SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia.

Acute myeloid leukemia (AML) cells rely on phospho-signaling pathways to gain unlimited proliferation potential. Here, we use domain-focused CRISPR screening and identify the nuclear phosphatase SCP4 as a dependency in AML, yet this enzyme is dispensable in normal hematopoietic progenitor cells. Using CRISPR exon scanning and gene complementation assays, we show that the catalytic function of SCP4 is essential in AML. Through mass spectrometry analysis of affinity-purified complexes, we identify the kinase paralogs STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. We show that STK35 and PDIK1L function catalytically and redundantly in the same pathway as SCP4 to maintain AML proliferation and to support amino acid biosynthesis and transport. We provide evidence that SCP4 regulates STK35/PDIK1L through two distinct mechanisms: catalytic removal of inhibitory phosphorylation and by promoting kinase stability. Our findings reveal a phosphatase-kinase signaling complex that supports the pathogenesis of AML.

Ssu72 phosphatase directly binds to ZAP-70, thereby providing fine-tuning of TCR signaling and preventing spontaneous inflammation.

ZAP-70 is required for the initiation of T cell receptor (TCR) signaling, and Ssu72 is a phosphatase that regulates RNA polymerase II activity in the nucleus. However, the mechanism by which ZAP-70 regulates the fine-tuning of TCR signaling remains elusive. Here, we found that Ssu72 contributed to the fine-tuning of TCR signaling by acting as tyrosine phosphatase for ZAP-70. Affinity purification-mass spectrometry and an in vitro assay demonstrated specific interaction between Ssu72 and ZAP-70 in T cells. Upon TCR stimulation, Ssu72-deficient T cells increased the phosphorylation of ZAP-70 and downstream molecules and exhibited hyperresponsiveness, which was restored by reducing ZAP-70 phosphorylation. In vitro assay demonstrated that recombinant Ssu72 reduced tyrosine phosphorylation of ZAP-70 via phosphatase activity. Cd4-CreSsu72fl/fl mice showed a defect in the thymic development of invariant natural killer T cells and reductions in CD4+ and CD8+ T cell numbers in the periphery but more CD44hiCD62Llo memory T cells and fewer CD44loCD62Lhi naïve T cells, compared with wild-type mice. Furthermore, Cd4-CreSsu72fl/fl mice developed spontaneous inflammation at 6 mo. In conclusion, Ssu72 phosphatase regulates the fine-tuning of TCR signaling by binding to ZAP-70 and regulating its tyrosine phosphorylation, thereby preventing spontaneous inflammation.

Phlpp1 is induced by estrogen in osteoclasts and its loss in Ctsk-expressing cells does not protect against ovariectomy-induced bone loss.

Prior studies demonstrated that deletion of the protein phosphatase Phlpp1 in Ctsk-Cre expressing cells enhances bone mass, characterized by diminished osteoclast activity and increased coupling to bone formation. Due to non-specific expression of Ctsk-Cre, the definitive mechanism for this observation was unclear. To further define the role of bone resorbing osteoclasts, we performed ovariectomy (Ovx) and Sham surgeries on Phlpp1 cKOCtsk and WT mice. Micro-CT analyses confirmed enhanced bone mass of Phlpp1 cKOCtsk Sham females. In contrast, Ovx induced bone loss in both groups, with no difference between Phlpp1 cKOCtsk and WT mice. Histomorphometry demonstrated that Ovx mice lacked differences in osteoclasts per bone surface, suggesting that estradiol (E2) is required for Phlpp1 deficiency to have an effect. We performed high throughput unbiased transcriptional profiling of Phlpp1 cKOCtsk osteoclasts and identified 290 differentially expressed genes. By cross-referencing these differentially expressed genes with all estrogen response element (ERE) containing genes, we identified IGFBP4 as potential estrogen-dependent target of Phlpp1. E2 induced PHLPP1 expression, but reduced IGFBP4 levels. Moreover, genetic deletion or chemical inhibition of Phlpp1 was correlated with IGFBP4 levels. We then assessed IGFBP4 expression by osteoclasts in vivo within intact 12-week-old females. Modest IGFBP4 immunohistochemical staining of TRAP+ osteoclasts within WT females was observed. In contrast, TRAP+ bone lining cells within intact Phlpp1 cKOCtsk females robustly expressed IGFBP4, but levels were diminished within TRAP+ bone lining cells following Ovx. These results demonstrate that effects of Phlpp1 conditional deficiency are lost following Ovx, potentially due to estrogen-dependent regulation of IGFBP4.

