PIWI-mediated control of tissue-specific transposons is essential for somatic cell differentiation.

PIWI proteins are known as mediators of transposon silencing in animal germlines but are also found in adult pluripotent stem cells of highly regenerative animals, where they are essential for regeneration. Study of the nuclear PIWI protein SMEDWI-2 in the planarian somatic stem cell system reveals an intricate interplay between transposons and cell differentiation in which a subset of transposons is inevitably activated during cell differentiation, and the PIWI protein is required to regain control. Absence of SMEDWI-2 leads to tissue-specific transposon derepression related to cell-type-specific chromatin remodeling events and in addition causes reduced accessibility of lineage-specific genes and defective cell differentiation, resulting in fatal tissue dysfunction. Finally, we show that additional PIWI proteins provide a stem-cell-specific second layer of protection in planarian neoblasts. These findings reveal a far-reaching role of PIWI proteins and PIWI-interacting RNAs (piRNAs) in stem cell biology and cell differentiation.

Beyond identity: Understanding the contribution of the 5' nucleotide of the antisense strand to RNAi activity.

In both the pharmaceutical and agricultural fields, RNA-based products have capitalized upon the mechanism of RNA interference for targeted reduction of gene expression to improve phenotypes and traits. Reduction in gene expression by RNAi is the result of a small interfering RNA (siRNA) molecule binding to an ARGONAUTE (AGO) protein and directing the effector complex to a homologous region of a target gene's mRNA. siRNAs properties that govern RNA-AGO association have been studied in detail. The siRNA 5' nucleotide (nt) identity has been demonstrated in plants to be an important property responsible for directing association of endogenous small RNAs with different AGO effector proteins. However, it has not been investigated whether the 5' nt identity is an efficacious determinant for topically-applied chemically synthesized siRNAs. In this study, we employed a sandpaper abrasion method to study the silencing efficacies of topically-applied 21 base-pair siRNA duplexes. The MAGNESIUM CHELATASE and GREEN FLUORESCENT PROTEIN genes were selected as endogenous and transgenic gene targets, respectively, to assess the molecular and phenotypic effects of gene silencing. Collections of siRNA variants with different 5' nt identities and different pairing states between the 5' antisense nt and its match in the sense strand of the siRNA duplex were tested for their silencing efficacy. Our results suggest a flexibility in the 5' nt requirement for topically applied siRNA duplexes in planta and highlight the similarity of 5' thermodynamic rules governing topical siRNA efficacy across plants and animals.

Ago1 Affects the Virulence of the Fungal Plant Pathogen Zymoseptoria tritici.

In host-pathogen interactions RNA interference (RNAi) has emerged as a pivotal mechanism to modify both, the immune responses of the host as well as the pathogenicity and virulence of the pathogen. In addition, in some fungi RNAi is also known to affect chromosome biology via its effect on chromatin conformation. Previous studies reported no effect of the RNAi machinery on the virulence of the fungal plant pathogen Zymoseptoria tritici however the role of RNAi is still poorly understood in this species. Herein, we elucidate whether the RNAi machinery is conserved within the genus Zymoseptoria. Moreover, we conduct functional analyses of Argonaute and Dicer-like proteins and test if the RNAi machinery affects chromosome stability. We show that the RNAi machinery is conserved among closely related Zymoseptoria species while an exceptional pattern of allelic diversity was possibly caused by introgression. The deletion of Ago1 reduced the ability of the fungus to produce asexual propagules in planta in a quantitative matter. Chromosome stability of the accessory chromosome of Z. tritici was not prominently affected by the RNAi machinery. These results indicate, in contrast to previous finding, a role of the RNAi pathway during host infection, but not in the stability of accessory chromosomes in Z. tritici.

Tissue-Specific Knockdown of Genes of the Argonaute Family Modulates Lifespan and Radioresistance in Drosophila Melanogaster.

