Dynamic DNA N 6-adenine methylation (6mA) governs the encystment process, showcased in the unicellular eukaryote Pseudocohnilembus persalinus.

The formation of resting cysts commonly found in unicellular eukaryotes is a complex and highly regulated survival strategy against environmental stress that involves drastic physiological and biochemical changes. Although most studies have focused on the morphology and structure of cysts, little is known about the molecular mechanisms that control this process. Recent studies indicate that DNA N 6-adenine methylation (6mA) could be dynamically changing in response to external stimuli; however, its potential role in the regulation of cyst formation remains unknown. We used the ciliate Pseudocohnilembus persalinus, which can be easily induced to form cysts to investigate the dynamic pattern of 6mA in trophonts and cysts. Single-molecule real-time (SMRT) sequencing reveals high levels of 6mA in trophonts that decrease in cysts, along with a conversion of symmetric 6mA to asymmetric 6mA. Further analysis shows that 6mA, a mark of active transcription, is involved in altering the expression of encystment-related genes through changes in 6mA levels and 6mA symmetric-to-asymmetric conversion. Most importantly, we show that reducing 6mA levels by knocking down the DNA 6mA methyltransferase PpAMT1 accelerates cyst formation. Taken together, we characterize the genome-wide 6mA landscape in P. persalinus and provide insights into the role of 6mA in gene regulation under environmental stress in eukaryotes. We propose that 6mA acts as a mark of active transcription to regulate the encystment process along with symmetric-to-asymmetric conversion, providing important information for understanding the molecular response to environmental cues from the perspective of 6mA modification.

Small RNA-mediated genome rearrangement pathways in ciliates.

Most eukaryotes employ a combination of transcriptional and post-transcriptional silencing mechanisms to suppress transposons, yet ciliates employ a more extreme approach. They separate germline and somatic functions into distinct nuclei, enabling the elimination of transposons from the active somatic genome through diverse small RNA-mediated genome rearrangement pathways during sexual processes.

Chromatin remodeling is required for sRNA-guided DNA elimination in Paramecium.

Small RNAs mediate the silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. The unicellular ciliate Paramecium is a model to study dynamic genome organization in eukaryotic cells, given its unique feature of nuclear dimorphism. Here, the formation of the somatic macronucleus during sexual reproduction requires eliminating thousands of transposon remnants (IESs) and transposable elements scattered throughout the germline micronuclear genome. The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries. Here we show that IES recognition and precise excision are facilitated by recruiting ISWI1, a Paramecium homolog of the chromatin remodeler ISWI. ISWI1 knockdown substantially inhibits DNA elimination, quantitatively similar to development-specific sRNA gene knockdowns but with much greater aberrant IES excision at alternative boundaries. We also identify key development-specific sRNA biogenesis and transport proteins, Ptiwi01 and Ptiwi09, as ISWI1 cofactors in our co-immunoprecipitation studies. Nucleosome profiling indicates that increased nucleosome density correlates with the requirement for ISWI1 and other proteins necessary for IES excision. We propose that chromatin remodeling together with small RNAs is essential for efficient and precise DNA elimination in Paramecium.

GTSF1 is required for transposon silencing in the unicellular eukaryote Paramecium tetraurelia.

The PIWI-interacting RNA (piRNA) pathway is crucial for transposon repression and the maintenance of genomic integrity. Gametocyte-specific factor 1 (GTSF1), a PIWI-associated protein indispensable for transposon repression, has been recently shown to potentiate the catalytic activity of PIWI in many metazoans. Whether the requirement of GTSF1 extends to PIWI proteins beyond metazoans is unknown. In this study, we identified a homolog of GTSF1 in the unicellular eukaryote Paramecium tetraurelia (PtGtsf1) and found that its role as a PIWI-cofactor is conserved. PtGtsf1 interacts with PIWI (Ptiwi09) and Polycomb Repressive Complex 2 and is essential for PIWI-dependent DNA elimination of transposons during sexual development. PtGtsf1 is crucial for the degradation of PIWI-bound small RNAs that recognize the organism's own genomic sequences. Without PtGtsf1, self-matching small RNAs are not degraded and results in an accumulation of H3K9me3 and H3K27me3, which may disturb transposon recognition. Our results demonstrate that the PIWI-GTSF1 interaction also exists in unicellular eukaryotes with a role in transposon silencing.

Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum.

Unicellular eukaryotes represent tremendous evolutionary diversity. However, the molecular mechanisms underlying this diversity remain largely unexplored, partly due to a limitation of genetic tools to only a few model species. Paramecium caudatum is a well-known unicellular eukaryote with an unexpectedly large germline genome, of which only two percent is retained in the somatic genome following sexual processes, revealing extensive DNA elimination. However, further progress in understanding the molecular mechanisms governing this process is hampered by a lack of suitable genetic tools. Here, we report the successful application of gene knockdown and protein localization methods to interrogate the function of both housekeeping and developmentally regulated genes in P. caudatum. Using these methods, we achieved the expected phenotypes upon RNAi by feeding, and determined the localization of these proteins by microinjection of fusion constructs containing fluorescent protein or antibody tags. Lastly, we used these methods to reveal that P. caudatum PiggyMac, a domesticated piggyBac transposase, is essential for sexual development, and is likely to be an active transposase directly involved in DNA cleavage. The application of these methods lays the groundwork for future studies of gene function in P. caudatum and can be used to answer important biological questions in the future.

Developmental mRNA clearance by PIWI-bound endo-siRNAs in Paramecium.

The clearance of untranslated mRNAs by Argonaute proteins is essential for embryonic development in metazoans. However, it is currently unknown whether similar processes exist in unicellular eukaryotes. The ciliate Paramecium tetraurelia harbors a vast array of PIWI-clade Argonautes involved in various small RNA (sRNA) pathways, many of which have not yet been investigated. Here, we investigate the function of a PIWI protein, Ptiwi08, whose expression is limited to a narrow time window during development, concomitant with the start of zygotic transcription. We show that Ptiwi08 acts in an endogenous small interfering RNA (endo-siRNA) pathway involved in the clearance of untranslated mRNAs. These endo-siRNAs are found in clusters that are strictly antisense to their target mRNAs and are a subset of siRNA-producing clusters (SRCs). Furthermore, the endo-siRNAs are 2'-O-methylated by Hen1 and require Dcr1 for their biogenesis. Our findings suggest that sRNA-mediated developmental mRNA clearance extends beyond metazoans and may be a more widespread mechanism than previously anticipated.

A small RNA-guided PRC2 complex eliminates DNA as an extreme form of transposon silencing.

In animal germlines, transposons are silenced at the transcriptional or post-transcriptional level to prevent deleterious expression. Ciliates employ a more direct approach by physically eliminating transposons from their soma, utilizing piRNAs to recognize transposons and imprecisely excise them. Ancient, mutated transposons often do not require piRNAs and are precisely eliminated. Here, we characterize the Polycomb Repressive Complex 2 (PRC2) in Paramecium and demonstrate its involvement in the removal of transposons and transposon-derived DNA. Our results reveal a striking difference between the elimination of new and ancient transposons at the chromatin level and show that the complex may be guided by Piwi-bound small RNAs (sRNAs). We propose that imprecise elimination in ciliates originates from an ancient transposon silencing mechanism, much like in plants and metazoans, through sRNAs, repressive methylation marks, and heterochromatin formation. However, it is taken a step further by eliminating DNA as an extreme form of transposon silencing.