Identification of Factors Regulating Poly(A) Tail Synthesis and Maturation

ABSTRACT Posttranscriptional maturation of the 3′ end of eukaryotic pre-mRNAs occurs as a three-step pathway involving site-specific cleavage, polymerization of a poly(A) tail, and trimming of the newly synthesized tail to its mature length. While most of the factors essential for catalyzing these reactions have been identified, those that regulate them remain to be characterized. Previously, we demonstrated that the yeast protein Pbp1p associates with poly(A)-binding protein (Pab1p) and controls the extent of mRNA polyadenylation. To further elucidate the function of Pbp1p, we conducted a two-hybrid screen to identify factors with which it interacts. Five genes encoding putative Pbp1p-interacting proteins were identified, including (i) FIR1/PIP1 and UFD1/PIP3, genes encoding factors previously implicated in mRNA 3′-end processing; (ii) PBP1 itself, confirming directed two-hybrid results and suggesting that Pbp1p can multimerize; (iii) DIG1, encoding a mitogen-activated protein kinase-associated protein; and (iv) PBP4 (YDL053C), a previously uncharacterized gene. In vitro polyadenylation reactions utilizing extracts derived from fir1Δ and pbp1Δ cells and from cells lacking the Fir1p interactor, Ref2p, demonstrated that Pbp1p, Fir1p, and Ref2p are all required for the formation of a normal-length poly(A) tail on precleaved CYC1 pre-mRNA. Kinetic analyses of the respective polyadenylation reactions indicated that Pbp1p is a negative regulator of poly(A) nuclease (PAN) activity and that Fir1p and Ref2p are, respectively, a positive regulator and a negative regulator of poly(A) synthesis. We suggest a model in which these three factors and Ufd1p are part of a regulatory complex that exploits Pab1p to link cleavage and polyadenylation factors of CFIA and CFIB (cleavage factors IA and IB) to the polyadenylation factors of CPF (cleavage and polyadenylation factor).

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Yeast Pab1 interacts with Rna15 and participates in the control of the poly(A) tail length in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.3694 ◽

1997 ◽

Vol 17 (7) ◽

pp. 3694-3701 ◽

Cited By ~ 78

Author(s):

N Amrani ◽

M Minet ◽

M Le Gouar ◽

F Lacroute ◽

F Wyers

Keyword(s):

Wild Type Strain ◽

Tail Length ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Wild Type ◽

Cleavage And Polyadenylation ◽

Two Hybrid ◽

Polyadenylation Factor

In Saccharomyces cerevisiae, the single poly(A) binding protein, Pab1, is the major ribonucleoprotein associated with the poly(A) tails of mRNAs in both the nucleus and the cytoplasm. We found that Pab1 interacts with Rna15 in two-hybrid assays and in coimmunoprecipitation experiments. Overexpression of PAB1 partially but specifically suppressed the rna15-2 mutation in vivo. RNA15 codes for a component of the cleavage and polyadenylation factor CF I, one of the four factors needed for pre-mRNA 3'-end processing. We show that Pab1 and CF I copurify in anion-exchange chromatography. These data suggest that Pab1 is physically associated with CF I. Extracts from a thermosensitive pab1 mutant and from a wild-type strain immunoneutralized for Pab1 showed normal cleavage activity but a large increase in poly(A) tail length. A normal tail length was restored by adding recombinant Pab1 to the mutant extract. The longer poly(A) tails were not due to an inhibition of exonuclease activities. Pab1 has previously been implicated in the regulation of translation initiation and in cytoplasmic mRNA stability. Our data indicate that Pab1 is also a part of the 3'-end RNA-processing complex and thus participates in the control of the poly(A) tail lengths during the polyadenylation reaction.

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Flumequine-Mediated Upregulation of p38 MAPK and JNK Results in Melanogenesis in B16F10 Cells and Zebrafish Larvae

Biomolecules ◽

10.3390/biom9100596 ◽

2019 ◽

Vol 9 (10) ◽

pp. 596 ◽

Cited By ~ 4

Author(s):

Wisurumuni Arachchilage Hasitha Maduranga Karunarathne ◽

Ilandarage Menu Neelaka Molagoda ◽

Myung Sook Kim ◽

Yung Hyun Choi ◽

Matan Oren ◽

...