Genome-wide characterization and expression analysis of TOPP-type protein phosphatases in soybean (Glycine max L.) reveal the role of GmTOPP13 in drought tolerance.

BACKGROUND: In response to various abiotic stressors such as drought, many plants engage different protein phosphatases linked to several physiological and developmental processes. However, comprehensive analysis of this gene family is lacking for soybean. OBJECTIVE: This study was performed to identify the TOPP-type protein phosphatase family in soybean and investigate the gene's role under drought stress. METHODS: Soybean genome sequences and transcriptome data were downloaded from the Phytozome v.12, and the microarray data were downloaded from NCBI GEO datasets GSE49537. Expression profiles of GmTOPP13 were obtained based on qRT-PCR results. GmTOPP13 gene was transformed into tobacco plants via Agrobacterium mediated method, and the drought tolerance was analyzed by water deficit assay. RESULTS: 15 GmTOPP genes were identified in the soybean genome database (GmTOPP1-15). GmTOPP genes were distributed on 9 of 20 chromosomes, with similar exon-intron structure and motifs arrangement. All GmTOPPs contained Metallophos and STPPase_N domains as well as the core catalytic sites. Cis-regulatory element analysis predicted that GmTOPPs were widely involved in plant development, stress and hormone response in soybean. Expression profiles showed that GmTOPPs expressed in different tissues and exhibited divergent expression patterns in leaf and root in response to drought stimulus. Moreover, GmTOPP13 gene was isolated and expression pattern analysis indicated that this gene was highly expressed in seed, root, leaf and other tissues detected, and intensively induced upon PEG6000 treatment. In addition, overexpression of GmTOPP13 gene enhanced the drought tolerance in tobacco plants. The transgenic tobacco plants showed regulation of stress-responsive genes including CAT, SOD, ERD10B and TIP during drought stress. CONCLUSIONS: This study provides valuable information for the study of GmTOPP gene family in soybean, and lays a foundation for further functional studies of GmTOPP13 gene under drought and other abiotic stresses.

Ctdnep1 and Eps8L2 regulate dorsal actin cables for nuclear positioning during cell migration.

Cells actively position their nuclei within the cytoplasm for multiple cellular and physiological functions.1-3 Consequently, nuclear mispositioning is usually associated with cell dysfunction and disease, from muscular disorders to cancer metastasis.4-7 Different cell types position their nuclei away from the leading edge during cell migration.8-11 In migrating fibroblasts, nuclear positioning is driven by an actin retrograde flow originated at the leading edge that drives dorsal actin cables away from the leading edge. The dorsal actin cables connect to the nuclear envelope by the linker of nucleoskeleton and cytoskeleton (LINC) complex on transmembrane actin-associated nuclear (TAN) lines.12-14 Dorsal actin cables are required for the formation of TAN lines. How dorsal actin cables are organized to promote TAN lines formation is unknown. Here, we report a role for Ctdnep1/Dullard, a nuclear envelope phosphatase,15-22 and the actin regulator Eps8L223-25 on nuclear positioning and cell migration. We demonstrate that Ctdnep1 and Eps8L2 directly interact, and this interaction is important for nuclear positioning and cell migration. We also show that Ctdnep1 and Eps8L2 are involved in the formation and thickness of dorsal actin cables required for TAN lines engagement during nuclear movement. We propose that Ctdnep1-Eps8L2 interaction regulates dorsal actin cables for nuclear movement during cell migration.

PLPP/CIN-mediated NF2-serine 10 dephosphorylation regulates F-actin stability and Mdm2 degradation in an activity-dependent manner.

Neurofibromin 2 (NF2, also known as merlin) is a tumor suppressor protein encoded by the neurofibromatosis type 2 gene NF2. NF2 is also an actin-binding protein that functions in an intrinsic signaling network critical for actin dynamics. Although protein kinase A (PKA)-mediated NF2-serin (S) 10 phosphorylation stabilizes filamentous actin (F-actin), the underlying mechanisms of NF2-S10 dephosphorylation and the role of NF2 in seizures have been elusive. Here, we demonstrate that pyridoxal-5'-phosphate phosphatase/chronophin (PLPP/CIN) dephosphorylated NF2-S10 site as well as cofilin-S3 site. In addition, NF2-S10 dephosphorylation reversely regulated murine double minute-2 (Mdm2) and postsynaptic density 95 (PSD95) degradations in an activity-dependent manner, which increased seizure intensity and its progression in response to kainic acid (KA). In addition, NF2 knockdown facilitated seizure intensity and its progress through F-actin instability independent of cofilin-mediated actin dynamics. Therefore, we suggest that PLPP/CIN may be a potential therapeutic target for epileptogenesis and NF2-associated diseases.

