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Peter S Klein - One of the best experts on this subject based on the ideXlab platform.
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cell cycle remodeling and zygotic gene activation at the Midblastula transition
Advances in Experimental Medicine and Biology, 2017Co-Authors: Jennifer Skirkanich, Maomao Zhang, Michael A Lampson, Peter S KleinAbstract:Following fertilization, vertebrate embryos delay large-scale activation of the zygotic genome from several hours in fish and amphibians to several days in mammals. Externally developing embryos also undergo synchronous and extraordinarily rapid cell divisions that are accelerated by promiscuous licensing of DNA replication origins, absence of gap phases and cell cycle checkpoints, and preloading of the egg with maternal RNAs and proteins needed to drive early development. After a species-specific number of cell divisions, the cell cycle slows and becomes asynchronous, gap phases appear, checkpoint functions are acquired, and large-scale zygotic gene activation begins. These events, along with clearance of maternal RNAs and proteins, define the maternal to zygotic transition and are coordinated at a developmental milestone termed the Midblastula transition (MBT). Despite the relative quiescence of the zygotic genome in vertebrate embryos, genes required for clearance of maternal RNAs and for the initial steps in mesoderm induction are robustly transcribed before MBT. The coordination and timing of the MBT depends on a mechanism that senses the ratio of nuclear to cytoplasmic content as well as mechanisms that are independent of the nuclear–cytoplasm ratio. Changes in chromatin architecture anticipate zygotic gene activation, and maternal transcription factors identified as regulators of pluripotency play critical roles in kick-starting the transition from the proliferative, pluripotent state of the early embryo to the more lineage-committed phase of development after the MBT. This chapter describes the regulation of the cell cycle and the activation of zygotic gene expression before and after the MBT in vertebrate embryos.
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β catenin tcf regulated transcription prior to the Midblastula transition
Development, 2002Co-Authors: Jing Yang, Rachel S Darken, Paul A Wilson, Peter S KleinAbstract:Following fertilization, the zygotic genome in many organisms is quiescent until the Midblastula transition (MBT), when large-scale transcription begins. In Xenopus embryos, for example, transcription is believed to be repressed until the twelfth cell division. Thus, although dorsal-ventral patterning begins during the first cell cycle, little attention has been given to transcriptional regulation in pre-MBT development. We present evidence that regulated transcription begins during early cleavage stages and that the β -catenin—Tcf complex is required for the transcription of the Xenopus nodal genes Xnr5 and Xnr6 as early as the 256-cell stage. Moreover, inhibition of β-catenin/Tcf function can block dorsal development, but only if the inhibition begins early and is maintained throughout pre-MBT stages. Dorsal development can be rescued in ventralized embryos if Tcf-dependent transcription is activated prior to MBT, but activation of Tcf after MBT cannot rescue ventralized embryos, suggesting that β -catenin/Tcf-dependent transcription is required prior to MBT for dorsal-ventral patterning in Xenopus.
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β-Catenin/Tcf-regulated transcription prior to the Midblastula transition
Development, 2002Co-Authors: Jing Yang, Rachel S Darken, Paul A Wilson, Peter S KleinAbstract:Following fertilization, the zygotic genome in many organisms is quiescent until the Midblastula transition (MBT), when large-scale transcription begins. In Xenopus embryos, for example, transcription is believed to be repressed until the twelfth cell division. Thus, although dorsal-ventral patterning begins during the first cell cycle, little attention has been given to transcriptional regulation in pre-MBT development. We present evidence that regulated transcription begins during early cleavage stages and that the β -catenin—Tcf complex is required for the transcription of the Xenopus nodal genes Xnr5 and Xnr6 as early as the 256-cell stage. Moreover, inhibition of β-catenin/Tcf function can block dorsal development, but only if the inhibition begins early and is maintained throughout pre-MBT stages. Dorsal development can be rescued in ventralized embryos if Tcf-dependent transcription is activated prior to MBT, but activation of Tcf after MBT cannot rescue ventralized embryos, suggesting that β -catenin/Tcf-dependent transcription is required prior to MBT for dorsal-ventral patterning in Xenopus.
