Two Piwis with Ago-like functions silence somatic genes at the chromatin level

Abstract

Most sRNA biogenesis mechanisms involve either RNAseIII cleavage or ping-pong amplification by different Piwi proteins harboring slicer activity. Here, we follow thequestion why the mechanism of transgene-induced silencing in the ciliate Paramecium needs both Dicer activity and two Ptiwi proteins. This pathway involves primary siRNAs produced from non-translatable transgenes and secondary siRNAs from endogenous remote loci. Our data does not indicate any signatures from ping-pongamplification but Dicer cleavage of long dsRNA. We show that Ptiwi13 and 14 have different preferences for primary and secondary siRNAs but do not load them mutually exclusive. Both Piwis enrich for antisense RNAs and Ptiwi14 loaded siRNAs show a 5′-U signature. Both Ptiwis show in addition a general preference for Uridine-rich sRNAs along the entire sRNA length. Our data indicates both Ptiwis and 2′-O-methylation to contribute to strand selection of Dicer cleaved siRNAs. This unexpected function of two distinct vegetative Piwis extends the increasing knowledge of the diversity of Piwi functions in diverse silencing pathways. As both Ptiwis show differential subcellular localization, Ptiwi13 in the cytoplasm and Ptiwi14 in thevegetative macronucleus, we conclude that cytosolic and nuclear silencing factors are necessary for efficient chromatin silencing.

Characterization of the RNA-interference pathway as a tool for reverse genetic analysis in the nascent phototrophic endosymbiosis, Paramecium bursaria

“…A feeding-induced siRNA-based RNAi pathway has been validated as a tool for gene knock-down in the non-photo-endosymbiotic ciliate, P. tetraurelia [33,34,36,48]. To establish the presence of a comparable pathway in P. bursaria, combined genomic and transcriptomic analyses were employed to identify putative homologues for all previously characterized P. tetraurelia RNAi protein components [33,45] (figure 1). We found that P. bursaria encodes a total of five Dicer or Dicer-like endonucleases (Dcr1, Dcr2/3-electronic supplementary material, dataset S1; Dcl1/2, Dcl3/4 and Dcl5-electronic supplementary material, dataset S2), three RdRPs (Rdr1/4, Rdr2 and Rdr3-electronic supplementary material, dataset S3), six AGO-Piwi components (PiwiA1, PiwiA2, PiwiB, PiwiC1, PiwiC2 and PiwiDelectronic supplementary material, dataset S4), a single Paramecium-specific Pds1 (Pds1-electronic supplementary material, dataset S5), and two nucleotidyl transferase (Cid1/3 and Cid2-electronic supplementary material, dataset S6) genes.…”

“…In ciliates, multiple whole genome duplication (WGD) events have led to the rapid expansion of gene families encoding RNAi components [38][39][40], resulting in a subsequent diversification of protein function. Example functions include transposon elimination, nuclear rearrangement and transcriptional regulation [32,35,[41][42][43][44][45]. Elegant investigation of the non-photo-endosymbiotic model system, Paramecium tetraurelia, has identified three distinct classes of RNAi pathway in Paramecium.…”

Characterisation of the RNA-interference pathway as a Tool for Genetics in the Nascent Phototrophic Endosymbiosis,Paramecium bursaria

“…tetraurelia RNAi protein components 33,45 (Figure 1). We found that P. bursaria encodes a total of five Dicer or Dicer-like endonucleases (Dcr1, Dcr2/3 -Dataset S1; Dcl1/2, Dcl3/4 and Dcl5 -Dataset S2), three RdRPs (Rdr1/4, Rdr2 and Rdr3 -Dataset S3), six AGO-Piwi components (PiwiA1, PiwiA2, PiwiB, PiwiC1, PiwiC2 and PiwiD-Dataset S4), a single Paramecium-specific Pds1 (Pds1 -Dataset S5), and two nucleotidyl transferase (Cid1/3 and Cid2 -Dataset S6)…”

“…A feeding-induced siRNA-based RNAi pathway has been validated as a tool for gene knock-down in the non-photo-endosymbiotic ciliate, P. tetraurelia 33,34,36,48 . To establish the presence of a comparable pathway in P. bursaria, combined genomic-and transcriptomic-analyses were employed to identify putative homologues for all previously characterised P. tetraurelia RNAi protein components 33,45 ( Figure 1). We found that P. bursaria encodes a total of five Dicer or Dicer-like endonucleases (Dcr1, Dcr2/3 -Dataset S1; Dcl1/2, Dcl3/4 and Dcl5 -Dataset S2), three RdRPs (Rdr1/4, Rdr2 and Rdr3 -Dataset S3), six AGO-Piwi components (PiwiA1, PiwiA2, PiwiB, PiwiC1, PiwiC2 and PiwiD-Dataset S4), a single Paramecium-specific Pds1 (Pds1 -Dataset S5), and two nucleotidyl transferase (Cid1/3 and Cid2 -Dataset S6) genes.…”

Broad domains of histone marks in the highly compact Paramecium macronuclear genome

“…For antibody against P. tetraurelia RBP1, the peptide SPHYTSHTNSPSPSYRSS-C was used for rabbit immunisation. Purification and testing of specificity by Western blots and immunostaining was carried out as described recently [18]. Since there are some amino acid differences in the N-terminal tail of the Paramecium H3P1 to Human H3 (Suppl.…”