Gene name - Cyclin B
Cytological map position - 59A
Function - Regulatory subunit of cyclin dependent kinase - G2-M cyclin
Keywords - cell cycle
Symbol - CycB
Genetic map position - 2-
Classification - Cyclin B
Cellular location - nuclear
|Recent literature||Gupte, T. M. (2015). Mitochondrial fragmentation due to inhibition of fusion increases Cyclin B through mitochondrial superoxide radicals. PLoS One 10: e0126829. PubMed ID: 26000631
During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly interesting events are first, mitochondrial hyperfusion during the G1-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented between late G2- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. This study bypassed the 'mitotic mitochondrial fragmentation' checkpoint by inducing fragmented mitochondrial morphology and then measuring the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophila S2R+ cell and cells in the pouch region of wing imaginal disc of Drosophila larvae it was shown that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. These experiments suggest that the inhibition of mitochondrial fusion increases superoxide radical content and leads to the upregulation of cyclin B that culminates in the observed changes in the cell cycle. Evidence is provided for the importance of mitochondrial superoxide in this process. These results provide an insight into the need for mitofusin-degradation during mitosis and also help in understanding the mechanism by which mitofusins may function as tumor suppressors.
|Kotov, A. A., Olenkina, O. M., Kibanov, M. V. and Olenina, L. V. (2016). RNA helicase Belle (DDX3) is essential for male germline stem cell maintenance and division in Drosophila. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 26876306
The present study showed that RNA helicase Belle (DDX3) was required intrinsically for mitotic progression and survival of germline stem cells (GSCs) and spermatogonial cells in the Drosophila melanogaster testes. Deficiency of Belle in the male germline resulted in a strong germ cell loss phenotype. Early germ cells are lost through cell death, whereas somatic hub and cyst cell populations are maintained. The observed phenotype is related to that of the human Sertoli Cell-Only Syndrome caused by the loss of DBY (DDX3) expression in the human testes and results in a complete lack of germ cells with preservation of somatic Sertoli cells. This study found the hallmarks of mitotic G2 delay in early germ cells of the larval testes of bel mutants. Both mitotic cyclins, A and B, are markedly reduced in the gonads of bel mutants. Transcription levels of cycB and cycA decrease significantly in the testes of hypomorph bel mutants. Overexpression of Cyclin B in the germline partially rescues germ cell survival, mitotic progression and fertility in the bel-RNAi knockdown testes. Taken together, these results suggest that a role of Belle in GSC maintenance and regulation of early germ cell divisions is associated with the expression control of mitotic cyclins.
|Flora, P., Schowalter, S., Wong-Deyrup, S., DeGennaro, M., Nasrallah, M. A. and Rangan, P. (2018). Transient transcriptional silencing alters the cell cycle to promote germline stem cell differentiation in Drosophila. Dev Biol 434(1): 84-95. PubMed ID: 29198563
Transcriptional silencing is a conserved process used by embryonic germ cells to repress somatic fate and maintain totipotency and immortality. In Drosophila, this transcriptional silencing is mediated by polar granule component (pgc). This study shows that in the adult ovary, pgc is required for timely germline stem cell (GSC) differentiation. Pgc is expressed transiently in the immediate GSC daughter (pre-cystoblast), where it mediates a pulse of transcriptional silencing. This transcriptional silencing mediated by pgc indirectly promotes the accumulation of Cyclin B (CycB) and cell cycle progression into late-G2 phase, when the differentiation factor bag of marbles (bam) is expressed. Pgc mediated accumulation of CycB is also required for heterochromatin deposition, which protects the germ line genome against selfish DNA elements. These results suggest that transient transcriptional silencing in the pre-cystoblast 're-programs' it away from self-renewal and toward the gamete differentiation program (Flora, 2018).
|Sechi, S., Frappaolo, A., Karimpour-Ghahnavieh, A., Gottardo, M., Burla, R., Di Francesco, L., Szafer-Glusman, E., Schinina, E., Fuller, M. T., Saggio, I., Riparbelli, M. G., Callaini, G. and Giansanti, M. G. (2019). Drosophila Doublefault protein coordinates multiple events during male meiosis by controlling mRNA translation. Development 146(22). PubMed ID: 31645358
During the extended prophase of Drosophila gametogenesis, spermatocytes undergo robust gene transcription and store many transcripts in the cytoplasm in a repressed state, until translational activation of select mRNAs in later steps of spermatogenesis. This study characterized the Drosophila Doublefault (Dbf) protein as a C2H2 zinc-finger protein, primarily expressed in testes, that is required for normal meiotic division and spermiogenesis. Loss of Dbf causes premature centriole disengagement and affects spindle structure, chromosome segregation and cytokinesis. Dbf interacts with the RNA-binding protein Syncrip/hnRNPQ, a key regulator of localized translation in Drosophila. It is proposed that the pleiotropic effects of dbf loss-of-function mutants are associated with the requirement of dbf function for translation of specific transcripts in spermatocytes. In agreement with this hypothesis, Dbf protein binds cyclin B mRNA and is essential for translation of cyclin B in mature spermatocytes.