J C Smith - One of the best experts on this subject based on the ideXlab platform.
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titration of four replication factors is essential for the xenopus laevis Midblastula transition
Science, 2013Co-Authors: Clara Collart, J C Smith, George E Allen, Charles R Bradshaw, Philip ZegermanAbstract:The rapid, reductive early divisions of many metazoan embryos are followed by the Midblastula transition (MBT), during which the cell cycle elongates and zygotic transcription begins. It has been proposed that the increasing nuclear to cytoplasmic (N/C) ratio is critical for controlling the events of the MBT. We show that four DNA replication factors—Cut5, RecQ4, Treslin, and Drf1—are limiting for replication initiation at increasing N/C ratios in vitro and in vivo in Xenopus laevis. The levels of these factors regulate multiple events of the MBT, including the slowing of the cell cycle, the onset of zygotic transcription, and the developmental activation of the kinase Chk1. This work provides a mechanism for how the N/C ratio controls the MBT and shows that the regulation of replication initiation is fundamental for normal embryogenesis.
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the Midblastula transition defines the onset of y rna dependent dna replication in xenopus laevis
Molecular and Cellular Biology, 2011Co-Authors: J C Smith, Clara Collart, Christo P Christov, Torsten KrudeAbstract:Noncoding Y RNAs are essential for the initiation of chromosomal DNA replication in mammalian cell extracts, but their role in this process during early vertebrate development is unknown. Here, we use antisense morpholino nucleotides (MOs) to investigate Y RNA function in Xenopus laevis and zebrafish embryos. We show that embryos in which Y RNA function is inhibited by MOs develop normally until the Midblastula transition (MBT) but then fail to replicate their DNA and die before gastrulation. Consistent with this observation, Y RNA function is not required for DNA replication in Xenopus egg extracts but is required for replication in a post-MBT cell line. Y RNAs do not bind chromatin in karyomeres before MBT, but they associate with interphase nuclei after MBT in an origin recognition complex (ORC)-dependent manner. Y RNA-specific MOs inhibit the association of Y RNAs with ORC, Cdt1, and HMGA1a proteins, suggesting that these molecular associations are essential for Y RNA function in DNA replication. The MBT is thus a transition point between Y RNA-independent and Y RNA-dependent control of vertebrate DNA replication. Our data suggest that in vertebrates Y RNAs function as a developmentally regulated layer of control over the evolutionarily conserved eukaryotic DNA replication machinery.
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smicl is required for phosphorylation of rna polymerase ii and affects 3 end processing of rna at the Midblastula transition in xenopus
Development, 2009Co-Authors: Clara Collart, Joana M Ramis, Thomas A Down, J C SmithAbstract:Smicl (Smad-interacting CPSF 30-like) is an unusual protein that interacts with transcription factors as well as with the cleavage and polyadenylation specificity factor (CPSF). Previous work has shown that Smicl is expressed maternally in the Xenopus embryo and is later required for transcription of Chordin . In this paper we search for additional targets of Smicl. We identify many genes whose onset of expression at the Midblastula transition (MBT) requires Smicl and is correlated with the translocation of Smicl from cytoplasm to nucleus. At least one such gene, Xiro1 , is regulated via 3′-end processing. In searching for a general mechanism by which Smicl might regulate gene expression at the MBT, we have discovered that it interacts with the tail of Rpb1, the largest subunit of RNA polymerase II. Our results show that Smicl is required for the phosphorylation of the Rpb1 tail at serine 2 of the repeated heptapeptide YSPTSPS. This site becomes hyperphosphorylated at the MBT, thus allowing the docking of proteins required for elongation of transcription and RNA processing. Our work links the onset of zygotic gene expression in the Xenopus embryo with the translocation of Smicl from cytoplasm to nucleus, the phosphorylation of Rpb1 and the 3′-end processing of newly transcribed mRNAs.
