Biology subjects

Swart, E. C.

Publications and source records attributed to Swart, E. C..

6 recordsLinked to original sources

bioRxiv2024

Two paralogous PHD finger proteins participate inParamecium tetraurelia's natural genome editing

The unicellular eukaryote Paramecium tetraurelia contains functionally distinct nuclei: germline micronuclei (MICs) and a somatic macronucleus (MAC). During sexual reproduction, the MIC genome is reorganized into a new MAC genome and the old MAC is lost. Almost 45,000 unique Internal Eliminated Sequences (IESs) distributed throughout the genome require precise excision to guarantee a functional new MAC genome. Here, we characterize a pair of paralogous PHD finger proteins involved in DNA elimination. DevPF1, the early-expressed paralog, is present in only some of the gametic and post-zygotic nuclei during meiosis. Both DevPF1 and DevPF2 localize in the new developing MACs, where IESs excision occurs. In DevPF2 knockdown (KD) long IESs are preferentially retained and late-expressed small RNAs decrease; no length preference for retained IESs was observed in DevPF1-KD and development-specific small RNAs were abolished. The expression of at least two genes from the new MAC with roles in genome reorganization seems to be influenced by DevPF1- and DevPF2-KD. Thus, both PHD fingers are crucial for new MAC genome development, with distinct functions, potentially via regulation of non-coding and coding transcription in the MICs and new MACs.

molecular biology↗

bioRxiv2023

How did UGA codon translation as tryptophan evolve in certain ciliates? A critique of Kachale et al. 2023 Nature

Ciliates are a widespread clade of microbial eukaryotes with the greatest diversity of nuclear genetic codes (at least eight) following a recent addition1. All non-standard ciliate genetic codes involve stop codon reassignments1,2,3. Two of these codes are ambiguous1-3, with "stop" codons either translated or terminating translation depending on their context2,3. Ambiguous genetic codes have arisen not only in ciliates, but also independently in trypanosomatids from the genus Blastocrithidia4 and an alveolate species from the genus Amoebophrya5. Two ambiguous genetic codes in ciliates share translation of UGA "stop" codons as tryptophan with Blastocrithidia and the Amoebophrya species. tRNA genes with complementary anticodons to reassigned UAA and UAG stop codons have invariably been found in ciliate species that translate these codons1,2. Furthermore, though a UGA-cognate tRNACysUCA was reported in Euplotes6, a ciliate genus that translates UGA as cysteine, vexingly, no nuclear genome-encoded tRNATrpUCA has been found in ciliate species with UGA tryptophan codons. Recently, Kachale et al. provided evidence for UGA translation as tryptophan in Blastocrithidia nonstop and the ciliate Condylostoma magnum using 4 base pair anticodon stem (AS) near-cognate tryptophan tRNATrpCCAs, rather than the typical 5 base pair stem tRNAs7. New tRNA data we report from additional ciliates bolsters this hypothesis. Kachale et al. also hypothesised that a particular amino acid substitution in the key stop codon recognition protein, eRF1 (eukaryotic Release Factor 1), favours translation of UGA as tryptophan instead of termination7. Contrary to Kachale et al, we propose such substitutions favouring reduced eRF1 competition enhancing "stop" codon translation do not need to occur concomitantly with tRNA alterations or acquisitions to evolve new genetic codes via stop codon reassignment. We report multiple instances of the substitution investigated in Kachale et al. 2023 that have not led to UGA translation, and multiple ciliate species with UGA tryptophan translation but without the substitution, indicating it is not necessary. Consistent with the ambiguous intermediate hypothesis for genetic code evolution, experimental evidence and our observations suggest continued potential ciliate eRF1-tRNA competition.