|Okazaki, R., Yamazoe, K. and Inoue, Y. H. (2020). Nuclear Export of Cyclin B Mediated by the Nup62 Complex Is Required for Meiotic Initiation in Drosophila Males. Cells 9(2). PubMed ID: 31979075
The central channel of the nuclear pore complex plays an important role in the selective transport of proteins between the nucleus and cytoplasm. Previous studies have demonstrated that the depletion of the Nup62 complex, constructing the nuclear pore channel in premeiotic Drosophila cells, resulted in the absence of meiotic cells. This study attempted to understand the mechanism underlying the cell cycle arrest before meiosis. dsRNAs was induced against the nucleoporin mRNAs using the Gal4/UAS system in Drosophila. The cell cycle of the Nup62-depleted cells was arrested before meiosis without CDK1 activation. The ectopic over-expression of CycB, but not constitutively active CDK1, resulted in partial rescue from the arrest. CycB continued to exist in the nuclei of Nup62-depleted cells and cells depleted of exportin encoded by emb. Protein complexes containing CycB, Emb, and Nup62 were observed in premeiotic spermatocytes. CycB, which had temporally entered the nucleus, was associated with Emb, and the complex was transported back to the cytoplasm through the central channel, interacting with the Nup62 complex. It is proposed that CycB is exported with Emb through the channel interacting with the Nup62 complex before the onset of meiosis. The nuclear export ensures the modification and formation of sufficient CycB-CDK1 in the cytoplasm.
|Mouawad, R., Himadewi, P., Kadiyala, D. and Arnosti, D. N. (2020). Selective repression of the Drosophila cyclin B promoter by retinoblastoma and E2F proteins. Biochim Biophys Acta Gene Regul Mech 1863(7): 194549. PubMed ID: 32275964
The Cyclin B1 gene encodes a G2/M cyclin that is deregulated in various human cancers, however, the transcriptional regulation of this gene is incompletely understood. The E2F and retinoblastoma family of proteins are involved in this gene's regulation, but there is disagreement on which of the E2F and retinoblastoma proteins interact with the promoter to regulate this gene. This study dissected the promoter region of the Drosophila CycB gene and studied the role of Rbf and E2F factors in its regulation. This gene exhibits remarkable features that distinguish it from G1/S regulated promoters, such as PCNA. The promoter is comprised of modular elements with dedicated repressor and activator functions, including a segment spanning the first intron that interferes with a 5' activator element. A highly active minimal promoter (-464, +100) is repressed by the Rbf1 retinoblastoma protein, but much more potently repressed by the Rbf2 protein, which has been linked in other studies to control of cell growth genes. Unlike many other cell-cycle genes, which are activated by E2F1 and repressed by E2F2, CycB is potently activated by E2F2, and repressed by E2F1. Although the bulk of Rbf binding is associated with a region 5' of the core promoter, E2F and retinoblastoma proteins functionally interact with the basal promoter region, in part through a conserved E2F site at -80 bp. The specific regulatory requirements of this late cell cycle promoter appear to be linked to the unique activities of E2F and retinoblastoma family members acting on a complex cis-regulatory circuit.
Cyclin B, along with its dimerization partner cdc2, plays a significant role in cell division in Drosophila, but this fact is easy to overlook for several reasons. First, mutational studies can be misleading, with respect to the vital role played by Cyclin B during cell division. A biological fail safe mechanism appears to exist between Cyclin B and Cyclin A: a single mutational deficiency in either of these two cyclins involved in the G2-M transition can be compensated for by the unmutated cyclin. One might reasonably suppose that because Cyclin B is thus replaceable, it is less than essential to the process. The compensation is not 100% effective however; mitotic spindles are abnormal and progression through mitosis is delayed in cyclin B deficient embryos (Knoblich, 1993).
Two other factors mitigate against finding a substantial role for Cyclin B in Drosophila development: first, maternal cyclins are involved in development through mitosis 14, at which time they are degraded. Second, and occuring after this time, the dynamics of String protein limit the rate of mitosis. Despite these limiting factors, Cyclins A and B are produced in G2 both before and after cell cycle 14.