Small RNAs are essential to coordinate many cellular processes, including the regulation of gene expression patterns, the prevention of genomic instability, and the suppression of the mutagenic transposon activity. These processes determine the aging, longevity, and sensitivity of cells and an organism to stress factors (particularly, ionizing radiation). The biogenesis and activity of small RNAs are provided by proteins of the Argonaute family. These proteins participate in the processing of small RNA precursors and the formation of an RNA-induced silencing complex. However, the role of Argonaute proteins in regulating lifespan and radioresistance remains poorly explored. We studied the effect of knockdown of Argonaute genes (AGO1, AGO2, AGO3, piwi) in various tissues on the Drosophila melanogaster lifespan and survival after the γ-irradiation at a dose of 700 Gy. In most cases, these parameters are reduced or did not change significantly in flies with tissue-specific RNA interference. Surprisingly, piwi knockdown in both the fat body and the nervous system causes a lifespan increase. But changes in radioresistance depend on the tissue in which the gene was knocked out. In addition, analysis of changes in retrotransposon levels and expression of stress response genes allow us to determine associated molecular mechanisms.

PIWIL1 governs the crosstalk of cancer cell metabolism and immunosuppressive microenvironment in hepatocellular carcinoma.

Altered energy metabolism of cancer cells shapes the immune cell response in the tumor microenvironment that facilitates tumor progression. Herein, we reported the novel of tumor cell-expressed Piwi Like RNA-Mediated Gene Silencing 1 (PIWIL1) in mediating the crosstalk of fatty acid metabolism and immune response of human hepatocellular carcinoma (HCC). PIWIL1 expression in HCC was increased compared to normal hepatic tissues and was positively correlated with the proliferation rate of HCC cell lines. PIWIL1 overexpression accelerated in vitro proliferation and in vivo growth of HCC tumors, while PIWIL1 knockdown showed opposite effects. PIWIL1 increased oxygen consumption and energy production via fatty acid metabolism without altering aerobic glycolysis. Inhibition of fatty acid metabolism abolished PIWIL1-induced HCC proliferation and growth. RNA-seq analysis revealed that immune system regulation might be involved, which was echoed by the experimental observation that PIWIL1-overexpressing HCC cells attracted myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment. MDSCs depletion reduced the proliferation and growth of PIWIL1-overexpressing HCC tumors. Complement C3, whose secretion was induced by PIWIL1 in HCC cells, mediates the interaction of HCC cells with MDSCs by activated p38 MAPK signaling in MDSCs, which in turn initiated expression of immunosuppressive cytokine IL10. Neutralizing IL10 secretion reduced the immunosuppressive activity of MDSCs in the microenvironment of PIWIL1-overexpressing HCC. Taken together, our study unraveled the critical role of PIWIL1 in initiating the interaction of cancer cell metabolism and immune cell response in HCC. Tumor cells-expressed PIWIL1 may be a potential target for the development of novel HCC treatment.

Structure-guided screening strategy combining surface plasmon resonance with nuclear magnetic resonance for identification of small-molecule Argonaute 2 inhibitors.

Argonaute (AGO) proteins are the key component of the RNA interference machinery that suppresses gene expression by forming an RNA-induced silencing complex (RISC) with microRNAs (miRNAs). Each miRNA is involved in various cellular processes, such as development, differentiation, tumorigenesis, and viral infection. Thus, molecules that regulate miRNA function are expected to have therapeutic potential. In addition, the biogenesis of miRNA is a multistep process involving various proteins, although the complete pathway remains to be elucidated. Therefore, identification of molecules that can specifically modulate each step will help understand the mechanism of gene suppression. To date, several AGO2 inhibitors have been identified. However, these molecules were identified through a single screening method, and no studies have specifically evaluated a combinatorial strategy. Here, we demonstrated a combinatorial screening (SCR) approach comprising an in silico molecular docking study, surface plasmon resonance (SPR) analysis, and nuclear magnetic resonance (NMR) analysis, focusing on the strong binding between the 5'-terminal phosphate of RNA and the AGO2 middle (MID) domain. By combining SPR and NMR, we identified binding modes of amino acid residues binding to AGO2. First, using a large chemical library (over 6,000,000 compounds), 171 compounds with acidic functional groups were screened using in silico SCR. Next, we constructed an SPR inhibition system that could analyze only the 5'-terminal binding site of RNA, and nine molecules that strongly bound to the AGO2 MID domain were selected. Finally, using NMR, three molecules that bound to the desired site were identified. The RISC inhibitory ability of the "hit" compounds was analyzed in human cell lysate, and all three hit compounds strongly inhibited the binding between double-stranded RNA and AGO2.

Transcriptional repression of Myc underlies the tumour suppressor function of AGO1 in Drosophila.