COP1 promotes ABA-induced stomatal closure by modulating the abundance of ABI/HAB and AHG3 phosphatases.

Plant stomata play a crucial role in leaf function, controlling water transpiration in response to environmental stresses and modulating the gas exchange necessary for photosynthesis. The phytohormone abscisic acid (ABA) promotes stomatal closure and inhibits light-induced stomatal opening. The Arabidopsis thaliana E3 ubiquitin ligase COP1 functions in ABA-mediated stomatal closure. However, the underlying molecular mechanisms are still not fully understood. Yeast two-hybrid assays were used to identify ABA signaling components that interact with COP1, and biochemical, molecular and genetic studies were carried out to elucidate the regulatory role of COP1 in ABA signaling. The cop1 mutants are hyposensitive to ABA-triggered stomatal closure under light and dark conditions. COP1 interacts with and ubiquitinates the Arabidopsis clade A type 2C phosphatases (PP2Cs) ABI/HAB group and AHG3, thus triggering their degradation. Abscisic acid enhances the COP1-mediated degradation of these PP2Cs. Mutations in ABI1 and AHG3 partly rescue the cop1 stomatal phenotype and the phosphorylation level of OST1, a crucial SnRK2-type kinase in ABA signaling. Our data indicate that COP1 is part of a novel signaling pathway promoting ABA-mediated stomatal closure by regulating the stability of a subset of the Clade A PP2Cs. These findings provide novel insights into the interplay between ABA and the light signaling component in the modulation of stomatal movement.

FORGETTER2 protein phosphatase and phospholipase D modulate heat stress memory in Arabidopsis.

Plants can mitigate environmental stress conditions through acclimation. In the case of fluctuating stress conditions such as high temperatures, maintaining a stress memory enables a more efficient response upon recurring stress. In a genetic screen for Arabidopsis thaliana mutants impaired in the memory of heat stress (HS) we have isolated the FORGETTER2 (FGT2) gene, which encodes a type 2C protein phosphatase (PP2C) of the D-clade. Fgt2 mutants acquire thermotolerance normally; however, they are defective in the memory of HS. FGT2 interacts with phospholipase D α2 (PLDα2), which is involved in the metabolism of membrane phospholipids and is also required for HS memory. In summary, we have uncovered a previously unknown component of HS memory and identified the FGT2 protein phosphatase and PLDα2 as crucial players, suggesting that phosphatidic acid-dependent signaling or membrane composition dynamics underlie HS memory.

Specificity determinants of phosphoprotein phosphatases controlling kinetochore functions.

Kinetochores are instrumental for accurate chromosome segregation by binding to microtubules in order to move chromosomes and by delaying anaphase onset through the spindle assembly checkpoint (SAC). Dynamic phosphorylation of kinetochore components is key to control these activities and is tightly regulated by temporal and spatial recruitment of kinases and phosphoprotein phosphatases (PPPs). Here we focus on PP1, PP2A-B56 and PP2A-B55, three PPPs that are important regulators of mitosis. Despite the fact that these PPPs share a very similar active site, they target unique ser/thr phosphorylation sites to control kinetochore function. Specificity is in part achieved by PPPs binding to short linear motifs (SLiMs) that guide their substrate specificity. SLiMs bind to conserved pockets on PPPs and are degenerate in nature, giving rise to a range of binding affinities. These SLiMs control the assembly of numerous substrate specifying complexes and their position and binding strength allow PPPs to target specific phosphorylation sites. In addition, the activity of PPPs is regulated by mitotic kinases and inhibitors, either directly at the activity level or through affecting PPP-SLiM interactions. Here, we discuss recent progress in understanding the regulation of PPP specificity and activity and how this controls kinetochore biology.

PTEN and PHLPP crosstalk in cancer cells and in TGFß-activated stem cells.