Keyword(s):

P38 Mapk ◽

Negative Regulator ◽

Mitogen Activated Protein Kinase ◽

Melanin Content ◽

Melanin Pigmentation ◽

Zebrafish Larvae ◽

B16f10 Cells ◽

Mitogen Activated Protein

Flumequine is a well-known second generation quinolone antibiotic that induces phototoxicity. However, the effect of flumequine on skin melanogenesis is unclear. Therefore, we, for the first time, investigated whether flumequine regulates melanogenesis. The present study showed that flumequine slightly inhibited in vitro mushroom tyrosinase activity but significantly increased extracellular and intracellular melanin content in B16F10 cells and promoted the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. Additionally, flumequine remarkably increased melanin pigmentation in zebrafish larvae without any toxicity. We also found that flumequine stimulated p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation; inhibition of p38 MAPK and JNK resulted in significant downregulation of extracellular and intracellular melanin content in B16F10 cells and pigmentation of zebrafish larvae accompanied with suppression of MITF and tyrosinase expression, indicating that flumequine-mediated p38 and JNK promote melanogenesis in vitro and in vivo. According to the molecular docking prediction, flumequine targeted dual-specificity MAPK phosphatase 16 (DUSP16), which is a major negative regulator of p38 MAPK and JNK. Our findings demonstrate that flumequine induces an increase in melanin content in B16F10 cells and zebrafish larvae by activating p38 MAPK and JNK. These data show the potential of flumequine for use as an anti-vitiligo agent.

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Rck2 Kinase Is a Substrate for the Osmotic Stress-Activated Mitogen-Activated Protein Kinase Hog1

Molecular and Cellular Biology ◽

10.1128/mcb.20.11.3887-3895.2000 ◽

2000 ◽

Vol 20 (11) ◽

pp. 3887-3895 ◽

Cited By ~ 113

Author(s):

Elizabeth Bilsland-Marchesan ◽

Joaquín Ariño ◽

Haruo Saito ◽

Per Sunnerhagen ◽

Francesc Posas

Keyword(s):

Protein Kinase ◽

Osmotic Stress ◽

Mitogen Activated Protein Kinase ◽

Yeast Cells ◽

Dependent Manner ◽

Mapk Activation ◽

Mitogen Activated Protein ◽

Two Hybrid

ABSTRACT Exposure of yeast cells to increases in extracellular osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK). Activation of Hog1 MAPK results in induction of a set of osmoadaptive responses, which allow cells to survive in high-osmolarity environments. Little is known about how the MAPK activation results in induction of these responses, mainly because no direct substrates for Hog1 have been reported. We conducted a two-hybrid screening using Hog1 as a bait to identify substrates for the MAPK, and the Rck2 protein kinase was identified as an interactor for Hog1. Both two-hybrid analyses and coprecipitation assays demonstrated that Hog1 binds strongly to the C-terminal region of Rck2. Upon osmotic stress, Rck2 was phosphorylated in vivo in a Hog1-dependent manner. Furthermore, purified Hog1 was able to phosphorylate Rck2 when activated both in vivo and in vitro. Rck2 phosphorylation occurred specifically at Ser519, a residue located within the C-terminal putative autoinhibitory domain. Interestingly, phosphorylation at Ser519 by Hog1 resulted in an increase of Rck2 kinase activity. Overexpression of Rck2 partially suppressed the osmosensitive phenotype of hog1Δ and pbs2Δ cells, suggesting that Rck2 is acting downstream of Hog1. Consistently, growth arrest caused by hyperactivation of the Hog1 MAPK was abolished by deletion of the RCK2 gene. Furthermore, overexpression of a catalytically impaired (presumably dominant inhibitory) Rck2 kinase resulted in a decrease of osmotolerance in wild-type cells but not in hog1Δ cells. Taken together, our data suggest that Rck2 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK upon osmotic stress.