Here, we report novel tumour suppressor activity for the Drosophila Argonaute family RNA-binding protein AGO1, a component of the miRNA-dependent RNA-induced silencing complex (RISC). The mechanism for growth inhibition does not, however, involve canonical roles as part of the RISC; rather, AGO1 controls cell and tissue growth by functioning as a direct transcriptional repressor of the master regulator of growth, Myc. AGO1 depletion in wing imaginal discs drives a significant increase in ribosome biogenesis, nucleolar expansion and cell growth in a manner dependent on Myc abundance. Moreover, increased Myc promoter activity and elevated Myc mRNA in AGO1-depleted animals requires RNA polymerase II transcription. Further support for transcriptional AGO1 functions is provided by physical interaction with the RNA polymerase II transcriptional machinery (chromatin remodelling factors and Mediator Complex), punctate nuclear localisation in euchromatic regions and overlap with Polycomb Group transcriptional silencing loci. Moreover, significant AGO1 enrichment is observed on the Myc promoter and AGO1 interacts with the Myc transcriptional activator Psi. Together, our data show that Drosophila AGO1 functions outside of the RISC to repress Myc transcription and inhibit developmental cell and tissue growth.This article has an associated 'The people behind the papers' interview.

The people behind the papers - Olga Zaytseva and Leonie Quinn.

Unregulated cell proliferation can be disastrous for development and underlies the progression of cancers throughout the lifespan. A new paper in Development dissects the molecular regulation of a key cell proliferation promoter (and infamous oncogene) Myc, using Drosophila as a model system. We caught up with Olga Zaytseva, recent PhD graduate and one of the paper's first authors, and her supervisor Leonie Quinn, Associate Professor at the John Curtin School of Medical Research in Canberra, to find out more.

Effects of Chemical Modifications on siRNA Strand Selection in Mammalian Cells.

Small interfering RNAs (siRNAs) enable efficient gene silencing through RNA interference (RNAi) mechanisms. The RNAi machinery relies on an RNA-guided nuclease, Argonaute-2 (Ago2), which preferentially selects a single strand from an siRNA duplex. Complementarity between the selected strand and an RNA target strand leads to silencing through cleavage. The U.S. Food and Drug Administration's recent approval of two siRNA drugs has reignited optimism for RNAi therapeutics. Despite this recent success in the field, off-target effects are still a major concern; however, chemical modifications have shown promise in mitigating some off-target gene silencing. To evaluate the impact of novel chemical modifications on strand selection, we developed a quantitative polymerase chain reaction-based assay that is compatible with several pre-existing siRNA libraries and was used to characterize chemically modified siRNAs. siRNAs bearing azobenzene and propargyl modifications at the central region of the passenger strand significantly improved strand selection. On the other hand, folic acid-modified siRNAs improved strand selection best when placed at the 3' terminus. This study highlights the development and utility of a convenient method to evaluate the impact that novel chemical modifications have on strand-specific gene silencing of siRNAs.

Expression of TNRC6 (GW182) Proteins Is Not Necessary for Gene Silencing by Fully Complementary RNA Duplexes.

The trinucleotide repeat containing 6 (TNRC6) family of proteins are core components of RNA interference (RNAi) and consist of three paralogs (TNRC6A, TNRC6B, and TNRC6C). The TNRC6 paralogs associate with argonaute (AGO) protein, the core RNAi factor, and bridge its interactions with other proteins. We obtained TNRC6A and TNRC6B single and double knockout cell lines to investigate how the TNRC6 paralogs contribute to RNAi. We found that TNRC6 proteins are not required for gene silencing when duplex RNAs are fully complementary. TNRC6 expression was necessary for regulation by a microRNA. TNRC6A, but not TNRC6B, expression was necessary for transcriptional activation by a duplex RNA targeting a gene promoter. By contrast, AGO2 is required for all three gene expression pathways. TNRC6A can affect the Dicer localization in cytoplasm versus the nucleus, but none of the three TNRC6 paralogs was necessary for nuclear localization of AGO2. Our data suggest that the roles of the TNRC6 paralogs differ in some details and that TNRC6 is not required for clinical therapeutic silencing mechanisms that involve fully complementary duplex RNAs.

Downregulation of mTOR Signaling Increases Stem Cell Population Telomere Length during Starvation of Immortal Planarians.