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nuclear accumulation of smad complexes occurs only after the Midblastula transition in xenopus
Development, 2007Co-Authors: Yasushi Saka, Anja I H Hagemann, Olaf Piepenburg, J C SmithAbstract:Activin and the Nodal-related proteins induce mesendodermal tissues during Xenopus development. These signals act through specific receptors to cause the phosphorylation, at their carboxyl termini, of Smad2 and Smad3. The phosphorylated Smad proteins form heteromeric complexes with Smad4 and translocate into the nucleus to activate the transcription, after the Midblastula transition, of target genes such as Xbra and goosecoid ( gsc ). In this paper we use bimolecular fluorescence complementation (BiFC) to study complex formation between Smad proteins both in vivo and in response to exogenous proteins. The technique has allowed us to detect Smad2-Smad4 heteromeric interactions during normal Xenopus development and Smad2 and Smad4 homo- and heteromers in isolated Xenopus blastomeres. Smad2-Smad2 and Smad2-Smad4 complexes accumulate rapidly in the nuclei of responding cells following Activin treatment, whereas Smad4 homomeric complexes remain cytoplasmic. When cells divide, Smad2-Smad4 complexes associate with chromatin, even in the absence of ligand. Our observation that Smad2-Smad4 complexes accumulate in the nucleus only after the Midblastula transition, irrespective of the stage at which cells were treated with Activin, may shed light on the mechanisms of developmental timing.
Jill C. Sible - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation of claspin is triggered by the nucleocytoplasmic ratio at the xenopus laevis Midblastula transition
Developmental Biology, 2011Co-Authors: Tetsuya Gotoh, Takeo Kishimoto, Jill C. SibleAbstract:At the Xenopus Midblastula transition (MBT), cell cycles lengthen, and checkpoints that respond to damaged or unreplicated DNA are established. The MBT is triggered by a critical nucleocytoplasmic (N/C) ratio; however, the molecular basis for its initiation remains unknown. In egg extracts, activation of Chk1 checkpoint kinase requires the adaptor protein Claspin, which recruits Chk1 for phosphorylation by ATR. At the MBT in embryos, Chk1 is transiently activated to lengthen the cell cycle. We show that Xenopus Claspin is phosphorylated at the MBT at both DNA replication checkpoint-dependent and -independent sites. Further, in egg extracts, Claspin phosphorylation depends on a threshold N/C ratio, but occurs even when ATR is inhibited. Not all phosphorylation that occurs at the MBT is reproduced in egg extracts. Our results identify Claspin as the most upstream molecule in the signaling pathway that responds to the N/C ratio and indicate that Claspin may also respond to an independent timer to trigger the MBT and activation of cell cycle checkpoints.
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altered expression of chk1 disrupts cell cycle remodeling at the Midblastula transition in xenopus laevis embryos
Cell Cycle, 2004Co-Authors: Matthew J Petrus, Ayesha D Carter, Dayna E Wilhelm, Monica Murakami, Nicholas C Kappas, Brian N Wroble, Jill C. SibleAbstract:Studies in several model systems, including Xenopus laevis oocytes and embryos, have indicated that the checkpoint kinase, Chk1, is required for early development, even in the absence of damaged or unreplicated DNA. Chk1 is transiently activated at the Midblastula transition (MBT) in Xenopus, a time when the cell cycle remodels from rapid embryonic cleavage cycles to longer, more regulated somatic cell cycles. To better understand the role of Chk1 in cell cycle remodeling, mRNA encoding Chk1 was microinjected into 1-cell stage embryos, and the effects on both the MBT and on the expression of several cell cycle regulators were examined. Zygotic transcription, a hallmark of the MBT that depends upon the nucleocytoplasmic (N/C) ratio, was blocked, as was degradation of maternal cyclin E, an event of the MBT that occurs independent of the N/C ratio. Levels of mitotic cyclins were elevated throughout early development, consistent with cell cycle arrest at G2/M. In these embryos, Cdc25A level was low, whereas C...