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Ptc1, a Type 2C Ser/Thr Phosphatase, Inactivates the HOG Pathway by Dephosphorylating the Mitogen-Activated Protein Kinase Hog1

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.51-60.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 51-60 ◽

Cited By ~ 136

Author(s):

Janel Warmka ◽

Jennifer Hanneman ◽

Ji Lee ◽

Dipesh Amin ◽

Irene Ota

Keyword(s):

Protein Kinase ◽

Osmotic Stress ◽

Metal Ion ◽

Mapk Pathway ◽

Negative Regulator ◽

Mitogen Activated Protein Kinase ◽

Hog Pathway ◽

Mitogen Activated Protein

ABSTRACT The HOG (high-osmolarity glycerol) mitogen-activated protein kinase (MAPK) pathway regulates the osmotic stress response in the yeast Saccharomyces cerevisiae. Three type 2C Ser/Thr phosphatases (PTCs), Ptc1, Ptc2, and Ptc3, have been isolated as negative regulators of this pathway. Previously, multicopy expression of PTC1 and PTC3 was shown to suppress lethality of the sln1Δ strain due to hyperactivation of the HOG pathway. In this work, we show thatPTC2 also suppresses sln1Δ lethality. Furthermore, the phosphatase activity of these PTCs was needed for suppression, as mutation of a conserved Asp residue, likely to coordinate a metal ion, inactivated PTCs. Further analysis of Ptc1 function in vivo showed that it inactivates the MAPK, Hog1, but not the MEK, Pbs2. In the wild type, Hog1 kinase activity increased transiently, ∼12-fold in response to osmotic stress, while overexpression of PTC1 limited activation to ∼3-fold. In contrast, overexpression of PTC1 did not inhibit phosphorylation of Hog1 Tyr in the phosphorylation lip, suggesting that Ptc1 does not act on Pbs2. Deletion of PTC1 also strongly affected Hog1, leading to high basal Hog1 activity and sustained Hog1 activity in response to osmotic stress, the latter being consistent with a role for Ptc1 in adaptation. In vitro, Ptc1 but not the metal binding site mutant, Ptc1D58N, inactivated Hog1 by dephosphorylating the phosphothreonine but not the phosphotyrosine residue in the phosphorylation lip. Consistent with its role as a negative regulator of Hog1, which accumulates in the nucleus upon activation, Ptc1 was found in both the nucleus and the cytoplasm. Thus, one function of Ptc1 is to inactivate Hog1.

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Interaction of the E1A Oncoprotein with Yak1p, a Novel Regulator of Yeast Pseudohyphal Differentiation, and Related Mammalian Kinases

Molecular Biology of the Cell ◽

10.1091/mbc.12.3.699 ◽

2001 ◽

Vol 12 (3) ◽

pp. 699-710 ◽

Cited By ~ 44

Author(s):

Zhiying Zhang ◽

M. Mitchell Smith ◽

Joe S. Mymryk

Keyword(s):

Protein Kinase ◽

Signaling Pathways ◽

Mammalian Cells ◽

Terminal Region ◽

Negative Regulator ◽

Mitogen Activated Protein Kinase ◽

Adenovirus E1a ◽

Mitogen Activated Protein ◽

Pseudohyphal Differentiation

The C-terminal portion of adenovirus E1A suppresses ras-induced metastasis and tumorigenicity in mammalian cells; however, little is known about the mechanisms by which this occurs. In the simple eukaryote Saccharomyces cerevisiae, Ras2p, the homolog of mammalian h-ras, regulates mitogen-activated protein kinase (MAPK) and cyclic AMP-dependent protein kinase A (cAMP/PKA) signaling pathways to control differentiation from the yeast form to the pseudohyphal form. When expressed in yeast, the C-terminal region of E1A induced pseudohyphal differentiation, and this was independent of both the MAPK and cAMP/PKA signaling pathways. Using the yeast two-hybrid system, we identified an interaction between the C-terminal region of E1A and Yak1p, a yeast dual-specificity serine/threonine protein kinase that functions as a negative regulator of growth. E1A also physically interacts with Dyrk1A and Dyrk1B, two mammalian homologs of Yak1p, and stimulates their kinase activity in vitro. We further demonstrate that Yak1p is required in yeast to mediate pseudohyphal differentiation induced by Ras2p-regulated signaling pathways. However, pseudohyphal differentiation induced by the C-terminal region of E1A is largely independent of Yak1p. These data suggest that mammalian Yak1p-related kinases may be targeted by the E1A oncogene to modulate cell growth.