Akt kinase regulates several cellular processes, among them growth, proliferation and survival, and has been correlated to neoplastic disease. We report here crosstalk between several Akt regulatory phosphatases that controls the level of the activated form (phosphorylated) of Akt and affects tumor cell aggressiveness. In prostate cancer cell lines, we observed that transient transfection of PTEN decreased the endogenous level of PHLPPs and in contrast, the transient transfection of PHLPPs decreased the endogenous level of PTEN. Furthermore, silencing of PTEN by siRNA resulted in increased PHLPP levels. This phenomenon was not seen in non-transformed cells or in prostate stem cells. This crosstalk promoted cancer cell invasion and was controlled by epigenetically regulated processes where activation of miRs (miR-190 and miR214), the polycomb group of proteins and DNA methylation were involved. The purinergic P2X4 receptor, which has been shown to have a role in wound healing, was identified to be the mediator of this crosstalk. We also studied prostate stem cells and found this crosstalk in the TGFß1-activated epithelial-mesenchymal transition (EMT). The crosstalk seemed to be a natural part of EMT. In summary, we identify a crosstalk between Akt phosphatases which is not present in non-transformed prostate cells but occurs in cancer cells and stem cells transformed by TGFß-1. This crosstalk is important for cellular invasion. BACKGROUND: Phosphatases regulate the Akt oncogene. RESULTS: Crosstalk between Akt phosphatases in prostate cancer cells and in TGF-ß1 activated stem cells but not in non-transformed cells. CONCLUSION: This back-up mechanism facilitates invasive migration of prostate stem and cancer cells. SIGNIFICANCE: Characterization of Akt regulation may lead to a better understanding of tumor development and to novel strategies for treatment.

Structure and function of the co-chaperone protein phosphatase 5 in cancer.

Protein phosphatase 5 (PP5) is a serine/threonine protein phosphatase that regulates many cellular functions including steroid hormone signaling, stress response, proliferation, apoptosis, and DNA repair. PP5 is also a co-chaperone of the heat shock protein 90 molecular chaperone machinery that assists in regulation of cellular signaling pathways essential for cell survival and growth. PP5 plays a significant role in survival and propagation of multiple cancers, which makes it a promising target for cancer therapy. Though there are several naturally occurring PP5 inhibitors, none is specific for PP5. Here, we review the roles of PP5 in cancer progression and survival and discuss the unique features of the PP5 structure that differentiate it from other phosphoprotein phosphatase (PPP) family members and make it an attractive therapeutic target.

Cofilin dysregulation alters actin turnover in frataxin-deficient neurons.

Abnormalities in actin cytoskeleton have been linked to Friedreich's ataxia (FRDA), an inherited peripheral neuropathy characterised by an early loss of neurons in dorsal root ganglia (DRG) among other clinical symptoms. Despite all efforts to date, we still do not fully understand the molecular events that contribute to the lack of sensory neurons in FRDA. We studied the adult neuronal growth cone (GC) at the cellular and molecular level to decipher the connection between frataxin and actin cytoskeleton in DRG neurons of the well-characterised YG8R Friedreich's ataxia mouse model. Immunofluorescence studies in primary cultures of DRG from YG8R mice showed neurons with fewer and smaller GCs than controls, associated with an inhibition of neurite growth. In frataxin-deficient neurons, we also observed an increase in the filamentous (F)-actin/monomeric (G)-actin ratio (F/G-actin ratio) in axons and GCs linked to dysregulation of two crucial modulators of filamentous actin turnover, cofilin-1 and the actin-related protein (ARP) 2/3 complex. We show how the activation of cofilin is due to the increase in chronophin (CIN), a cofilin-activating phosphatase. Thus cofilin emerges, for the first time, as a link between frataxin deficiency and actin cytoskeleton alterations.

Dullard-mediated Smad1/5/8 inhibition controls mouse cardiac neural crest cells condensation and outflow tract septation.

The establishment of separated pulmonary and systemic circulation in vertebrates, via cardiac outflow tract (OFT) septation, is a sensitive developmental process accounting for 10% of all congenital anomalies. Neural Crest Cells (NCC) colonising the heart condensate along the primitive endocardial tube and force its scission into two tubes. Here, we show that NCC aggregation progressively decreases along the OFT distal-proximal axis following a BMP signalling gradient. Dullard, a nuclear phosphatase, tunes the BMP gradient amplitude and prevents NCC premature condensation. Dullard maintains transcriptional programs providing NCC with mesenchymal traits. It attenuates the expression of the aggregation factor Sema3c and conversely promotes that of the epithelial-mesenchymal transition driver Twist1. Altogether, Dullard-mediated fine-tuning of BMP signalling ensures the timed and progressive zipper-like closure of the OFT by the NCC and prevents the formation of a heart carrying the congenital abnormalities defining the tetralogy of Fallot.

Role of SSH1 in colorectal cancer prognosis and tumor progression.

