Silencing-associated and meiosis-specific small RNA pathways in Paramecium tetraurelia

Abstract

Distinct small RNA pathways are involved in the two types of homology-dependent effects described in Paramecium tetraurelia, as shown by a functional analysis of Dicer and Dicer-like genes and by the sequencing of small RNAs. The siRNAs that mediate post-transcriptional gene silencing when cells are fed with double-stranded RNA (dsRNA) were found to comprise two subclasses. DCR1-dependent cleavage of the inducing dsRNA generates ∼23-nt primary siRNAs from both strands, while a different subclass of ∼24-nt RNAs, characterized by a short untemplated poly-A tail, is strictly antisense to the targeted mRNA, suggestive of secondary siRNAs that depend on an RNA-dependent RNA polymerase. An entirely distinct pathway is responsible for homology-dependent regulation of developmental genome rearrangements after sexual reproduction. During early meiosis, the DCL2 and DCL3 genes are required for the production of a highly complex population of ∼25-nt scnRNAs from all types of germline sequences, including both strands of exons, introns, intergenic regions, transposons and Internal Eliminated Sequences. A prominent 5′-UNG signature, and a minor fraction showing the complementary signature at positions 21–23, indicate that scnRNAs are cleaved from dsRNA precursors as duplexes with 2-nt 3′ overhangs at both ends, followed by preferential stabilization of the 5′-UNG strand.

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.…”

“…Moreover, imprecise loss of generally repetitive germ line DNA is associated with chromosome fragmentation (21). Maternal inheritance of rearrangement patterns from the old to the new MAC involves a trans-nuclear genome comparison mediated by different populations of noncoding RNAs (re-viewed in reference 13), including constitutive maternal transcripts originating from the old MAC (22) and 25-nucleotide (nt) scan RNAs (scnRNAs) produced during MIC meiosis by a specialized RNA interference (RNAi) machinery (23).…”

“…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).…”

Subtraction by addition: domesticated transposases in programmed DNA elimination: Figure 1.

“…Further characterization of these RNAidirected mechanisms demonstrated that both Tetrahymena and Paramecium begin early in their development to decipher what sequences should be eliminated, well before the transposases are expressed. At the start of meiosis, the germline MIC genome is bidirectionally transcribed (Chalker and Yao 2001), and the resulting dsRNA transcripts are processed into 25-to 30-nucleotide (nt) small RNAs (depending on the species) by Dicerlike ribonucleases (Malone et al 2005;Mochizuki and Gorovsky 2005;Lepere et al 2008Lepere et al , 2009). Later in development, these meiosis-specific small RNAs move to the developing somatic MACs and, at least in Tetrahymena, direct heterochromatin modifications to IESs.…”

Small RNA-mediated regulation of DNA dosage in the ciliate Oxytricha

“…The ciliate Tetrahymena expresses diverse small RNA classes that are derived from either pseudogene clusters, protein coding genes, or telomeres (Couvillion et al 2009;Farley and Collins 2017). Both Tetrahymena and Paramecium express a meiosis-specific, Dicer-dependent class of small RNAs called scanRNAs that derive from the germline micronucleus (Mochizuki et al 2002;Lepère et al 2009). Irrespective of their biological function, the endogenous siRNAs usually act at the transcriptional or post-transcriptional level to silence their target genes.…”