Reduction of caloric intake delays and prevents age-associated diseases and extends the life span in many organisms. It may be that these benefits are due to positive effects of caloric restriction on stem cell function. We use the planarian model Schmidtea mediterranea, an immortal animal that adapts to long periods of starvation by shrinking in size, to investigate the effects of starvation on telomere length. We show that the longest telomeres are a general signature of planarian adult stem cells. We also observe that starvation leads to an enrichment of stem cells with the longest telomeres and that this enrichment is dependent on mTOR signaling. We propose that one important effect of starvation for the rejuvenation of the adult stem cell pool is through increasing the median telomere length in somatic stem cells. Such a mechanism has broad implications for how dietary effects on aging are mediated at the whole-organism level.

Argonaute-2 is associated to brown adipose tissue activation.

MicroRNAs (miRNAs) are important modulators of thermogenic brown adipose tissue (BAT). They have been involved in its differentiation and hence its functioning. While different regulators of the miRNA machinery have been shown to be essential for BAT differentiation, little is known about their implication in BAT activation. The aim of this work was to evaluate the role of AGO2, the chief miRNA mediator, in BAT activation. We took advantage of two non-genetic models of BAT activation to analyze the miRNA machinery and miRNA expression in BAT. We used principal component analysis (PCA) to obtain an overview of miRNA expression according to the BAT activation state. In vitro, we examined AGO2 expression during brown adipocyte differentiation and activation. Finally, we downregulated AGO2 to reveal its potential role in the thermogenic function of brown adipocytes. PCA analysis allowed to cluster animals on their miRNA signature in active BAT. Moreover, hierarchical clustering showed a positive correlation between global upregulation of miRNA expression and active BAT. Consistently, the miRNA machinery, particularly AGO2, was upregulated in vivo in active BAT and in vitro in mature brown adipocytes. Finally, the partial loss-of-function of AGO2 in mature brown adipocytes is sufficient to lead to a diminished expression of UCP1 associated to a decreased uncoupled respiration. Therefore, our study shows the potential contribution of AGO2 in BAT activation. Since BAT is a calorie-burning tissue these data have a translational potential in terms of therapeutic target in the field of altered fuel homeostasis associated to obesity and diabetes.

Effect of Sugar 2',4'-Modifications on Gene Silencing Activity of siRNA Duplexes.

In this study, we explore the effect of a library of 2'-, 4'-, and 2',4'-modified uridine nucleosides and their impact on silencing firefly luciferase and on down-regulated in renal cell carcinoma (DRR) gene targets. The modifications studied were 2'-F-ribose, 2'-F-arabinose, 2'-OMe-ribose, 2'-F,4'-OMe-ribose, 2'-F,4'-OMe-arabinose, and 2'-OMe,4'-F-ribose. We found that 2',4'-modifications are well tolerated within A-form RNA duplexes, leading to virtually no change in melting temperature as assessed by UV thermal melting. The impact of the dual (2',4') modification was assessed by comparing gene silencing ability to 2'- or 4'- (singly) modified siRNA counterparts. siRNAs with (2',4')-modified overhangs generally outperformed the native siRNA as well as siRNAs with a 2'- or 4'-modified overhang, suggesting that 2',4'-modified nucleotides interact favorably with Argonaute protein's PAZ domain. Among the most active siRNAs were those with 2'-F,4'-OMe-ribose or 2'-F,4'-OMe-arabinose at the overhangs. When modifications were placed at both overhangs and internal positions, a duplex with the 2'-F (internal) and 2'-F,4'-OMe (overhang) combination was found to be the most potent, followed by the duplex with 2'-OMe (internal) and 2',4'-diOMe (overhang) modifications. Given the nuclease resistance exhibited by 2',4'-modified siRNAs, particularly when the modification is placed at or near the overhangs, these findings may allow the creation of superior siRNAs for therapy.

Re-Engineering RNA Molecules into Therapeutic Agents.