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Ser-3 is important for regulating Mos interaction with and stimulation of mitogen-activated protein kinase kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.4727 ◽

1995 ◽

Vol 15 (9) ◽

pp. 4727-4734 ◽

Cited By ~ 18

Author(s):

M Chen ◽

J A Cooper

Keyword(s):

Protein Kinase ◽

Catalytic Domain ◽

Mitogen Activated Protein Kinase ◽

Early Embryos ◽

Mitogen Activated Protein ◽

Hybrid Test ◽

Two Hybrid ◽

Autophosphorylation Site ◽

Protein Kinase Kinase

Mos is a germ cell-specific serine/threonine protein kinase that activates mitogen-activated protein kinase (MAPK) through MAPK kinase (MKK). In Xenopus oocytes, Mos synthesis is required for progesterone-induced activation of MAPK and maturation promoting factor. Injection of Mos or active MAPK causes mitotic arrest in early embryos, suggesting that Mos also acts via MKK and MAPK to induce the arrest of unfertilized eggs in metaphase of meiosis II. We have investigated whether Mos activity is regulated by phosphorylation. Previous studies have identified Ser-3 as the principal autophosphorylation site. We show that Mos interacts with the catalytic domain of MKK in a Saccharomyces cerevisiae two-hybrid test. Acidic substitutions of the sites phosphorylated by Mos in MKK reduce the interaction, implying that the complex may dissociate after phosphorylation of MKK by Mos. Furthermore, the Mos-MKK interaction requires Mos kinase activity, suggesting that Mos autophosphorylation may be involved in the interaction. Substitution of Ser-3 of Mos with Ala reduces the interaction with MKK and also reduces both the activation of MKK by Mos in vitro and cleavage arrest induced by Mos fusion protein in Xenopus embryos. By contrast, substitution of Ser-3 by Glu, an acidic amino acid that mimics phosphoserine, fosters the Mos interaction with MKK and permits activation of MKK in vitro and Mos-induced cleavage arrest. Moreover, the Glu-3 substitution increases the interaction of a kinase-inactive Mos mutant with MKK. Taken together, these results suggest that an important step in Mos activation involves the phosphorylation at Ser-3, which promotes Mos interaction with and activation of MKK.

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Tollip Regulates Proinflammatory Responses to Interleukin-1 and Lipopolysaccharide

Molecular and Cellular Biology ◽

10.1128/mcb.26.3.735-742.2006 ◽

2006 ◽

Vol 26 (3) ◽

pp. 735-742 ◽

Cited By ~ 128

Author(s):

Arnaud Didierlaurent ◽

Brian Brissoni ◽

Dominique Velin ◽

Natalia Aebi ◽

Aubry Tardivel ◽

...

Keyword(s):

Interleukin 1 ◽

Mapk Signaling ◽

Negative Regulator ◽

Mitogen Activated Protein Kinase ◽

Necrosis Factor Alpha ◽

Mitogen Activated Protein ◽

Proinflammatory Responses ◽

Tlr4 Ligand ◽

Factor Alpha

ABSTRACT Activation of interleukin-1 (IL-1) receptor (IL-1R), Toll-like receptor 2 (TLR2), and TLR4 triggers NF-κB and mitogen-activated protein kinase (MAPK)-dependent signaling, thereby initiating immune responses. Tollip has been implicated as a negative regulator of NF-κB signaling triggered by these receptors in in vitro studies. Here, deficient mice were used to determine the physiological contribution of Tollip to immunity. NF-κB, as well as MAPK, signaling appeared normal in Tollip-deficient cells stimulated with IL-1β or the TLR4 ligand lipopolysaccharide (LPS). Similarly, IL-1β- and TLR-driven activation of dendritic cells and lymphocytes was indistinguishable from wild-type cells. In contrast, the production of the proinflammatory cytokines, IL-6 and tumor necrosis factor alpha was significantly reduced after IL-1β and LPS treatment at low doses but not at lethal doses of LPS. Tollip therefore controls the magnitude of inflammatory cytokine production in response to IL-1β and LPS.