BACKGROUND AND AIM: Slingshot 1 protein (SSH1) plays a critical role in cytoskeleton dynamic regulation. Increasing evidence suggest that SSH1 expression is upregulated in several cancers and relates to tumor progression and drug resistance. Here, we evaluated the role of SSH1 in colorectal cancer (CRC) development and its prognostic value in patients with CRC. METHODS: SSH1 expression was examined by quantitative real-time polymerase chain reaction, western blot analysis, or immunohistochemistry. The association between SSH1 expression and clinical characteristics and prognosis was evaluated. Stable SSH1 knockdown cells were used for in vitro assays and xenograft models. Correlation between SSH1 expression and epithelial-mesenchymal transition (EMT) was analyzed by western blot and online data analysis. RESULTS: SSH1 expression was upregulated in cancer tissue compared with paired non-cancerous tissue in patients with CRC. SSH1 expression level in CRC tissue was associated with tumor stage, lymph node metastasis, and correlated with poor prognosis as indicated by univariate and multivariate analyses. In vitro, loss of SSH1 impaired colony formation, migration, and invasion of CRC cells. In vivo data suggest that SSH1 could promote the progression and metastasis of CRC. Interestingly, E-cadherin, ZEB1, and Snail, which are markers of EMT, had a significant expression correlation with SSH1. CONCLUSIONS: SSH1 expression is associated with CRC progression and predicts poor prognosis. SSH1 may promote CRC tumor progression by regulating EMT.

PP2A and microtubules function in 5-aminolevulinic acid-mediated H2 O2 signaling in Arabidopsis guard cells.

5-aminolevulinic acid (ALA), a plant growth regulator with great application potential in agriculture and horticulture, induces stomatal opening and inhibits stomatal closure by decreasing guard cell H2 O2 . However, the mechanisms behind ALA-decreased H2 O2 in guard cells are not fully understood. Here, using type 2A protein phosphatase (PP2A) inhibitors, microtubule-stabilizing/disrupting drugs and green fluorescent protein-tagged α-tubulin 6 transgenic Arabidopsis (GFP-TUA6), we find that PP2A and cortical microtubules (MTs) are involved in ALA-regulated stomatal movement. Then, we analyze stomatal responses of Arabidopsis overexpressing C2 catalytic subunit of PP2A (PP2A-C2) and pp2a-c2 mutant to ALA and abscisic acid (ABA) under both light and dark conditions, and show that PP2A-C2 participates in ALA-induced stomatal movement. Furthermore, using pharmacological methods and confocal studies, we reveal that PP2A and MTs function upstream and downstream, respectively, of H2 O2 in guard cell signaling. Finally, we demonstrate the role of H2 O2 -mediated microtubule arrangement in ALA inhibiting ABA-induced stomatal closure. Our findings indicate that MTs regulated by PP2A-mediated H2 O2 decreasing play an important role in ALA guard cell signaling, revealing new insights into stomatal movement regulation.

Identification of PP2A and S6 Kinase as Modifiers of Leucine-Rich Repeat Kinase-Induced Neurotoxicity.

Mutations in LRRK2 are currently recognized as the most common monogenetic cause of Parkinsonism. The elevation of kinase activity of LRRK2 that frequently accompanies its mutations is widely thought to contribute to its toxicity. Accordingly, many groups have developed LRRK2-specific kinase inhibitors as a potential therapeutic strategy. Given that protein phosphorylation is a reversible event, we sought to elucidate the phosphatase(s) that can reverse LRRK2-mediated phosphorylation, with the view that targeting this phosphatase(s) may similarly be beneficial. Using an unbiased RNAi phosphatase screen conducted in a Drosophila LRRK2 model, we identified PP2A as a genetic modulator of LRRK2-induced neurotoxicity. Further, we also identified ribosomal S6 kinase (S6K), a target of PP2A, as a novel regulator of LRRK2 function. Finally, we showed that modulation of PP2A or S6K activities ameliorates LRRK2-associated disease phenotype in Drosophila.

Protein phosphatase 6 is a key factor regulating spermatogenesis.

Protein phosphatase 6 (PP6) is a member of the PP2A-like subfamily, which plays a critical role in many fundamental cellular processes. We recently reported that PP6 is essential for female fertility. Here, we report that PP6 is involved in meiotic recombination and that germ cell-specific deletion of PP6 by Stra8-Cre causes defective spermatogenesis. The PP6-deficient spermatocytes were arrested at the pachytene stage and defects in DSB repair and crossover formation were observed, indicating that PP6 facilitated meiotic double-stranded breaks (DSB) repair. Further investigations revealed that depletion of PP6 in the germ cells affected chromatin relaxation, which was dependent on MAPK pathway activity, consequently preventing programmed DSB repair factors from being recruited to proper positions on the chromatin. Taken together, our results demonstrate that PP6 has an important role in meiotic recombination and male fertility.

