The nuclear PIWI-interacting protein Gtsf1 controls the selective degradation of small RNAs inParamecium

Abstract

Ciliates undergo developmentally programmed genome elimination, in which small RNAs direct the removal of target DNA segments, including transposable elements. At each sexual generation, the development of the macronucleus (MAC) requires massive and reproducible elimination of a large proportion of the germline micronuclear (MIC) genome, leading to a highly streamlined somatic MAC genome. 25-nt long scnRNAs are produced from the entire germline MIC genome during meiosis, and this initial complex small RNA population is then transported to the maternal MAC, where selection of scnRNAs corresponding to germline (MIC)-specific sequences is thought to take place. Selected scnRNAs, loaded onto the PIWI protein Ptiwi09, guide the deposition of histone H3 post-translational modifications (H3K9me3 and H3K27me3) onto transposable elements in the developing macronucleus, ultimately triggering their specific elimination. How germline-specific MIC scnRNAs are selected remains to be determined. Here, we provide important mechanistic insights into the scnRNA selection pathway by identifying aParameciumhomolog of Gametocyte specific factor 1 (Gtsf1) as essential for the selective degradation of scnRNAs corresponding to retained somatic MAC sequences. Consistently, we also show that Gtsf1 is exclusively localized in the maternal macronucleus and associates with the scnRNA-binding protein Ptiwi09. Furthermore, Gtsf1 is necessary for DNA elimination and correct H3K9me3 and H3K27me3 localization in the new developing macronucleus, demonstrating that the scnRNA selection process is important for genome elimination. We propose that Gtsf1 is required for the coordinated degradation of Ptiwi09-scnRNA complexes that pair with nascent RNA transcribed from the maternal MAC genome, similarly to the mechanism suggested for microRNA target-directed degradation in metazoans.

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

“…Revisiting previous KD experiments with additional replicates would be worthwhile to explore the extent of batch-to-batch IRS and expression variance for other KDs. Noteworthy, variability in IES retention across replicates has recently been shown for GTSF1 (Charmant et al, 2023; Wang et al, 2023), suggesting this phenomenon is not restricted to DevPF1 and DevPF2 . In general, KD experiments are challenging to tightly control for reproducibility, and more effort should be invested in generating knockouts in Paramecium , as established in Tetrahymena (Chalker, 2012).…”

“…Variable new MAC enrichment by a sucrose gradient might introduce variation into the IRS analysis, as fragments of the parental MAC add unexcised IES sequences, diluting the effect of IES retention (Charmant et al, 2023). Fluorescence-activated nuclear sorting (FANS) enables better nuclear separation in Paramecium (Charmant et al, 2023; Guérin et al, 2017) and should be able to eliminate most of such variation. Additionally, microinjection of DNA into macronuclei before RNAi experiments can be used to control for contaminating DNA from old MAC fragments.…”

“…iesRNAs are proposed to derive from dsRNAs transcribed from excised IESs (Allen et al, 2017;Sandoval et al, 2014). Hence, failed excision of IESs in DevPF1-or DevPF2-KD contributes to reduced iesRNA levels, as has consistently been observed for many other KDs of genes involved in Paramecium genome editing (Charmant et al, 2023;de Vanssay et al, 2020;Ignarski et al, 2014;Maliszewska-Olejniczak et al, 2015;Singh et al, 2022;Wang et al, 2023). The lack of scnRNAs in the DevPF1-KD cannot explain the absence of iesRNAs, as these accumulate even if the preceding scnRNA production is blocked (Sandoval et al, 2014).…”

“…The copyright holder for this preprint this version posted January 23, 2024. ; https://doi.org/10.1101/2024.01.23.576875 doi: bioRxiv preprint short dsRNAs, removing one of the two strands, and stabilize single-stranded scnRNAs throughout the selection process in the parental MAC and targeting of MIC-specific sequences in the new MACs (Bouhouche et al, 2011;Furrer et al, 2017). In the parental MAC, Gtsf1 was recently proposed to promote ubiquitination and subsequent degradation of the Ptiwi01/09 complexes harboring MAC-matching scnRNAs (Charmant et al, 2023;Wang et al, 2023). In the new MACs, the putative transcription elongation factor TFIIS4 was proposed to promote non-coding transcription required for scanning the developing genome (Maliszewska-Olejniczak et al, 2015).…”

Small‐RNA‐guided histone modifications and somatic genome elimination in ciliates

“…Interestingly, recent findings have identified roles for proteins traditionally associated with different functions in other organisms in scnRNA selection in ciliates, such as PRC2 and its cofactors (Mir o-Pina et al, 2023;C. Wang et al, 2022) and Gtsf1 (Charmant et al, 2023;C. Wang, Lv, et al, 2023).…”