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loss of xchk1 function triggers apoptosis after the Midblastula transition in xenopus laevis embryos
Mechanisms of Development, 2003Co-Authors: Ayesha D Carter, Jill C. SibleAbstract:Abstract Prior to the Midblastula transition (MBT), Xenopus laevis embryos do not engage cell cycle checkpoints, although overexpression of the kinase XChk1 arrests cell divisions. At the MBT, XChk1 transiently activates and promotes cell cycle lengthening. In this study, endogenous XChk1 was inhibited by the expression of dominant-negative XChk1 (DN-XChk1). Development appeared normal until the early gastrula stage, when cells lost attachments and chromatin condensed. TUNEL and caspase assays indicated these embryos died by apoptosis during gastrulation. Embryos with unreplicated DNA likewise died by apoptosis. Embryos expressing DN-XChk1 proceeded through additional rapid rounds of DNA replication but initiated zygotic transcription on schedule. Therefore, XChk1 is essential in the early Xenopus embryo for cell cycle remodeling and for survival after the MBT.
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Zygotic Transcription Is Required to Block a Maternal Program of Apoptosis in Xenopus Embryos
Developmental Biology, 1997Co-Authors: Jill C. Sible, Andrea L Lewellyn, Julie A Anderson, James L MallerAbstract:Abstract At the Midblastula transition duringXenopusdevelopment, the cell cycle is remodeled, and zygotic transcription is initiated. Additionally, cyclin E1 is degraded at the Midblastula transition independently of protein synthesis, the number of cell cycles, and the nuclear-to-cytoplasmic ratio. In the studies reported here, cell cycles were delayed by transient inhibition of protein synthesis with cycloheximide (100 μg/ml) prior to the Midblastula transition. Even after reaccumulation of mitotic cyclins and resumption of cell divisions, cycloheximide-treated embryos did not resume DNA synthesis, failed to initiate transcription, and synchronously became apoptotic before the gastrula stage. These results were independent of the stage at which embryos were treated or the duration of treatment. Inhibition of zygotic transcription with α-amanitin also induced apoptosis. These data suggest that a developmental checkpoint at the Midblastula transition is maternally regulated and can trigger apoptosis. Apoptosis induced by cycloheximide or α-amanitin was blocked by injection of RNA encodingXenopusBcl-2, suggesting that this maternal program is normally blocked by expression of an apoptotic inhibitor. Embryos pulsed with lower doses of cycloheximide (10 μg/ml) delayed development prior to the Midblastula transition but resumed DNA synthesis, initiated transcription, and gastrulated normally. This indicates that the apoptotic response is initiated only when delayed embryos are unable to support initiation of zygotic transcription.
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a role for cyclin e cdk2 in the timing of the Midblastula transition in xenopus embryos
Developmental Biology, 1997Co-Authors: Rebecca S Hartley, Andrea L Lewellyn, Jill C. Sible, James L MallerAbstract:Abstract DuringXenopusdevelopment, the early cell cycles consist of rapid oscillations between DNA synthesis and mitosis until completion of the 12th mitotic division. Then the cycle lengthens and becomes asynchronous, zygotic transcription begins, and G phases are established, a period known as the Midblastula transition (MBT). Some aspects of the MBT, such as zygotic transcription, depend on acquisition of a threshold nuclear to cytoplasmic (N/C) ratio, whereas others, such as maternal cyclin E degradation, are independent of nuclear events and appear to be controlled by an autonomous maternal timer. To investigate the function of cyclin E during the early cycles, cyclin E/Cdk2 kinase activity was specifically inhibited in fertilized eggs by a truncated form of theXenopusCdk inhibitor, Xic1 (Δ34Xic1). Δ34Xic1 caused lengthening of the embryonic cell cycles that correlated with increased levels of mitotic cyclins. However, DNA synthesis was not inhibited. Several hallmarks of the MBT were delayed for several hours in Δ34Xic1-injected embryos, including the disappearance of cyclins E and A, the initiation of zygotic transcription, and the reappearance of phosphotyrosine on Cdc2. In both control and Δ34Xic1-injected embryos, cyclin E was degraded after the 12th mitotic division as zygotic transcription began, but experiments with α-amanitin show that cyclin E degradation is not dependent on zygotic transcription. Thus, the length of the early cycles and the timing of maternal cyclin degradation depend upon cyclin E/Cdk2 activity. Neither oscillations in cyclin E/Cdk2 activity during the early cycles nor the disappearance of cyclin E at the MBT were dependent on protein synthesis. These data suggest that cyclin E/Cdk2 is directly linked to an autonomous maternal timer that drives the early embryonic cell cycles until the MBT.
