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

Mason, Victor C.; Wang, Chundi; Nowacki, Mariusz

doi:10.1016/j.celrep.2023.112213

Cited by 3 publications

(6 citation statements)

References 56 publications

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“…One could imagine that the scanning process leads to the differential 2'-O-methylation of MAC-and MIC-scnRNAs: MIC-scnRNAs would be 2'-O-methylated, which would protect them from degradation, while MAC-scanRNA would not. Data from a recent study contradicts this hypothesis, because HEN1 KD during Paramecium autogamy did not impair scnRNA selection (Solberg et al, 2023). Thus, 2'-O-methylation of scnRNAs does not appear to be involved in the selection process.…”

Section: Discussionmentioning

confidence: 93%

“…While Rdr1 plays a role in the dsRNA-induced RNAi pathway, Rdr2 was previously shown to be required for all known types of siRNAs in Paramecium, including endogenous siRNAs from constitutive SRCs (Carradec et al, 2015;Karunanithi et al, 2019). A separate study reported that developmental-specific SRCs are bound to the Ptiwi08 protein and appear to be Dicer 1-dependent (Solberg et al, 2023). The biological roles of the 23-nt siRNAs produced from the developmental-specific SRCs and of Rdr2 remain unclear.…”

Section: Discussionmentioning

confidence: 99%

“…The biological roles of the 23-nt siRNAs produced from the developmental-specific SRCs and of Rdr2 remain unclear. Further analyses will be needed to determine whether, as hypothesized in Solberg et al, 2023, developmental-specific SRCs regulate aberrant, untranslated mRNAs during development, or instead play a role in other processes such as gene copy number control during development, or act as an antiviral defense mechanism.…”

Section: Discussionmentioning

confidence: 99%

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Non-catalytic function of PRC2 in the control of small RNA dynamics during programmed genome elimination inParamecium

Miró-Pina

Arnaiz

Vanssay

et al. 2023

Preprint

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To limit transposable element (TE) mobilization, most eukaryotes have evolved small RNAs to silence TE activity via homology-dependent mechanisms. Small RNAs, 20-30 nucleotides in length, bind to PIWI proteins and guide them to nascent transcripts by sequence complementarity, triggering the recruitment of histone methyltransferase enzymes on chromatin to repress the transcriptional activity of TEs and other repeats. In the ciliate Paramecium tetraurelia, 25-nt scnRNAs corresponding to TEs recruit Polycomb Repressive Complex 2 (PRC2), and trigger their elimination during the formation of the somatic nucleus. Here, we sequenced sRNAs during the entire sexual cycle with unprecedented precision. Our data confirmed that scnRNAs are produced from the entire germline genome, from TEs and non-TE sequences, during meiosis. Non-TE scnRNAs are selectively degraded, which results in the specific selection of TE-scnRNAs. We provide important mechanistic insight into the scnRNA selection pathway by identifying PRC2 and its cofactors as essential for the selective degradation of non-TE-scnRNAs. Our findings reveal a new mechanism for PRC2 that involves a non-methyltransferase function for regulating small RNA dynamics during development.

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Section: Discussionmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Non-catalytic function of PRC2 in the control of small RNA dynamics during programmed genome elimination inParamecium

Miró-Pina

Arnaiz

Vanssay

et al. 2023

Preprint

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“…Although GFP-tagging has become the standard for protein localization studies in ciliates, GFP is a rather large tag and is not as commonly used for protein purification or other assays into protein function. Several studies have successfully utilized the smaller Flag-HA tag (33 amino acids containing both Flag and HA tags) for a variety of such assays 25 , 45 , 46 . We therefore also generated Flag-HA fusion constructs by inserting Flag-HA after the start codon ATG of the RPB1 gene.…”

Section: Resultsmentioning

confidence: 99%

“…During sexual reproduction, the germline genome undergoes large-scale DNA elimination and reorganization to form a new somatic genome through a series of genome rearrangement processes 21 – 23 . Studies into these processes have provided insights into transposon silencing, epigenetics and various chromatin-related processes and have made many contributions to cell and molecular biology 16 , 24 , 25 .…”

Section: Introductionmentioning

confidence: 99%

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

Gao,

Solberg,

Wang

et al. 2024

Commun Biol

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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.

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Dynamic DNAN⁶-adenine methylation (6mA) governs the encystment process, showcased in the unicellular eukaryotePseudocohnilembus persalinus

Liu,

Niu,

et al. 2024

Genome Res.

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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 DNAN6-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 ciliatePseudocohnilembus 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 inP. persalinusand 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.

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

Cited by 3 publications

References 56 publications

Non-catalytic function of PRC2 in the control of small RNA dynamics during programmed genome elimination inParamecium

Non-catalytic function of PRC2 in the control of small RNA dynamics during programmed genome elimination inParamecium

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

Dynamic DNAN⁶-adenine methylation (6mA) governs the encystment process, showcased in the unicellular eukaryotePseudocohnilembus persalinus

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

Cited by 3 publications

References 56 publications

Non-catalytic function of PRC2 in the control of small RNA dynamics during programmed genome elimination inParamecium

Non-catalytic function of PRC2 in the control of small RNA dynamics during programmed genome elimination inParamecium

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

Dynamic DNAN6-adenine methylation (6mA) governs the encystment process, showcased in the unicellular eukaryotePseudocohnilembus persalinus

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Product

Resources

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Dynamic DNAN⁶-adenine methylation (6mA) governs the encystment process, showcased in the unicellular eukaryotePseudocohnilembus persalinus