philosophie Zoologique. While Lamarck embraced the notion of the inheritance of acquired characters, he did not invent it [1]. New phenomena discovered recently offer molecular pathways for the transmission of several acquired characters. Ciliates have long provided model systems to study phenomena that bypass traditional modes of inheritance. RNA, normally thought of as a conduit in gene expression, displays a novel mode of action in ciliated protozoa. For example, maternal RNA templates provide both an organizing guide for DNA rearrangements in Oxytricha and a template that can transmit spontaneous mutations that may arise during somatic growth to the next generation, providing two such mechanisms of so-called Lamarckian inheritance. This suggests that the somatic ciliate genome is really an "epigenome", formed through templates and signals arising from the previous generation. This review will discuss these new biological roles for RNA, including noncoding "template" RNA molecules. The evolutionary consequences of viable mechanisms in ciliates to transmit acquired characters may create an additional store of heritable variation that contributes to the cosmopolitan success of this diverse lineage of microbial eukaryotes.

The germline genome of ciliates is extensively rearranged during development of a new somatic macronucleus from the germline micronucleus, a process that follows sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) and multicopy transposons are eliminated, whereas cellular genes are amplified to w800 n. For a subset of IESs, introduction of the IES sequence into the maternal (prezygotic) macronucleus specifically inhibits excision of the homologous IES in the developing zygotic macronucleus. This and other homology-dependent maternal effects have suggested that rearrangement patterns are epigenetically determined by an RNAmediated, trans-nuclear comparison, involving the RNA interference pathway, of germline and somatic genomes. Results: We report the identification of novel developmentally regulated RNA binding proteins, Nowa1p and Nowa2p, which are required for the survival of sexual progeny. Green fluorescent protein (GFP) fusions show that Nowa1p accumulates into the maternal macronucleus shortly before meiosis of germline micronuclei and is later transported to developing macronuclei. No-wa1p/2p depletion impairs the elimination of transposons and of those IESs that are controlled by maternal effects, confirming the existence of distinct IES classes.

The germline genome of ciliates is extensively rearranged during development of a new somatic macronucleus from the germline micronucleus, a process that follows sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) and multicopy transposons are eliminated, whereas cellular genes are amplified to w800 n. For a subset of IESs, introduction of the IES sequence into the maternal (prezygotic) macronucleus specifically inhibits excision of the homologous IES in the developing zygotic macronucleus. This and other homology-dependent maternal effects have suggested that rearrangement patterns are epigenetically determined by an RNAmediated, trans-nuclear comparison, involving the RNA interference pathway, of germline and somatic genomes. Results: We report the identification of novel developmentally regulated RNA binding proteins, Nowa1p and Nowa2p, which are required for the survival of sexual progeny. Green fluorescent protein (GFP) fusions show that Nowa1p accumulates into the maternal macronucleus shortly before meiosis of germline micronuclei and is later transported to developing macronuclei. No-wa1p/2p depletion impairs the elimination of transposons and of those IESs that are controlled by maternal effects, confirming the existence of distinct IES classes.

intimately involved in developmental DNA elimination processes [S7-S9] and can explain the non-Mendelian heredity of alternative The frequencies of dinucleotides for the megabase chromosome sequence were calculated taking into account the frequencies of rearrangement patterns [S10, S11]. At least in plants, DNA methylation on cytosine residues and each individual nucleotide and are given as the ratio of observed to expected frequencies, f obs/exp . We found both CpG and TpA dinu-histone H3 methylation on K9 are interdependent processes (each can be both cause and consequence of the other) and together cleotides to be underrepresented with respect to their expected frequencies (ratios of 0.4 and 0.84, respectively). In contrast, the ensure heterochromatin formation [S12, S13]. The emerging picture is that in organisms that do not methylate DNA, histone H3 methyla-reciprocal GpC and ApT frequencies did not deviate from expected values, nor did the frequencies for any of the other dinucleotides.

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.

Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: A small, germline micronucleus and a massive, vegetative macronucleus. The genome within Stylonychia's macronucleus has a very unusual architecture, comprised variably and highly amplified "nanochromosomes," each usually encoding a single gene with a minimal amount of surrounding noncoding DNA. As only a tiny fraction of the Stylonychia genes has been sequenced, and to promote research using this organism, we sequenced its macronuclear genome. We report the analysis of the 50.2-Mb draft S. lemnae macronuclear genome assembly, containing in excess of 16,000 complete nanochromosomes, assembled as less than 20,000 contigs. We found considerable conservation of fundamental genomic properties between S. lemnae and its close relative, Oxytricha trifallax, including nanochromosomal gene synteny, alternative fragmentation, and copy number. Protein domain searches in Stylonychia revealed two new telomere-binding protein homologs and the presence of linker histones. Among the diverse histone variants of S. lemnae and O. trifallax, we found divergent, coexpressed variants corresponding to four of the five core nucleosomal proteins (H1.2, H2A.6, H2B.4, and H3.7) suggesting that these ciliates may possess specialized nucleosomes involved in genome processing during nuclear differentiation. The assembly of the S. lemnae macronuclear genome demonstrates that largely complete, well-assembled highly fragmented genomes of similar size and complexity may be produced from one library and lane of Illumina HiSeq 2000 shotgun sequencing. The provision of the S. lemnae macronuclear genome sets the stage for future detailed experimental studies of chromatin-mediated, RNA-guided developmental genome rearrangements.

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The Oxytricha trifallax mitochondrial genome contains the largest sequenced ciliate mitochondrial chromosome (~70 kb) plus a ~5 kb linear plasmid bearing, mitochondrial telomeres. We identify two new ciliate split genes (rps3 and nad2) as well as four new mitochondrial genes (ribosomal small subunit protein genes: rps-2, 7, 8, 10), previously undetected in ciliates due to their extreme divergence. The increased size of the Oxytricha mitochondrial genome relative to other ciliates is primarily a consequence of terminal expansions, rather than the retention of ancestral mitochondrial genes. Successive segmental duplications, visible in one of the two Oxytricha mitochondrial subterminal regions, appear to have contributed to the genome expansion. Consistent with pseudogene formation and decay, the subtermini possess shorter, more loosely packed ORFs than the remainder of the genome. The mitochondrial plasmid shares a 251 bp region with 82% identity to the mitochondrial chromosome, suggesting that it most likely integrated into the chromosome at least once. This region on the chromosome is also close to the end of the most terminal member of a series of duplications, hinting at a possible association between the plasmid and the duplications. The presence of mitochondrial telomeres on the mitochondrial plasmid suggests that such plasmids may be a vehicle for Downloaded from 2 lateral transfer of telomeric sequences between mitochondrial genomes. We conjecture that the extreme divergence observed in ciliate mitochondrial genomes may be due, in part, to repeated invasions by relatively error-prone DNA polymerase-bearing mobile elements.

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, whereas 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 as powerful transgenerational carriers of epigenetic information for genome programming.

RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa. Maternal RNA templates provide both an organizing guide for DNA rearrangements and a template that can transport somatic mutations to the next generation. This opportunity for RNAmediated genome rearrangement and DNA repair is profound in the ciliate Oxytricha, which deletes 95% of its germline genome during development in a process that severely fragments its chromosomes and then sorts and reorders the hundreds of thousands of pieces remaining. Oxytricha's somatic nuclear genome is therefore an epigenome formed through RNA templates and signals arising from the previous generation. Furthermore, this mechanism of RNA-mediated epigenetic inheritance can function across multiple generations, and the discovery of maternal template RNA molecules has revealed new biological roles for RNA and has hinted at the power of RNA molecules to sculpt genomic information in cells.