Daniel Levy - One of the best experts on this subject based on the ideXlab platform.
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Altering the levels of nuclear import factors in early Xenopus laevis embryos affects later development.
PLOS ONE, 2019Co-Authors: Predrag Jevtić, Richik Nilay Mukherjee, Pan Chen, Daniel LevyAbstract:More than just a container for DNA, the nuclear envelope carries out a wide variety of critical and highly regulated cellular functions. One of these functions is nuclear import, and in this study we investigate how altering the levels of nuclear transport factors impacts developmental progression and organismal size. During early Xenopus laevis embryogenesis, the timing of a key developmental event, the Midblastula transition (MBT), is sensitive to nuclear import factor levels. How might altering nuclear import factors and MBT timing in the early embryo affect downstream development of the organism? We microinjected X. laevis two-cell embryos with mRNA to increase levels of importin α or NTF2, resulting in differential amounts of nuclear import factors in the two halves of the embryo. Compared to controls, these embryos exhibited delayed gastrulation, curved neural plates, and bent tadpoles with different sized eyes. Furthermore, embryos microinjected with NTF2 developed into smaller froglets compared to control microinjected embryos. We propose that altering nuclear import factors and nuclear size affects MBT timing, cell size, and cell number, subsequently disrupting later development. Thus, altering nuclear import factors early in development can affect function and size at the organismal level.
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Altering nuclear import in early Xenopus laevis embryos affects later development
bioRxiv, 2019Co-Authors: Predrag Jevtić, Daniel LevyAbstract:More than just a container for DNA, the nucleus carries out a wide variety of critical and highly regulated cellular functions. One of these functions is nuclear import, and in this study we investigate how altering nuclear import impacts developmental progression and organismal size. During early Xenopus laevis embryogenesis, the timing of a key developmental event, the Midblastula transition (MBT), is sensitive to nuclear import factor levels. How might altering nuclear import and MBT timing in the early embryo affect downstream development of the organism? We microinjected X. laevis two-cell embryos to increase levels of importin α or NTF2, resulting in differential amounts of nuclear import factors in the two halves of the embryo. Compared to controls, these embryos exhibited delayed gastrulation, curved neural plates, and bent tadpoles with different sized eyes. Furthermore, embryos microinjected with NTF2 developed into smaller froglets compared to control microinjected embryos. We propose that altering nuclear import and size affects MBT timing, cell size, and cell number, subsequently disrupting later development. Thus, altering nuclear import early in development can affect function and size at the organismal level.
