Non-catalytic function of PRC2 in the control of small RNA dynamics during programmed genome elimination inParamecium

Abstract

To limit transposable element (TE) mobilization, most eukaryotes have evolved small RNAs to silence TE activity via homology-dependent mechanisms. Small RNAs, 20-30 nucleotides in length, bind to PIWI proteins and guide them to nascent transcripts by sequence complementarity, triggering the recruitment of histone methyltransferase enzymes on chromatin to repress the transcriptional activity of TEs and other repeats. In the ciliate Paramecium tetraurelia, 25-nt scnRNAs corresponding to TEs recruit Polycomb Repressive Complex 2 (PRC2), and trigger their elimination during the formation of the somatic nucleus. Here, we sequenced sRNAs during the entire sexual cycle with unprecedented precision. Our data confirmed that scnRNAs are produced from the entire germline genome, from TEs and non-TE sequences, during meiosis. Non-TE scnRNAs are selectively degraded, which results in the specific selection of TE-scnRNAs. We provide important mechanistic insight into the scnRNA selection pathway by identifying PRC2 and its cofactors as essential for the selective degradation of non-TE-scnRNAs. Our findings reveal a new mechanism for PRC2 that involves a non-methyltransferase function for regulating small RNA dynamics during development.

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

“…We extended the scnRNA analysis by sequencing total small RNAs in the 20- to 30-nt range from Gtsf1-depleted cells at several time points (from T=0 to T=35 hours after the onset of autogamy) (Figure S1; Figure S5). In Gtsf1-depleted cells, the 25-nt scnRNAs are produced from the whole MIC genome at the beginning of the sexual cycle (T=0 hours), as in control RNAi conditions, consistent with previous work (Miró-Pina et al, 2023) (Figure 6B-C). This can be clearly observed when examining the mapping of 25-nt scnRNAs across an individual genomic region (Figure 6B), where MAC sequences, as well as MIC sequences (IES and OES), are covered.…”

“…To immunoprecipitate Gtsf1, we prepared nuclear extracts from control Paramecium cells and cells expressing the functional fusion protein. As expected, immunofluorescence experiments confirmed that Gtsf1, like Ptiwi09 (Bouhouche et al, 2011; Miró-Pina et al, 2023), localizes in the maternal macronucleus during meiosis (Figure 2D). Immunoprecipitation (IP) of the FLAG tag in triplicates for control and Gtsf1 (Figure 2E, Figure S1) was followed by MS (Figure 2F and 2G).…”

“…We also identified PRC2 core components (Ezl1, Caf1, Eed) and PRC2 cofactors (Rf2 and Rf4) (Figure 1C, Table S1). These proteins were recently shown to be involved in the scnRNA selection process (Miró-Pina et al, 2023; Wang et al, 2022), and the Rf4 cofactor was further shown to physically interact with Ptiwi09 in the new developing MAC (Miró-Pina et al, 2022). Another Ptiwi09 interactor we identified was an uncharacterized, small protein (16 kDa) referred to as Gametocyte specific factor 1 (Gtsf1) (Figure 1C, Figure 2A, Table S1).…”

“…Thus, the vast majority of these small RNAs originate from distinct genomic hotspots. The logic is radically different in the ciliate Paramecium , in which the entire germline genome initially produces 25-nt scnRNAs from both TE and non-TE sequences (Lepere et al, 2009; Miró-Pina et al, 2023; Sandoval et al, 2014; Singh et al, 2014). In subsequent steps, the subpopulation of scnRNAs that correspond to TEs is selected to trigger the physical removal of TEs from the genome, a definitive form of TE silencing (Bouhouche et al, 2011; Furrer et al, 2017; Lepere et al, 2009, 2008).…”

“…25-nt long scnRNAs are produced during meiosis from the whole germline MIC genome, which is entirely transcribed by RNA polymerase II with the participation of a specialized Spt5/Spt4 elongation complex (Gruchota et al, 2017; Khurana et al, 2014; Mochizuki and Gorovsky, 2004; Owsian et al, 2022). scnRNA biogenesis relies on a developmental-specific RNA interference (RNAi) pathway that involves the Dicer-like proteins Dcl2 and Dcl3 (Lepere et al, 2009; Miró-Pina et al, 2023; Owsian et al, 2022; Sandoval et al, 2014; Singh et al, 2014). These scnRNAs then bind to Ptiwi01/09 proteins and are transported to the maternal MAC (Furrer et al, 2017), where selection of scnRNAs is believed to occur.…”