Identification of novel, functional long non-coding RNAs involved in programmed, large scale genome rearrangements.

Non-coding RNAs (ncRNAs) make up to ~98% percent of the transcriptome of a given organism. In recent years one relatively new class of ncRNAs, long non-coding RNAs (lncRNAs), were shown to be more than mere by-products of gene expression and regulation. The unicellular eukaryote Paramecium tetraurelia is a member of the ciliate phylum, an extremely heterogeneous group of organisms found in most bodies of water across the globe. A hallmark of ciliate genetics is nuclear dimorphism and programmed elimination of transposons and transposon-derived DNA elements, the latter of which is essential for the maintenance of the somatic genome. Paramecium and ciliates in general harbour a plethora of different ncRNA species, some of which drive the process of large scale genome rearrangements, including DNA elimination, during sexual development. Here, we identify and validate the first known functional lncRNAs in ciliates to date. Using deep-sequencing and subsequent bioinformatic processing and experimental validation, we show that Paramecium expresses at least 15 lncRNAs. These candidates were predicted by a highly conservative pipeline and informatic analyses hint at differential expression during development. Depletion of two lncRNAs, lnc1 and lnc15, resulted in clear phenotypes, decreased survival, morphological impairment and a global effect on DNA elimination

Twisted Tales: Insights into Genome Diversity of Ciliates Using Single-Cell ‘Omics

The emergence of robust single-cell ‘omics techniques enables studies of uncultivable species, allowing for the (re)discovery of diverse genomic features. In this study, we combine single-cell genomics and transcriptomics to explore genome evolution in ciliates (a \u3e 1 Gy old clade). Analysis of the data resulting from these single-cell ‘omics approaches show: 1) the description of the ciliates in the class Karyorelictea as “primitive”is inaccurate because their somatic macronuclei contain loci of varying copy number (i.e., they have been processed by genome rearrangements from the zygotic nucleus); 2) gene-sized somatic chromosomes exist in the class Litostomatea, consistent with Balbiani’s (1890) observation of giant chromosomes in this lineage; and 3) gene scrambling exists in the underexplored Postciliodesmatophora (the classes Heterotrichea and Karyorelictea, abbreviated here as the Po-clade), one of two major clades of ciliates. Together these data highlight the complex evolutionary patterns underlying germline genome architectures in ciliates and provide a basis for further exploration of principles of genome evolution in diverse microbial lineages

Nuclear Architecture and Patterns of Molecular Evolution Are Correlated in the Ciliate \u3cem\u3eChilodonella uncinata\u3c/em\u3e

The relationship between nuclear architecture and patterns of molecular evolution in lineages across the eukaryotic tree of life is not well understood, partly because molecular evolution is traditionally explored as changes in base pairs along a linear sequence without considering the context of nuclear position of chromosomes. The ciliate Chilodonella uncinata is an ideal system to address the relationship between nuclear architecture and patterns of molecular evolution as the somatic macronucleus of this ciliate is composed of a peripheral DNA-rich area (orthomere) and a DNA-poor central region (paramere) to form a “heteromeric” macronucleus. Moreover, because the somatic chromosomes of C. uncinata are highly processed into “gene-sized” chromosomes (i.e., nanochromosomes), we can assess fine-scale relationships between location and sequence evolution. By combining fluorescence microscopy and analyses of transcriptome data from C. uncinata , we find that highly expressed genes have the greatest codon usage bias and are enriched in DNA-poor regions. In contrast, genes with less biased sequences tend to be concentrated in DNA abundant areas, at least during vegetative growth. Our analyses are consistent with recent work in plants and animals where nuclear architecture plays a role in gene expression. At the same time, the unusual localization of nanochromosomes suggests that the highly structured nucleus in C. uncinata may create a “gene bank” that facilitates rapid changes in expression of genes required only in specific life history stages. By using “nonmodel” organisms like C. uncinata , we can explore the universality of eukaryotic features while also providing examples of novel properties (i.e., the presence of a gene bank) that build from these features

Characterization of the Life Cycle and Heteromeric Nature of the Macronucleus of the Ciliate \u3ci\u3eChilodonella uncinata\u3c/i\u3e Using Fuorescence Microscopy

Only a limited number of studies exist on the life cycles of nonmodel ciliates such as Chilodonella uncinata (Cl: Phyllopharyngea). The handful of papers on this taxon indicate the presence of a heteromeric macronucleus, marked by separate DNA-rich and DNA-poor regions. Here, we study the life cycle of C. uncinata using confocal laser scanning microscopy with 4′,6-diamidino-2- phenylindole staining, which allows us to differentiate nuclear dynamics of the micronucleus and the macronucleus during life-cycle stages. We photo-documented various stages and confirmed aspects of the development of the new macronucleus previously characterized by electron microscopy. We further reveal the heteromeric structure of the macronucleus with Z-stacks and three-dimensional (3D) reconstructions. We find no evidence for the presence of an endosome at the center of the macronucleus during vegetative growth. In addition to illustrating the life cycle of this ciliate, the approaches developed for this study will enable additional comparative analyses of nuclear dynamics using fluorescence microscopy