Functional specialization of Piwi proteins in Paramecium tetraurelia from post-transcriptional gene silencing to genome remodelling

Abstract

Proteins of the Argonaute family are small RNA carriers that guide regulatory complexes to their targets. The family comprises two major subclades. Members of the Ago subclade, which are present in most eukaryotic phyla, bind different classes of small RNAs and regulate gene expression at both transcriptional and post-transcriptional levels. Piwi subclade members appear to have been lost in plants and fungi and were mostly studied in metazoa, where they bind piRNAs and have essential roles in sexual reproduction. Their presence in ciliates, unicellular organisms harbouring both germline micronuclei and somatic macronuclei, offers an interesting perspective on the evolution of their functions. Here, we report phylogenetic and functional analyses of the 15 Piwi genes from Paramecium tetraurelia. We show that four constitutively expressed proteins are involved in siRNA pathways that mediate gene silencing throughout the life cycle. Two other proteins, specifically expressed during meiosis, are required for accumulation of scnRNAs during sexual reproduction and for programmed genome rearrangements during development of the somatic macronucleus. Our results indicate that Paramecium Piwi proteins have evolved to perform both vegetative and sexual functions through mechanisms ranging from post-transcriptional mRNA cleavage to epigenetic regulation of genome rearrangements.

Epigenetics of Ciliates

“…In Paramecium, two Argonaute proteins, Ptiwi01 and Ptiwi09, and two Dicer proteins, Dcl2 and Dcl3, were identified as the functional counterparts of Tetrahymena Twi1p and Dcl1p, respectively (Lepere et al 2009;Bouhouche et al 2011). Double knockdown of the genes encoding these Argonaute or Dicer proteins causes both the loss of 25-nt small RNAs and defects in DNA elimination.…”

“…7, steps g and h) in late stages of conjugation. This model is supported by the following observations: (1) In Tetrahymena, although scnRNAs complementary to both MDSs and the micronuclear-specific sequences (mostly IESs) are produced in the early conjugation stage, only the latter scnRNA population becomes gradually enriched in the mid-conjugation stages Aronica et al 2008); (2) in the midconjugation stages, after the bulk of scnRNA production, Argonaute proteins (Ptiwi01 and Ptiwi09 in Paramecium and Twi1p in Tetrahymena) localize initially in the parental macronucleus, then later move to the new macronucleus (Mochizuki et al 2002;Bouhouche et al 2011); and (3) the RNA-binding protein Nowa1/2, which is required for the elimination of maternally regulated IESs, is also translocated from the parental to new macronucleus in Paramecium (Nowacki et al 2005).…”

Strand-asymmetric endogenous Tetrahymena small RNA production requires a previously uncharacterized uridylyltransferase protein partner

“…It seems likely that the varied subunits of cellular Rdr complexes (RDRCs) contribute to the specialization of Rdr function in vivo, restricting the general activity of Rdr enzymes that is observed on single-stranded RNA templates in vitro (Voinnet 2008;Talsky and Collins 2010). 1 Ciliated protozoa have a large complexity of RNA silencing machinery relative to other single-celled organisms, including numerous Piwi proteins that are the exclusive Argonaute family members encoded by ciliate genomes sequenced to date (Couvillion et al 2009;Bouhouche et al 2011). In addition to conjugationspecific silencing pathways that induce the transient heterochromatin formation and DNA elimination necessary for sexual reproduction, additional pathways produce sRNAs constitutively during culture expansion by vegetative growth (Duharcourt et al 2009;Mochizuki 2012).…”

Functional Study of Genes Essential for Autogamy and Nuclear Reorganization in Paramecium

“…Indeed, previously described genes involved in the control or the catalysis of programmed DNA rearrangements in the new MAC were shown to be induced during sexual reproduction (6,10,(23)(24)(25)(26). Our functional analysis, performed on a subset of 15 genes from expression clusters I and II, revealed that one gene (PTMB.186c), likely to be involved in the control of the cell cycle, is required for a normal outcome of autogamy.…”

“…Previous screens in P. tetraurelia uncovered the essential role of a developmentally regulated SUMO pathway likely to operate in the developing new MAC (24) and of Die5p, a nuclear protein of unknown function acting at a late step during DNA rearrangements (25). Essential genes encoding proteins involved in scnRNA biogenesis or transport were shown to be expressed specifically during sexual processes (10,23,26).…”