Rm62, a DEAD‐box RNA helicase,complexes with DSP1 in Drosophila embryos

Two main classes of proteins, Polycomb group (PcG) and Trithorax group (TrxG), play a key role in the regulation of homeotic genes. These proteins act in multimeric complexes to remodel chromatin. A third class of proteins named Enhancers of Trithorax and Polycomb (ETP) modulates the activity of TrxG and PcG, but their role remains largely unknown. We previously identified an HMGB‐like protein, DSP1 (Dorsal Switch Protein 1), which was classified as an ETP. Preliminary studies have revealed that DSP1 is involved in multimeric complexes. Here we identify a DEAD‐box RNA helicase, Rm62, as partner of DSP1 in a 250‐kDa complex. Coimmunoprecipitation assays performed on embryo extracts indicate that DSP1 and Rm62 are associated in 3‐ to 12‐h embryos. Furthermore, DSP1 and Rm62 colocalize on polytene chromosomes. Consistent with these results, a mutation in Rm62 enhances a null mutation of dsp1 and also mutations of trxG or PcG, suggesting that Rm62 has characteristics of an ETP. We show here for the first time that an RNA helicase is involved in the maintenance of homeotic genes. genesis 48:244–253, 2010. © 2010 Wiley‐Liss, Inc.     

Pepper protein phosphatase type 2C,CaADIP1 and its interacting partner CaRLP1 antagonistically regulate ABA signalling and drought response

Abscisic acid (ABA) is a key phytohormone that regulates plant growth and developmental processes, including seed germination and stomatal closing. Here, we report the identification and functional characterization of a novel type 2C protein phosphatase, CaADIP1 (Capsicum annuum A BA and D rought‐I nduced P rotein phosphatase 1). The expression of CaADIP1 was induced in pepper leaves by ABA, drought and NaCl treatments. Arabidopsis plants overexpressing CaADIP1 (CaADIP1‐OX) exhibited an ABA‐hyposensitive and drought‐susceptible phenotype. We used a yeast two‐hybrid screening assay to identify CaRLP1 (Capsicum annuum R CAR‐L ike P rotein 1), which interacts with CaADIP1 in the cytoplasm and nucleus. In contrast to CaADIP1‐OX plants, CaRLP1‐OX plants displayed an ABA‐hypersensitive and drought‐tolerant phenotype, which was characterized by low levels of transpirational water loss and increased expression of stress‐responsive genes relative to those of wild‐type plants. In CaADIP1‐OX/CaRLP1‐OX double transgenic plants, ectopic expression of the CaRLP1 gene led to strong suppression of CaADIP1‐induced ABA hyposensitivity during the germinative and post‐germinative stages, indicating that CaADIP1 and CaRLP1 act in the same signalling pathway and CaADIP1 functions downstream of CaRLP1. Our results indicate that CaADIP1 and its interacting partner CaRLP1 antagonistically regulate the ABA‐dependent defense signalling response to drought stress.     

Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs

Abscisic acid (ABA) is a key phytohormone involved in adaption to environmental stress and regulation of plant development. Clade A protein phosphatases type 2C (PP2Cs), such as HAB1, are key negative regulators of ABA signaling in Arabidopsis. To obtain further insight into regulation of HAB1 function by ABA, we have screened for HAB1‐interacting partners using a yeast two‐hybrid approach. Three proteins were identified, PYL5, PYL6 and PYL8, which belong to a 14‐member subfamily of the Bet v1‐like superfamily. HAB1–PYL5 interaction was confirmed using BiFC and co‐immunoprecipitation assays. PYL5 over‐expression led to a globally enhanced response to ABA, in contrast to the opposite phenotype reported for HAB1‐over‐expressing plants. F₂ plants that over‐expressed both HAB1 and PYL5 showed an enhanced response to ABA, indicating that PYL5 antagonizes HAB1 function. PYL5 and other members of its protein family inhibited HAB1, ABI1 and ABI2 phosphatase activity in an ABA‐dependent manner. Isothermal titration calorimetry revealed saturable binding of (+)ABA to PYL5, with K_d values of 1.1 μm or 38 nm in the absence or presence of the PP2C catalytic core of HAB1, respectively. Our work indicates that PYL5 is a cytosolic and nuclear ABA receptor that activates ABA signaling through direct inhibition of clade A PP2Cs. Moreover, we show that enhanced resistance to drought can be obtained through PYL5‐mediated inhibition of clade A PP2Cs.     