Efforts to chemically modify nucleic acids got underway merely a decade after the discovery of the DNA double helix and initially targeted nucleosides and nucleotides. The origins of three analogues that remain staples of modification strategies and figure prominently in FDA-approved nucleic acid therapeutics can be traced to the 1960s: 2'-deoxy-2'-fluoro-RNA (2'-F RNA), 2'- O-methyl-RNA (2'- OMe RNA), and the phosphorothioates (PS-DNA/RNA). Progress in nucleoside phosphoramidite-based solid phase oligonucleotide synthesis has gone hand in hand with the creation of second-generation (e.g., 2'- O-(2-methoxyethyl)-RNA, MOE-RNA) and third-generation (e.g., bicyclic nucleic acids, BNAs) analogues, giving rise to an expanding universe of modified nucleic acids. Thus, beyond site-specifically altered DNAs and RNAs with a modified base, sugar, and/or phosphate backbone moieties, nucleic acid chemists have created a host of conjugated oligonucleotides and artificial genetic polymers (XNAs). The search for oligonucleotides with therapeutic efficacy constitutes a significant driving force for these investigations. However, nanotechnology, diagnostics, synthetic biology and genetics, nucleic acid etiology, and basic research directed at the properties of native and artificial pairing systems have all stimulated the design of ever more diverse modifications. Modification of nucleic acids can affect pairing and chemical stability, conformation and interactions with a flurry of proteins and enzymes that play important roles in uptake, transport or processing of targets. Enhancement of metabolic stability is a central concern in the design of antisense, siRNA and aptamer oligonucleotides for therapeutic applications. In the antisense approach, uniformly modified oligonucleotides or so-called gapmers are used to target a specific RNA. The former may sterically block transcription or direct alternative splicing, whereas the latter feature a central PS window that elicits RNase H-mediated cleavage of the target. The key enzyme in RNA interference (RNAi) is Argonaute 2 (Ago2), a dynamic multidomain enzyme that binds multiple regions of the guide (antisense) and passenger (sense) siRNAs. The complexity of the individual interactions between Ago2 and the siRNA duplex provides significant challenges for chemical modification. Therefore, a uniform (the same modification throughout, e.g., antisense) or nearly uniform (e.g., aptamer) modification strategy is less useful in the pursuit of siRNA therapeutic leads. Instead, unique structural features and protein interactions of 5'-end (guide/Ago2MID domain), seed region, central region (cleavage site/Ago2 PIWI domain), and 3'-terminal nucleotides (guide/Ago2 PAZ domain) demand a more nuanced approach in the design of chemically modified siRNAs for therapeutic use. This Account summarizes current siRNA modification strategies with an emphasis on the regio-specific interactions between oligonucleotide and Ago2 and how these affect the choice of modification and optimization of siRNA efficacy. In addition to standard assays applied to measure the effects of modification on the stability of pairing and resistance against nuclease degradation, structural insights based on crystallographic data for modified RNAs alone and in complex with Ago2 from molecular modeling studies are a valuable guide in the design of siRNA therapeutics. Thus, this comprehensive approach is expected to result in accelerated generation of new siRNA-based therapies against various diseases, now that the first siRNA has obtained approval by the US FDA for treatment of hereditary hATTR amyloidosis.

PIWI proteins contribute to apoptosis during the UPR in human airway epithelial cells.

Small noncoding microRNAs (miRNAs) post-transcriptionally regulate a large portion of the human transcriptome. miRNAs have been shown to play an important role in the unfolded protein response (UPR), a cellular adaptive mechanism that is important in alleviating endoplasmic reticulum (ER) stress and promoting cell recovery. Another class of small noncoding RNAs, the Piwi-interacting RNAs (piRNAs) together with PIWI proteins, was originally shown to play a role as repressors of germline transposable elements. More recent studies, however, indicate that P-element induced WImpy proteins (PIWI proteins) and piRNAs also regulate mRNA levels in somatic tissues. Using genome-wide small RNA next generation sequencing, cell viability assays, and caspase activity assays in human airway epithelial cells, we demonstrate that ER stress specifically up-regulates total piRNA expression profiles, and these changes correlate with UPR-induced apoptosis as shown by up-regulation of two pro-apoptotic factor mRNAs, CHOP and NOXA. Furthermore, siRNA knockdown of PIWIL2 and PIWIL4, two proteins involved in piRNA function, attenuates UPR-related cell death, inhibits piRNA expression, and inhibits the up-regulation of CHOP and NOXA mRNA expression. Hence, we provide evidence that PIWIL2 and PIWIL4 proteins, and potentially the up-regulated piRNAs, constitute a novel epigenetic mechanism that control cellular fate during the UPR.

A dual signal amplification-assisted DNAzyme biosensor for ultrasensitive detection of Argonaute 2 activity.

We develop a new DNAzyme biosensor for Argonaute 2 (Ago2) assay based on target-initiated rolling circle amplification in combination with an 8-17 DNAzyme-mediated dual signal amplification strategy. This biosensor can achieve ultrahigh sensitivity with a detection limit of 0.35 pM, and it can be further used for the screening of Ago2 inhibitors and the accurate measurement of endogenous Ago2 from HeLa cell extracts.

