Biology subjects

Nischwitz, E.

Publications and source records attributed to Nischwitz, E..

7 recordsLinked to original sources

bioRxiv2025

An Evolutionarily Conserved N-terminal Domain of RRF-3 Governs GTSF-1 Binding in Nematodes

GTSF1 is an essential activating cofactor for PIWI proteins in many metazoans. In the nematode Caenorhabditis elegans, however, GTSF-1 does not bind PIWI, but associates with the RNA-dependent RNA polymerase RRF-3, supporting endo-siRNA (26G-RNA) biogenesis. Here, we demonstrate that this rewiring is deeply conserved across nematodes. For C. briggsae and Pristionchus pacificus we show that GTSF-1 interacts with RRF-3 and is essential for 26G-RNA production and fertility. We map this interaction to an N-terminal domain of RRF-3, termed the GTSF-1 interacting domain (GID), and show that the GTSF-1 zinc finger region alone is sufficient for binding. Mutagenesis identifies critical residues mediating this interaction and reveals that GTSF-1 stability depends on RRF-3. Other RdRPs possess GID-like domains, which we propose to bind GTSF-1-related proteins. Phylogenomic and structural analyses support GTSF-1-RRF-3 interactions across all major nematode lineages and map the shift in GTSF-1 activity to the last common nematode ancestor. We propose that binding of GTSF-1 induces conformational changes in RRF-3 that facilitate ERI complex assembly and activate RdRP function, paralleling its role as a PIWI activator.

molecular biology↗

bioRxiv2025

Crosstalk between and developmental dynamics of C. elegans Argonaute proteins

Gene regulation via Argonaute-bound small RNAs is a broadly conserved mechanism, present in all domains of life. The nematode Caenorhabditis elegans expresses several worm-specific Argonautes (WAGOs), which interact with the small RNAs known as 22G-RNAs. These WAGOs have roles in gene regulation, transposon-defence as well as in viral control and experimentally induced RNA interference. Despite many studies, direct relationships between WAGO targeting, as defined by 22G RNA sequences, and mRNA abundance are not clear. Also, the effects of developmental stage and WAGO-interconnectivity have been under-studied thus far. We studied these aspects for two germline-expressed WAGO proteins, WAGO-1 and WAGO-3. We show that WAGO-1 mostly affects 22G-RNA expression in gravid adult worms, while WAGO-3 predominantly affects 22G-RNA expression in embryos. Furthermore, we detect a link between WAGO-3 and the maternal 26G-RNA pathway governed by the Argonaute protein ERGO-1, and between WAGO-1 and the paternal 26G-RNA pathway governed by the Argonautes ALG-3/4. We also demonstrate that, globally speaking, loss of WAGO-1 or WAGO-3 does not result in upregulation of their target mRNAs, as defined by 22G-RNA complementarity. Finally, metagene analysis of 22G-RNA profiles suggests loss of one WAGO protein leads to shifts in WAGO 22G-RNA binding. Overall, we conclude that WAGO-1 and WAGO-3 are developmentally dynamic, are embedded in distinct regulatory networks, and that potential silencing of individual mRNAs by these WAGO proteins is hard to assess by simple loss-of-function studies. SummaryRNA interference describes silencing of genes via small RNAs coupled to an Argonaute protein. C. elegans expresses a class of secondary small RNAs known as 22G-RNAs which are coupled to Argonautes known as WAGOs (worm-specific Argonautes). We have sequenced 22G-RNA and mRNA populations from deletion mutants of two such WAGOs, WAGO-1 and WAGO-3, to investigate Argonaute crosstalk and the relationship between 22G-RNA and mRNA levels. We found that WAGO-1 and WAGO-3 engage different, complementary pathways, and that target silencing cannot be deduced simply from 22G-RNA complementarity.

genetics↗

bioRxiv2024

ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA

Type II interferon (IFN{gamma}) signaling is essential for innate immunity and critical for effective immunological checkpoint blockade in cancer immunotherapy. Genetic screen identification of post-transcriptional regulators of this pathway has been challenging since such factors are often essential for cell viability. Here, we utilize our inducible CRISPR/Cas9 approach to screen for key post-transcriptional regulators of IFN{gamma} signaling, and in this way identify ERH and the ERH-associated splicing and RNA export factors MAGOH, SRSF1, and ALYREF. Loss of these factors impairs post-transcriptional mRNA maturation of JAK2, a crucial kinase for IFN{gamma} signaling, resulting in abrogated JAK2 protein levels and diminished IFN{gamma} signaling. Further analysis highlights a critical role for ERH in preventing intron retention in AU-rich regions in specific transcripts, such as JAK2. This regulation is markedly different from previously described retention of GC-rich introns. Overall, these findings reveal that post-transcriptional JAK2 processing is a critical rate-limiting step for the IFN{gamma}-driven innate immune response.

cell 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↗

bioRxiv2023

piRNA processing by a trimeric Schlafen-domain nuclease

Transposable elements are genomic parasites that expand within and spread between genomes1. Piwi proteins control transposon activity, notably in the germline2,3. These proteins recognize their targets through small RNA co-factors named piRNAs, making piRNA biogenesis a key specificity-determining step in this crucial genome immunity system. While the processing of piRNA precursors is an essential step in this process, many molecular details of this process remain unknown. We identify a novel endoribonuclease, PUCH, that initiates piRNA processing in the nematode Caenorhabditis elegans. Genetic and biochemical studies show that PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), executes 5-end piRNA precursor cleavage. PUCH-mediated processing strictly requires an m7G-Cap and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds piRNA precursors4, and that this interaction enhances piRNA production in vivo. The identification of PUCH completes the repertoire of C. elegans piRNA biogenesis factors and uncovers a novel type of RNA endonuclease formed by three SLFL proteins. Mammalian Schlafen (Slfn) genes have been associated with immunity responses5, exposing a thus far unknown molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements.

molecular biology↗

bioRxiv2020

The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

Telomeres are bound by dedicated protein complexes, like shelterin in mammals, which protect telomeres from DNA damage. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to screen for proteins binding to C. elegans telomeres, and identified TEBP-1 and TEBP-2, two paralogs that associate to telomeres in vitro and in vivo. TEBP-1 and TEBP-2 are expressed in the germline and during embryogenesis. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a mortal germline, a phenotype characterized by transgenerational germline deterioration. Notably, tebp-1; tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. TEBP-1 and TEBP-2 form a telomeric complex with the known single-stranded telomere-binding proteins POT-1, POT-2, and MRT-1. Furthermore, we find that POT-1 bridges the double- stranded binders TEBP-1 and TEBP-2, with the single-stranded binders POT-2 and MRT-1. These results describe the first telomere-binding complex in C. elegans, with TEBP-1 and TEBP-2, two double-stranded telomere binders required for fertility and that mediate opposite telomere dynamics.

cell biology↗

bioRxiv2020

The RNA Polymerase II core subunit RPB-9 directs transcriptional elongation at piRNA loci in Caenorhabditis elegans

PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA Polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematode Caenorhabditis elegans. rpb-9 mutants fail to initiate heritable piRNA-mediated gene silencing. Furthermore, we show that RPB-9 is required to repress two DNA transposon families and a subset of somatic genes in the C. elegans germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis. We demonstrate that RPB-9 acts to promote transcriptional elongation/termination at endogenous piRNA loci. We conclude that as a part of its rapid evolution the piRNA pathway has co-opted another ancient machinery, this time for high-fidelity transcription.

genetics↗