The XRN1-regulated RNA helicase activity of YTHDC2 ensures mouse fertility independently of m6A recognition

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Helicase associated 2 domain is essential for helicase activity of RNA helicase A

RNA helicase A (RHA), a DExD/H box protein, plays critical roles in a wide variety of cellular or viral functions. RHA contains a conserved core helicase domain that is flanked by five other domains. Two double-stranded RNA binding domains (dsRBD1 and dsRBD2) are at the N-terminus, whereas HA2 (helicase associated 2), OB-fold (oligonucleotide- or oligosaccharide-binding fold), and RGG (repeats of arginine and glycine–glycine residues) domains are at the C-terminus. The role of these domains in the helicase activity of RHA is still elusive due to the difficulty of obtaining enzymatically active mutant RHA. Here, we purified a series of mutant RHAs containing deletions in either N-terminus or C-terminus. Analysis of these mutant RHAs reveals that the dsRBDs are not required for RNA unwinding, but can enhance the helicase activity by promoting the binding of RHA to substrate RNA. In contrast, deletion of C-terminal domains including RGG, OB-fold, and HA2 does not significantly affect the binding of RHA to substrate RNA. However, HA2 is essential for the RNA unwinding by RHA whereas the RGG and OB-fold are dispensable. The results indicate that the core helicase domain alone is not enough for RHA to execute the unwinding activity.     

The structure of Arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases.     

Protein phosphatase type 2A,PP2A,is involved in degradation of gp130

The interleukin-6 (IL-6) stimulates growth in cells such as multiple myeloma and B-cell plasmacytomas/hybridomas, while it inhibits growth in several myeloid leukemia cells. The IL-6 receptor has subunit called gp130. It was reported that Ser-782 of gp130 is phosphorylated by unidentified kinase(s) in cell extracts, and level of gp130 (S782A) transiently expressed on the cell surface of COS-7 is 6-times higher than that of the wild type. These results motivated us to analyze whether the phosphorylation of gp130 at Ser-782 is involved in its degradation or not. In this study, we demonstrated here that treatment of HepG2 cells with okadaic acid (OA), a potent inhibitor for PP2A, promotes phosphorylation of gp130 at Ser-782 and degradation of gp130. MG115, a proteasome inhibitor, suppressed this degradation. These effects of OA could not be replaced with tautomycetin (TC), an inhibitor for PP1. Purified PP2A dephosphorylated phospho-Ser-782 of gp130 in vitro. IL-6-induced activation of Stat3 was suppressed by preincubation of the cells with OA, suggesting that the IL-6 signaling pathway was blocked by OA through degradation of gp130. Taken together, present results strongly suggest that degradation of gp130 is regulated through a phosphorylation-dephosphorylation mechanism in which PP2A is crucially involved and that gp130 is a potential therapeutic target in cancers. (Mol Cell Biochem 269: 183–187, 2005)     

ABA inhibits myristoylation and induces shuttling of the RGLG1 E3 ligase to promote nuclear degradation of PP2CA

Hormone‐ and stress‐induced shuttling of signaling or regulatory proteins is an important cellular mechanism to modulate hormone signaling and cope with abiotic stress. Hormone‐induced ubiquitination plays a crucial role to determine the half‐life of key negative regulators of hormone signaling. For ABA signaling, the degradation of clade‐A PP 2Cs, such as PP 2 CA or ABI 1, is a complementary mechanism to PYR / PYL / RCAR ‐mediated inhibition of PP 2C activity. ABA promotes the degradation of PP 2 CA through the RGLG 1 E3 ligase, although it is not known how ABA enhances the interaction of RGLG 1 with PP 2 CA given that they are predominantly found in the plasma membrane and the nucleus, respectively. We demonstrate that ABA modifies the subcellular localization of RGLG 1 and promotes nuclear interaction with PP 2 CA . We found RGLG 1 is myristoylated in vivo , which facilitates its attachment to the plasma membrane. ABA inhibits the myristoylation of RGLG 1 through the downregulation of N‐myristoyltransferase 1 ( NMT 1 ) and promotes nuclear translocation of RGLG 1 in a cycloheximide‐insensitive manner. Enhanced nuclear recruitment of the E3 ligase was also promoted by increasing PP 2 CA protein levels and the formation of RGLG 1–receptor–phosphatase complexes. We show that RGLG 1 ^Gly2Ala mutated at the N‐terminal myristoylation site shows constitutive nuclear localization and causes an enhanced response to ABA and salt or osmotic stress. RGLG 1/5 can interact with certain monomeric ABA receptors, which facilitates the formation of nuclear complexes such as RGLG 1– PP 2 CA – PYL 8. In summary, we provide evidence that an E3 ligase can dynamically relocalize in response to both ABA and increased levels of its target, which reveals a mechanism to explain how ABA enhances RGLG 1– PP 2 CA interaction and hence PP 2 CA degradation.     