Multiple Functions of the Essential Gene PpV in Drosophila Early Development.

Protein phosphatase V (PpV) encodes the Drosophila homolog of the evolutionarily conserved Protein Phosphatase 6 (PP6). The physiological and developmental functions of PpV/PP6 have not been well characterized due to lack of a genetically defined mutant. Here, we identified a PpV non-sense mutation and describe multiple mutant phenotypes in oogenesis and early embryogenesis. Specifically, we found that the defects in chromosome segregation during nuclear cycles are related to AuroraA function, which is consistent with the interaction of PP6 and AuroraA in mammalian cells. Surprisingly, we also identified a PpV function specifically in blastoderm cell cycle but not in cell proliferation in the follicle epithelium or larval wing imaginal discs. Embryos from PpV germline clones frequently undergo an extra nuclear division cycle. By epistasis analysis, we found that PpV functions in parallel with tribbles, but independently of auroraA for the remodeling of the nuclear cycles. Taken together, this study reports novel developmental functions of PpV and provides a framework for further genetic analysis under physiological conditions.

Plasmodium berghei serine/threonine protein phosphatase PP5 plays a critical role in male gamete fertility.

Sexual development in malaria parasites involves multiple signal transduction pathways mediated by reversible protein phosphorylation. Here, we functionally characterised a protein phosphatase, Ser/Thr protein phosphatase 5 (PbPP5), during sexual development of the rodent malaria parasite Plasmodium berghei. The recombinant protein phosphatase domain displayed obvious protein phosphatase activity and was sensitive to PP1/PP2A inhibitors including cantharidic acid (IC50 = 122.2 nM), cantharidin (IC50 = 74.3 nM), endothall (IC50 = 365.5 nM) and okadaic acid (IC50 = 1.3 nM). PbPP5 was expressed in both blood stages and ookinetes with more prominent expression during sexual development. PbPP5 was localised in the cytoplasm of the parasite and highly concentrated beneath the parasite plasma membrane in free merozoites and ookinetes. Targeted deletion of the pbpp5 gene had no influence on asexual blood-stage parasite multiplication or the survival curve of the infected hosts. However, male gamete formation and fertility were severely affected, resulting in almost complete blockade of ookinete conversion and oocyst development in the Δpbpp5 lines. This sexual development defect was rescued by crossing Δpbpp5 with the female defective Δpbs47 parasite line, but not with the male defective Δpbs48/45 line, thus confirming the essential function of the pbpp5 gene in male gamete fertility. Furthermore, the aforementioned PP1/PP2A inhibitors all had inhibitory effects on exflagellation of male gametocytes and ookinete conversion. In particular, endothall, a selective inhibitor of PP2A, completely blocked exflagellation and ookinete conversion at ∼548.3 nM. This study elucidated an essential function of PbPP5 during male gamete development and fertility.

A genetics screen highlights emerging roles for CPL3, RST1 and URT1 in RNA metabolism and silencing.

Post-transcriptional gene silencing (PTGS) is a major mechanism regulating gene expression in higher eukaryotes. To identify novel players in PTGS, a forward genetics screen was performed on an Arabidopsis thaliana line overexpressing a strong growth-repressive gene, ETHYLENE RESPONSE FACTOR6 (ERF6). We identified six independent ethyl-methanesulfonate mutants rescuing the dwarfism of ERF6-overexpressing plants as a result of transgene silencing. Among the causative genes, ETHYLENE-INSENSITIVE5, SUPERKILLER2 and HASTY1 have previously been reported to inhibit PTGS. Notably, the three other causative genes have not, to date, been related to PTGS: UTP:RNA-URIDYLYLTRANSFERASE1 (URT1), C-TERMINAL DOMAIN PHOSPHATASE-LIKE3 (CPL3) and RESURRECTION1 (RST1). We show that these genes may participate in protecting the 3' end of transgene transcripts. We present a model in which URT1, CPL3 and RST1 are classified as PTGS suppressors, as compromisation of these genes provokes the accumulation of aberrant transcripts which, in turn, trigger the production of small interfering RNAs, initiating RNA silencing.