The transient Spt4-Spt5 complex as an upstream regulator of non-coding RNAs during development

Abstract

The Spt4-Spt5 complex is conserved and essential RNA polymerase elongation factor. To investigate the role of the Spt4-Spt5 complex in non-coding transcription during development, we used the unicellular model Paramecium tetraurelia. In this organism harboring both germline and somatic nuclei, massive transcription of the entire germline genome takes place during meiosis. This phenomenon starts a series of events mediated by different classes of non-coding RNAs that control developmentally programmed DNA elimination. We focused our study on Spt4, a small zinc-finger protein encoded in P. tetraurelia by two genes expressed constitutively and two genes expressed during meiosis. SPT4 genes are not essential in vegetative growth, but they are indispensable for sexual reproduction, even though genes from both expression families show functional redundancy. Silencing of the SPT4 genes resulted in the absence of double-stranded ncRNAs and reduced levels of scnRNAs – 25 nt-long sRNAs produced from these double-stranded precursors in the germline nucleus. Moreover, we observed that the presence of a germline-specific Spt4-Spt5m complex is necessary for transfer of the scnRNA-binding PIWI protein between the germline and somatic nucleus. Our study establishes that Spt4, together with Spt5m, is essential for expression of the germline genome and necessary for developmental genome rearrangements.

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

“…For the construction of in-frame 3xFLAG-HA-GTSF1 (pOC17) , 3xFLAG-PTIWI09 (pJG091, Owsian et al, 2022) fusion plasmids, 3xFLAG-HA or 3xFLAG tags that were codon-optimized for the P. tetraurelia genetic code were added to the 5’ of the gene. As a result, the tag is fused to the N-terminus of GTSF1 or of PTIWI09 .…”

“…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.…”

Paramecium epigenetics in development and proliferation

“…Starting with Paramecium meiosis, long double‐stranded RNA is transcribed by RNA‐polymerase in association with the MIC specific transcription factor complex SPT4‐SPT5 from MIC chromosomes (Gruchota et al, 2017; Owsian et al, 2022). Small dsRNAs are produced by two Dicer‐like proteins, Dcl2/3, with characteristics in a 5′ UNG signature, 3′ 2nt overhangs, and the precise length of 25nt (Lepère et al, 2009; Sandoval et al, 2014).…”

A SUMO E3 ligase promotes long non-coding RNA transcription to regulate small RNA-directed DNA elimination

“…The sexual reproduction-specific Spt4 and Spt5 paralogs are specifically involved in the MIC transcription in Paramecium (Owsian et al, 2022;Gruchota et al, 2017), while Tetrahymena genome does not encode such specialized Spt4/Spt5 paralogs. These micronuclear long noncoding RNAs (MIC-lncRNAs) are processed into small RNAs (~29-nt in Tetrahymena and 25-nt in Paramecium), called scnRNAs, by Dicer homologs (Malone et al, 2005;Mochizuki & Gorovsky, 2005;Lepère et al, 2009) and loaded into Piwi-clade Argonaute proteins (Mochizuki et al, 2002;Noto et al, 2010;Bouhouche et al, 2011).…”

“…During meiotic prophase in Tetrahymena , lncRNAs are transcribed bidirectionally in the MIC in a genome-wide manner by RNA polymerase II and dedicated conjugation-specific Mediator-associated proteins (Chalker & Yao, 2001; Schoeberl et al , 2012; Mochizuki & Gorovsky, 2004b; Tian et al , 2019; Garg et al , 2019). The sexual reproduction-specific Spt4 and Spt5 paralogs are specifically involved in the MIC transcription in Paramecium (Owsian et al , 2022; Gruchota et al , 2017), while Tetrahymena genome does not encode such specialized Spt4/Spt5 paralogs. These micronuclear long non-coding RNAs (MIC-lncRNAs) are processed into small RNAs (∼29-nt in Tetrahymena and 25-nt in Paramecium ), called scnRNAs, by Dicer homologs (Malone et al , 2005; Mochizuki & Gorovsky, 2005; Lepère et al , 2009) and loaded into Piwi-clade Argonaute proteins (Mochizuki et al , 2002; Noto et al , 2010; Bouhouche et al , 2011).…”