An Arabidopsis homolog of importin β1 is required for ABA response and drought tolerance

The import of proteins into the nucleus in response to drought is critical for mediating the reprogramming of gene expression that leads to drought tolerance. However, regulatory mechanisms involved in nuclear protein import remain largely unknown. Here, we have identified an Arabidopsis gene (AtKPNB1) as a homolog of human KPNB1 (importin β1). AtKPNB1 was expressed in multiple organs, and the protein was localized in the cytoplasm and nucleus. AtKPNB1 was able to facilitate nuclear import of a model protein. Null mutation of AtKPNB1 delayed development under normal growth conditions and increased sensitivity to abscisic acid (ABA) during seed germination and cotyledon development. Inactivation of AtKPNB1 increased stomatal closure in response to ABA, reduced the rate of water loss, and substantially enhanced drought tolerance. AtKPNB1 interacted with several importin α proteins, nucleoporin AtNUP62, and the Arabidopsis Ran proteins. Inactivation of AtKPNB1 did not affect the ABA responsiveness or the expression level or subcellular localization of ABI1, ABI2 or ABI5, key regulators of the ABA signaling pathway. Moreover, phenotypic analysis of epistasis revealed that AtKPNB1 modulates the ABA response and drought tolerance through a pathway that is independent of ABI1 and ABI5. Collectively, our results show that AtKPNB1 is an Arabidopsis importin β that functions in ABA signaling.     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.     

SIRT3, PP2A and TTP protein stability in the presence of TNF‐α on vincristine‐induced apoptosis of leukaemia cells

The contribution of vincristine (VCR)‐induced microtubule destabilization to evoke apoptosis in cancer cells remains to be resolved. Thus, we investigated the cytotoxic mechanism of VCR on U937 and HL‐60 human leukaemia cell lines. We discovered that VCR treatment resulted in the up‐regulation of TNF‐α expression and activation of the death receptor pathway, which evoked apoptosis of U937 cells. Moreover, VCR induced microtubule destabilization and mitotic arrest. VCR treatment down‐regulated SIRT3, and such down‐regulation caused mitochondrial ROS to initiate phosphorylation of p38 MAPK. p38 MAPK suppressed MID1‐modulated degradation of the protein phosphatase 2A (PP2A) catalytic subunit. The SIRT3‐ROS‐p38 MAPK‐PP2A axis inhibited tristetraprolin (TTP)‐controlled TNF‐α mRNA degradation, consequently, up‐regulating TNF‐α expression. Restoration of SIRT3 and TTP expression, or inhibition of the ROS‐p38 MAPK axis increased the survival of VCR‐treated cells and repressed TNF‐α up‐regulation. In contrast to suppression of the ROS‐p38 MAPK axis, overexpression of SIRT3 modestly inhibited the effect of VCR on microtubule destabilization and mitotic arrest in U937 cells. Apoptosis of HL‐60 cells, similarly, went through the same pathway. Collectively, our data indicate that the SIRT3‐ROS‐p38 MAPK‐PP2A‐TTP axis modulates TNF‐α expression, which triggers apoptosis of VCR‐treated U937 and HL‐60 cells. We also demonstrate that the apoptotic signalling is not affected by VCR‐elicited microtubule destabilization.     

A more cost effective and rapid high percentage germ‐line transmitting chimeric mouse generation procedure via microinjection of 2‐cell, 4‐cell,and 8‐cell embryos with ES and iPS cells

The long‐standing traditional method of delivering embryonic stem (ES) cells adjacent to the inner cell mass (ICM) of blastocysts to generate chimeras improved with the advent of laser‐ or Piezo assisted 8‐cell embryo microinjection. Building on this technology but omitting either the laser or the Piezo to penetrate the zona pellucida and making use of earlier embryonic stages (2‐cell and 4‐cell), we were able to significantly speed up and economize our ES cell microinjection and chimera production throughput. We demonstrate here that embryonic (ES) and induced pluripotent stem (iPS) cells can stay fully pluripotent when delivered into 2‐cell‐ and 4‐cell‐stage embryos, long before they would naturally be incorporated into the ICM of a blastocyst (E3.5) and give rise to high percentage and germline transmitting chimeras. genesis 48:394–399, 2010. © 2010 Wiley‐Liss, Inc.     

Altered modulation of lamin A/C‐HDAC2 interaction and p21 expression during oxidative stress response in HGPS

Defects in stress response are main determinants of cellular senescence and organism aging. In fibroblasts from patients affected by Hutchinson–Gilford progeria, a severe LMNA‐linked syndrome associated with bone resorption, cardiovascular disorders, and premature aging, we found altered modulation of CDKN1A, encoding p21, upon oxidative stress induction, and accumulation of senescence markers during stress recovery. In this context, we unraveled a dynamic interaction of lamin A/C with HDAC2, an histone deacetylase that regulates CDKN1A expression. In control skin fibroblasts, lamin A/C is part of a protein complex including HDAC2 and its histone substrates; protein interaction is reduced at the onset of DNA damage response and recovered after completion of DNA repair. This interplay parallels modulation of p21 expression and global histone acetylation, and it is disrupted by LMNAmutations leading to progeroid phenotypes. In fact, HGPS cells show impaired lamin A/C‐HDAC2 interplay and accumulation of p21 upon stress recovery. Collectively, these results link altered physical interaction between lamin A/C and HDAC2 to cellular and organism aging. The lamin A/C‐HDAC2 complex may be a novel therapeutic target to slow down progression of progeria symptoms.