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both nuclear size and dna amount contribute to Midblastula transition timing in xenopus laevis
Scientific Reports, 2017Co-Authors: Predrag Jevtic, Daniel LevyAbstract:During early Xenopus laevis embryogenesis both nuclear and cell volumes decrease with the nuclear-to-cytoplasmic (N/C) volume ratio reaching a maximum at the Midblastula transition (MBT). At the MBT, embryonic transcription is upregulated and cell cycles lengthen. Early studies demonstrated a role for the DNA-to-cytoplasmic ratio in the control of MBT timing. By altering nuclear size, we previously showed that the N/C volume ratio also contributes to proper MBT timing. Here we examine the relative contributions of nuclear size and DNA amount to MBT timing by simultaneously altering nuclear size and ploidy in X. laevis embryos. Compared to diploid embryos, haploids exhibited a delay in both zygotic gene expression and cell cycle lengthening, while diploid embryos with increased N/C volume ratios showed early expression of zygotic genes and premature lengthening of cell cycles. Interestingly, haploids with increased N/C volume ratios exhibited an intermediate effect on the timing of zygotic gene expression and cell cycle lengthening. Decreasing nuclear size in post-MBT haploid embryos caused a further delay in cell cycle lengthening and the expression of some zygotic genes. Our data suggest that both the N/C volume ratio and DNA amount contribute to the regulation of MBT timing with neither parameter being dominant.
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nuclear size scaling during xenopus early development contributes to Midblastula transition timing
Current Biology, 2015Co-Authors: Predrag Jevtic, Daniel LevyAbstract:Summary Early Xenopus laevis embryogenesis is a robust system for investigating mechanisms of developmental timing. After a series of rapid cell divisions with concomitant reductions in cell size, the first major developmental transition is the Midblastula transition (MBT), when zygotic transcription begins and cell cycles elongate [1–3]. Whereas the maintenance of a constant nuclear-to-cytoplasmic (N/C) volume ratio is a conserved cellular property [4–7], it has long been recognized that the N/C volume ratio changes dramatically during early Xenopus development [8]. We investigated how changes in nuclear size and the N/C volume ratio during early development contribute to the regulation of MBT timing. Whereas previous studies suggested a role for the N/C volume ratio in MBT timing [1, 9–13], none directly tested the effects of altering nuclear size. In this study, we first quantify blastomere and nuclear sizes in X. laevis embryos, demonstrating that the N/C volume ratio increases prior to the MBT. We then manipulate nuclear volume in embryos by microinjecting different nuclear scaling factors, including import proteins, lamins, and reticulons. Using this approach, we show that increasing the N/C volume ratio in pre-MBT embryos leads to premature activation of zygotic gene transcription and early onset of longer cell cycles. Conversely, decreasing the N/C volume ratio delays zygotic transcription and leads to additional rapid cell divisions. Whereas the DNA-to-cytoplasmic ratio has been implicated in MBT timing [1, 9–18], our data show that nuclear size also contributes to the regulation of MBT timing, demonstrating the functional significance of nuclear size during development.
Clara Collart - One of the best experts on this subject based on the ideXlab platform.
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titration of four replication factors is essential for the xenopus laevis Midblastula transition
Science, 2013Co-Authors: Clara Collart, J C Smith, George E Allen, Charles R Bradshaw, Philip ZegermanAbstract:The rapid, reductive early divisions of many metazoan embryos are followed by the Midblastula transition (MBT), during which the cell cycle elongates and zygotic transcription begins. It has been proposed that the increasing nuclear to cytoplasmic (N/C) ratio is critical for controlling the events of the MBT. We show that four DNA replication factors—Cut5, RecQ4, Treslin, and Drf1—are limiting for replication initiation at increasing N/C ratios in vitro and in vivo in Xenopus laevis. The levels of these factors regulate multiple events of the MBT, including the slowing of the cell cycle, the onset of zygotic transcription, and the developmental activation of the kinase Chk1. This work provides a mechanism for how the N/C ratio controls the MBT and shows that the regulation of replication initiation is fundamental for normal embryogenesis.