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Biphasic Activation of Aurora-A Kinase during the Meiosis I- Meiosis II Transition in Xenopus Oocytes

Molecular and Cellular Biology ◽

10.1128/mcb.23.5.1703-1716.2003 ◽

2003 ◽

Vol 23 (5) ◽

pp. 1703-1716 ◽

Cited By ~ 32

Author(s):

Chunqi Ma ◽

Cathy Cummings ◽

X. Johné Liu

Keyword(s):

Germinal Vesicle ◽

Mitogen Activated Protein Kinase ◽

Aurora A Kinase ◽

Aurora A ◽

Apparent Paradox ◽

Germinal Vesicle Breakdown ◽

Mitogen Activated Protein ◽

Mrna Polyadenylation ◽

Polyadenylation Factor ◽

Meiosis I

ABSTRACT Xenopus Aurora-A (also known as Eg2) is a member of the Aurora family of mitotic serine/threonine kinases. In Xenopus oocytes, Aurora-A phosphorylates and activates a cytoplasmic mRNA polyadenylation factor (CPEB) and therefore plays a pivotal role in MOS translation. However, hyperphosphorylation and activation of Aurora-A appear to be dependent on maturation-promoting factor (MPF) activation. To resolve this apparent paradox, we generated a constitutively activated Aurora-A by engineering a myristylation signal at its N terminus. Injection of Myr-Aurora-A mRNA induced germinal vesicle breakdown (GVBD) with the concomitant activation of MOS, mitogen-activated protein kinase, and MPF. Myr-Aurora-A-injected oocytes, however, appeared to arrest in meiosis I with high MPF activity and highly condensed, metaphase-like chromosomes but no organized microtubule spindles. No degradation of CPEB or cyclin B2 was observed following GVBD in Myr-Aurora-A-injected oocytes. In the presence of progesterone, the endogenous Aurora-A became hyperphosphorylated and activated at the time of MPF activation. Following GVBD, Aurora-A was gradually dephosphorylated and inactivated before it was hyperphosphorylated and activated again. This biphasic pattern of Aurora-A activation mirrored that of MPF activation and hence may explain meiosis I arrest by the constitutively activated Myr-Aurora-A.

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JSAP1, a Novel Jun N-Terminal Protein Kinase (JNK)-Binding Protein That Functions as a Scaffold Factor in the JNK Signaling Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7539 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7539-7548 ◽

Cited By ~ 195

Author(s):

Michihiko Ito ◽

Katsuji Yoshioka ◽

Mizuho Akechi ◽

Shinya Yamashita ◽

Nobuhiko Takamatsu ◽

...

Keyword(s):

Protein Kinase ◽

Mitogen Activated Protein Kinase ◽

Yeast Two Hybrid ◽

Terminal Protein ◽

Jnk Signaling ◽

Mapk Cascades ◽

Mitogen Activated Protein ◽

Jnk Signaling Pathway ◽

Two Hybrid

ABSTRACT The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38α), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.

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Lnk adaptor protein down-regulates specific Kit-induced signaling pathways in primary mast cells

Blood ◽

10.1182/blood-2008-05-154849 ◽

2008 ◽

Vol 112 (10) ◽

pp. 4039-4047 ◽

Cited By ~ 34

Author(s):

Clotilde Simon ◽

Elisabetta Dondi ◽

Amandine Chaix ◽

Paulo de Sepulveda ◽

Terrance J. Kubiseski ◽

...

Keyword(s):

Mast Cells ◽

Signaling Pathways ◽

Adaptor Protein ◽

Negative Regulator ◽

Mitogen Activated Protein Kinase ◽

Inhibitory Function ◽

Mitogen Activated Protein ◽

And Migration ◽

Carboxy Terminal

Abstract Stem cell factor (SCF) plays critical roles in proliferation, survival, migration, and function of hematopoietic progenitor and mast cells through binding to Kit receptor. Previous studies have implicated the adaptor protein Lnk as an important negative regulator of SCF signaling. However, the molecular mechanism underlying this regulation is unclear. Here, we showed that the Src homology 2 domain (SH2) of Lnk binds directly and preferentially to phosphorylated tyrosine 567 in Kit juxtamembrane domain. Using Lnk−/− bone marrow mast cells (BMMCs) transduced with different Lnk proteins, we demonstrated that Lnk down-regulates SCF-induced proliferation with attenuation of mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase signaling. Furthermore, we showed that Lnk−/− BMMCs displayed increased SCF-dependent migration compared with wild-type cells, revealing a novel Lnk-mediated inhibitory function. This correlated with enhanced Rac and p38 MAPK activation. Finally, we found that Lnk domains and carboxy-terminal tyrosine contribute differently to inhibition of in vitro expansion of hematopoietic progenitors. Altogether, our results demonstrate that Lnk, through its binding to Kit tyrosine 567, negatively modulates specific SCF-dependent signaling pathways involved in the proliferation and migration of primary hematopoietic cells.

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