Relationship between PIWIL4-Mediated H3K4me2 Demethylation and piRNA-Dependent DNA Methylation.

Retrotransposon genes are silenced by DNA methylation because of potential harm due to insertional mutagenesis. DNA methylation of retrotransposon genes is erased and re-established during male germ cell development. Both piRNA-dependent and piRNA-independent mechanisms are active during the re-establishment process, with the piRNA-independent mechanism occurring first. In this study, we analyzed the role of PIWIL4/MIWI2 in the modification of histone H3 and subsequent piRNA-dependent DNA methylation. Dimethylation at H3K4 is highly enriched at piRNA-dependent methylated regions and anti-correlated with de novo DNA methylation during the phase of piRNA-independent DNA methylation. In addition, PIWIL4, which binds the H3K4 demethylases KDM1A and KDM5B, is required for removing H3K4me2 marks. These data show that PIWIL4 plays important roles in histone modification and piRNA-dependent DNA methylation.

Small interfering RNA pathway contributes to antiviral immunity in Spodoptera frugiperda (Sf9) cells following Autographa californica multiple nucleopolyhedrovirus infection.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a well-known virus in the Baculoviridae family. Presence of the p35 gene in the AcMNPV genome as a suppressor of the short interfering RNA (siRNA) pathway is a strong reason for the importance of the siRNA pathway in the host cellular defense. Given that, here we explored the roles of Dicer-2 (Dcr2) and Argonaute 2 (Ago2) genes, key factors in the siRNA pathway in response to AcMNPV infection in Spodoptera frugiperda Sf9 cells. The results showed that the transcript levels of Dcr2 and Ago2 increased in response to AcMNPV infection particularly over 16 h post infection suggesting induction of the siRNA pathway. Reductions in the expression levels of Dcr2 and Ago2 by using specific dsRNAs in Sf9 cells modestly enhanced production of viral genomic DNA which indicated their role in the host antiviral defense. Using deep sequencing, our previous study showed a large number of small reads (siRNAs of ∼20 nucleotides) from AcMNPV-infected Sf9 cells that were mapped to some of the viral genes (hot spots). Down-regulation of Dcr2 in Sf9 cells resulted in enhanced expression levels of the selected virus hotspot genes (i.e. ORF-9 and ORF-148), while the transcript levels of virus cold spots (i.e. ORF-18 and ORF-25) with no or few siRNAs mapped to them did not change. Overexpression of AcMNPV p35 as a suppressor of RNAi and anti-apoptosis gene in Sf9 cells increased virus replication. Also, replication of mutant AcMNPV lacking the p35 gene was significantly increased in Sf9 cells with reduced transcript levels of Dcr2 and Ago2, highlighting the antiviral role of the siRNA pathway in Sf9 cells. Together, our results demonstrate that Dcr2 and Ago2 genes contribute in efficient antiviral response of Sf9 cells towards AcMNPV, and in turn, the AcMNPV p35 suppresses the siRNA pathway, besides being an antiapoptotic protein.

Prospectively Isolated Tetraspanin+ Neoblasts Are Adult Pluripotent Stem Cells Underlying Planaria Regeneration.

Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.

PIWIL2 suppresses Siah2-mediated degradation of HDAC3 and facilitates CK2α-mediated HDAC3 phosphorylation.

HDAC3 is involved in deacetylation of histone and non-histone proteins, having a key role in the regulation of gene transcription and also in the process of tumorgenesis. However, how HDAC3 is regulated in cancer remains largely unclear. Here, we showed that PIWIL2 can interact with HDAC3, leading to stabilization of HDAC3 from ubiquitin-mediated degradation by competitive association with E3 ubiquitin ligase Siah2. Furthermore, we found that expression of PIWIL2 enhanced HDAC3 activity via CK2α. PIWIL2 facilitated the interaction between HDAC3 and CK2α, thus exhibiting a promotion on the HDAC3 phosphorylation by CK2α. Further work showed that PIWIL2 could promote cell proliferation and suppress cell apoptosis via regulating HDAC3. Our present study firstly revealed that PIWIL2 can play a role in HDAC3-mediated epigenetic regulation on cancer cell proliferation and apoptosis. These findings provide a novel insight into the roles of PIWIL2 in tumorigenesis.