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the Midblastula transition defines the onset of y rna dependent dna replication in xenopus laevis
Molecular and Cellular Biology, 2011Co-Authors: J C Smith, Clara Collart, Christo P Christov, Torsten KrudeAbstract:Noncoding Y RNAs are essential for the initiation of chromosomal DNA replication in mammalian cell extracts, but their role in this process during early vertebrate development is unknown. Here, we use antisense morpholino nucleotides (MOs) to investigate Y RNA function in Xenopus laevis and zebrafish embryos. We show that embryos in which Y RNA function is inhibited by MOs develop normally until the Midblastula transition (MBT) but then fail to replicate their DNA and die before gastrulation. Consistent with this observation, Y RNA function is not required for DNA replication in Xenopus egg extracts but is required for replication in a post-MBT cell line. Y RNAs do not bind chromatin in karyomeres before MBT, but they associate with interphase nuclei after MBT in an origin recognition complex (ORC)-dependent manner. Y RNA-specific MOs inhibit the association of Y RNAs with ORC, Cdt1, and HMGA1a proteins, suggesting that these molecular associations are essential for Y RNA function in DNA replication. The MBT is thus a transition point between Y RNA-independent and Y RNA-dependent control of vertebrate DNA replication. Our data suggest that in vertebrates Y RNAs function as a developmentally regulated layer of control over the evolutionarily conserved eukaryotic DNA replication machinery.
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smicl is required for phosphorylation of rna polymerase ii and affects 3 end processing of rna at the Midblastula transition in xenopus
Development, 2009Co-Authors: Clara Collart, Joana M Ramis, Thomas A Down, J C SmithAbstract:Smicl (Smad-interacting CPSF 30-like) is an unusual protein that interacts with transcription factors as well as with the cleavage and polyadenylation specificity factor (CPSF). Previous work has shown that Smicl is expressed maternally in the Xenopus embryo and is later required for transcription of Chordin . In this paper we search for additional targets of Smicl. We identify many genes whose onset of expression at the Midblastula transition (MBT) requires Smicl and is correlated with the translocation of Smicl from cytoplasm to nucleus. At least one such gene, Xiro1 , is regulated via 3′-end processing. In searching for a general mechanism by which Smicl might regulate gene expression at the MBT, we have discovered that it interacts with the tail of Rpb1, the largest subunit of RNA polymerase II. Our results show that Smicl is required for the phosphorylation of the Rpb1 tail at serine 2 of the repeated heptapeptide YSPTSPS. This site becomes hyperphosphorylated at the MBT, thus allowing the docking of proteins required for elongation of transcription and RNA processing. Our work links the onset of zygotic gene expression in the Xenopus embryo with the translocation of Smicl from cytoplasm to nucleus, the phosphorylation of Rpb1 and the 3′-end processing of newly transcribed mRNAs.
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Smicl is required for phosphorylation of RNA polymerase II and affects 3′-end processing of RNA at the Midblastula transition in Xenopus
Development, 2009Co-Authors: Clara Collart, Joana M Ramis, Thomas A Down, James C. SmithAbstract:Smicl (Smad-interacting CPSF 30-like) is an unusual protein that interacts with transcription factors as well as with the cleavage and polyadenylation specificity factor (CPSF). Previous work has shown that Smicl is expressed maternally in the Xenopus embryo and is later required for transcription of Chordin . In this paper we search for additional targets of Smicl. We identify many genes whose onset of expression at the Midblastula transition (MBT) requires Smicl and is correlated with the translocation of Smicl from cytoplasm to nucleus. At least one such gene, Xiro1 , is regulated via 3′-end processing. In searching for a general mechanism by which Smicl might regulate gene expression at the MBT, we have discovered that it interacts with the tail of Rpb1, the largest subunit of RNA polymerase II. Our results show that Smicl is required for the phosphorylation of the Rpb1 tail at serine 2 of the repeated heptapeptide YSPTSPS. This site becomes hyperphosphorylated at the MBT, thus allowing the docking of proteins required for elongation of transcription and RNA processing. Our work links the onset of zygotic gene expression in the Xenopus embryo with the translocation of Smicl from cytoplasm to nucleus, the phosphorylation of Rpb1 and the 3′-end processing of newly transcribed mRNAs.