evolutionary biology↗

bioRxiv2023

ISWI1 complex proteins facilitate developmental genome editing in Paramecium

Chromatin remodeling is required for essential cellular processes, including DNA replication, DNA repair, and transcription regulation. The ciliate germline and soma are partitioned into two distinct nuclei within the same cell. During a massive editing process that forms a somatic genome, ciliates eliminate thousands of DNA sequences from a germline genome copy in the form of internal eliminated sequences (IESs). Recently we showed that the chromatin remodeler ISWI1 is required for somatic genome development in the ciliate Paramecium tetraurelia. Here we describe two paralogous proteins, ICOP1 and ICOP2, essential for DNA elimination. ICOP1 and ICOP2 are highly divergent from known proteins; the only domain detected showed distant homology to the WSD motif. We show that both ICOP1 and ICOP2 interact with the chromatin remodeler ISWI1. Upon ICOP knockdown, changes in alternative IES excision boundaries and nucleosome densities are similar to those observed for ISWI1 knockdown. We thus propose that a complex comprising ISWI1 and either or both ICOP1 and ICOP2 are needed for chromatin remodeling and accurate DNA elimination in Paramecium.

molecular biology↗

bioRxiv2022

RNA-mediated nucleosome depletion is required for elimination of transposon-derived DNA.

Small RNAs are known to mediate silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. Post-zygotic development of the Paramecium somatic genome requires elimination of thousands of transposon remnants (IESs) and transposable elements that are scattered throughout the germline genome (Garnier et al. 2004). The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries (Bouhouche et al. 2011; Furrer et al. 2017). Previous research suggests that small RNAs induce heterochromatin formation within IESs, which, in turn, is required for DNA elimination (Liu et al. 2007). Here we show that IES recognition and precise excision is facilitated by recruitment of a homolog of a chromatin remodeler ISWI, which depletes target genomic regions of nucleosomes, making the chromatin accessible for DNA cleavage. ISWI knockdown in Paramecium leads to pronounced inhibition of DNA elimination. Furthermore, nucleosome profiling indicates that ISWI is required for IES elimination in nucleosome-dense genomic regions, while other IESs do not require small RNAs or ISWI for excision. ISWI silencing notably also reduces DNA elimination precision, resulting in aberrant excision at alternative IES boundaries. In summary, we demonstrate that chromatin remodeling that increases DNA accessibility together with small RNAs are necessary for efficient and precise DNA elimination in Paramecium.

molecular biology↗

bioRxiv2021

The Blepharisma stoltei macronuclear genome: towards the origins of whole genome reorganization

Massive DNA excision occurs regularly in ciliates, ubiquitous microbial eukaryotes with somatic and germline nuclei in the same cell. Tens of thousands of internally eliminated sequences (IESs) scattered throughout a copy of the ciliate germline genome are deleted during development of the streamlined somatic genome. Blepharisma represents one of the two earliest diverging ciliate classes, and, unusually, has dual pathways of somatic nuclear development, making it ideal for investigating the functioning and evolution of these processes. Here, we report the somatic genome assembly of Blepharisma stoltei strain ATCC 30299 (41 Mb), arranged as numerous alternative telomere-capped minichromosomes. This genome encodes eight PiggyBac transposase homologs liberated from transposons. All are subject to purifying selection, but just one, the putative IES excisase, has a complete catalytic triad. We propose PiggyBac homologs were ancestral excisases that enabled evolution of extensive, natural genome editing.

genomics↗

bioRxiv2021

BleTIES: Annotation of natural genome editing in ciliates using long read sequencing

SummaryCiliates are single-celled eukaryotes that eliminate specific, interspersed DNA sequences (internally eliminated sequences, IESs) from their genomes during development. These are challenging to annotate and assemble because IES-containing sequences are much less abundant in the cell than those without, and IES sequences themselves often contain repetitive and low-complexity sequences. Long read sequencing technologies from Pacific Biosciences and Oxford Nanopore have the potential to reconstruct longer IESs than has been possible with short reads, and also the ability to detect correlations of neighboring element elimination. Here we present BleTIES, a software toolkit for detecting, assembling, and analyzing IESs using mapped long reads. Availability and implementationBleTIES is implemented in Python 3. Source code is available at https://github.com/Swart-lab/bleties (MIT license), and also distributed via Bioconda. ContactContact: kb.seah@tuebingen.mpg.de Supplementary informationBenchmarking of BleTIES with published sequence data.

bioinformatics↗