Linda Sperling

Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia

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.

Translational control of intron splicing in eukaryotes.

Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs. 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 cannot be efficiently predicted solely on the basis of sequence motifs. In multicellular eukaryotes, long introns are recognized through exon definition and most genes produce multiple mRNA variants through alternative splicing. The nonsense-mediated mRNA decay (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals. 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.

BioMart Central Portal: an open database network for the biological community

BioMart Central Portal is a first of its kind, community-driven effort to provide unified access to dozens of biological databases spanning genomics, proteomics, model organisms, cancer data, ontology information and more. Anybody can contribute an independently maintained resource to the Central Portal, allowing it to be exposed to and shared with the research community, and linking it with the other resources in the portal. Users can take advantage of the common interface to quickly utilize different sources without learning a new system for each. The system also simplifies cross-database searches that might otherwise require several complicated steps. Several integrated tools streamline common tasks, such as converting between ID formats and retrieving sequences. The combination of a wide variety of databases, an easy-to-use interface, robust programmatic access and the array of tools make Central Portal a one-stop shop for biological data querying. Here, we describe the structure of Central Portal and show example queries to demonstrate its capabilities. Database URL: http://central.biomart.org.

RNA interference by feeding in Paramecium

RNA interference can be induced very efficiently by feeding the ciliate Paramecium with bacteria engineered to express double-stranded RNA, opening the possibility of large-scale functional screening in this unicell.

Elucidation of Clathrin-Mediated Endocytosis in Tetrahymena Reveals an Evolutionarily Convergent Recruitment of Dynamin

Ciliates, although single-celled organisms, contain numerous subcellular structures and pathways usually associated with metazoans. How this cell biological complexity relates to the evolution of molecular elements is unclear, because features in these cells have been defined mainly at the morphological level. Among these ciliate features are structures resembling clathrin-coated, endocytic pits associated with plasma membrane invaginations called parasomal sacs. The combination of genome-wide sequencing in Tetrahymena thermophila with tools for gene expression and replacement has allowed us to examine this pathway in detail. Here we demonstrate that parasomal sacs are sites of clathrin-dependent endocytosis and that AP-2 localizes to these sites. Unexpectedly, endocytosis in Tetrahymena also involves a protein in the dynamin family, Drp1p (Dynamin-related protein 1). While phylogenetic analysis of AP subunits indicates a primitive origin for clathrin-mediated endocytosis, similar analysis of dynamin-related proteins suggests, strikingly, that the recruitment of dynamin-family proteins to the endocytic pathway occurred independently during the course of the ciliate and metazoan radiations. Consistent with this, our functional analysis suggests that the precise roles of dynamins in endocytosis, as well as the mechanisms of targeting, differ in metazoans and ciliates.

The Paramecium Germline Genome Provides a Niche for Intragenic Parasitic DNA: Evolutionary Dynamics of Internal Eliminated Sequences

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.

ParameciumDB in 2011: new tools and new data for functional and comparative genomics of the model ciliate Paramecium tetraurelia

ParameciumDB is a community model organism database built with the GMOD toolkit to integrate the genome and biology of the ciliate Paramecium tetraurelia. Over the last four years, post-genomic data from proteome and transcriptome studies has been incorporated along with predicted orthologs in 33 species, annotations from the community and publications from the scientific literature. Available tools include BioMart for complex queries, GBrowse2 for genome browsing, the Apollo genome editor for expert curation of gene models, a Blast server, a motif finder, and a wiki for protocols, nomenclature guidelines and other documentation. In-house tools have been developed for ontology browsing and evaluation of off-target RNAi matches. Now ready for next-generation deep sequencing data and the genomes of other Paramecium species, this open-access resource is available at http://paramecium.cgm.cnrs-gif.fr.

Cortical morphogenesis in Paramecium: A transcellular wave of protein phosphorylation involved in ciliary rootlet disassembly☆

In Paramecium, the morphogenesis of the cortex at cell division, which assures reconstruction of shape and surface pattern, has been shown to involve transcellular signals which spread across the cortex like a wave, originating principally from the oral apparatus. One of the events these signals control is the reorganization of the ciliary rootlets through a cycle of regression and regrowth. The ciliary rootlets are nucleated on the ciliary basal bodies and form a scaffold extending over the entire cell surface that is important in aligning the basal bodies and the unit territories organized around them in longitudinal rows. We present evidence that the mechanism underlying their reorganization is cell-cycle-dependent phosphorylation of the structural proteins which compose the ciliary rootlets. We have isolated the rootlets and prepared a polyclonal antibody against them. In situ immunofluorescence of dividing cells with the anti rootlet antibody, and with the monoclonal antibody MPM-2 specific for phosphoproteins shows that a wave of phosphorylation of the ciliary rootlets spreads across the cell at division and just precedes their regression. Two-dimensional Western blot analysis of cytoskeleton and isolated rootlets along with alkaline phosphatase treatment demonstrates that the rootlets are composed of phosphoproteins, while experiments with interphase and dividing cells provide direct evidence that hyperphosphorylation of these proteins at division brings about disassembly of the structure.

Paramecium genome survey : a pilot project

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

Genome-defence small RNAs exapted for epigenetic mating-type inheritance

In the ciliate Paramecium, transposable elements and their single-copy remnants are deleted during the development of somatic macronuclei from germline micronuclei, at each sexual generation. Deletions are targeted by scnRNAs, small RNAs produced from the germ line during meiosis that first scan the maternal macronuclear genome to identify missing sequences, and then allow the zygotic macronucleus to reproduce the same deletions. Here we show that this process accounts for the maternal inheritance of mating types in Paramecium tetraurelia, a long-standing problem in epigenetics. Mating type E depends on expression of the transmembrane protein mtA, and the default type O is determined during development by scnRNA-dependent excision of the mtA promoter. In the sibling species Paramecium septaurelia, mating type O is determined by coding-sequence deletions in a different gene, mtB, which is specifically required for mtA expression. These independently evolved mechanisms suggest frequent exaptation of the scnRNA pathway to regulate cellular genes and mediate transgenerational epigenetic inheritance of essential phenotypic polymorphisms.