The strongest evidence that Cyclin B is crucial to mitosis is provided by the measurement of cyclin levels throughout the process. During cycles 8-13 a progressive increase in the degradation of cyclins at mitosis leads to increasing oscillations of cdc2 kinase activity. Quantitative measurements indicate that less than 10% of Cyclin A and B are degraded at mitosis 5, and that about 30%, 60%, and 80% are degraded at mitoses 8, 11 and 13 respectively. It appears that cyclin synthesis is limiting for mitoses 10-13 (Edgar, 1994).
During interphase 14, programmed degradation of maternal String protein leads to inhibitory phosphorylation of cdc2 and cell cycle arrest. Subsequently, mitoses 14-16 are triggered by pulses of zygotic string transcription. The use of an N-terminally truncated Cyclin B in Drosophila provides evidence for the role of Cyclin B in timing mitosis. Cyclin A is degraded during metaphase and Cyclin B degradation occurs at approximately the metaphase-anaphase transition (Whitfield, 1990). The N-terminally truncated Cyclin B fails to degrade due to absence of a 'destruction box' in the truncated N-terminal region. The truncated Cyclin B results in mitotic delay at late anaphase (Rimmington, 1994). The gene fizzy, homologous to cdc20 in S. cerevisiae, is required for metaphase-anaphase transition in Drosophila, and is required for normal cyclin A and B degradation. It is suggested that fizzy functions to promote the ubiquitin-dependent proteolytic events that occur during mitosis (Dawson, 1995).
Looking to yeast and to vertebrates for clues as to the role of Cyclin B in mitosis, one finds a bewildering array of information. There appears to be a connection between G1-S cyclins and G2-M cyclins. Cdk2 kinase, the partner for G1-S cyclins, is a positive regulator of cdc2-Cyclin B complexes in Xenopus. When cdk2 kinase activity is inhibited by the cdk-specific inhibitor, a block to mitosis occurs, and inactive cdc2-cyclin B accumulates (Guadagno, 1996). Eukaryotic cells have evolved regulatory mechanisms to ensure the strict alternation of DNA replication and mitosis. Cdc2/cyclin B has a role in preventing re-replication of the genome before mitosis. A Xenopus homolog of S. pombe, cdc21, exhibits cell-cycle dependent chromatin binding and phosphorylation in association with S-phase control. Cdc21 remains bound to chromatin during the initiation of DNA replication and is displaced with the progression of the replication fork. Cytoplasmic cdc21 remains underphosphorylated but at the beginning of mitosis the entire pool of cdc21 is hyperphosphorylated, possibly by the cdc2/cyclin B kinase. These properties identify Xenopus cdc21 as a possible component of the DNA licensing factor (Coué, 1996). For more information about licensing factor see the DNA replication site.
Several targets of cdc2-Cyclin B kinase have been identified. The dimer cdc2-Cyclin B targets Wee1 kinase. Wee1 then inhibits cell division by phosphorylating cdc2. In each cell cycle, the mouse Wee1 kinase is phosphorylated at M-phase resulting in inactivation of Wee kinase. The N-terminal domain or entire molecule is extensively phosphorylated by the cdc2-Cyclin B dimer (Honda, 1995). Drosophila Cyclin B has a consensus cAMP-dependent protein kinase site. Evidence from Xenopus suggests that cyclin degradation and exit from mitosis requires Cyclin B/cdc2-dependent activation of the cAMP-PKA pathway. The concentration of cAMP and the activity of PKA decrease at the onset of mitosis and increase at the transition between mitosis and interphase (Grieco, 1996). Proteins of the mitotic apparatus are direct targets of cdc2-Cyclin B dimer.
A human homolog of Xenopus Eg5, (a kinesin-related motor protein) is implicated in the assembly and dynamics of the mitotic spindle. An evolutionarily conserved cdc2 phosphorylation site in HsEg5 is phosphorylated specifically during mitosis in HeLa cells and in vitro by p34cdc2/Cyclin B. Phosphorylation controls the association of this motor protein with the spindle apparatus (Blangy, 1995).
Cdc2/Cyclin B targets the ubiquitin proteins involved in cyclin destruction. Two specific components are required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase (termed the cyclosome) that purifies as part of an approximately 1500-kDa complex and is active only near the end of mitosis. The cyclosome has been separated from its ultimate upstream activator, cdc2, that activates the cyclosome complex by means of phosphorlyation (Lahav-Baratz, 1995).
The cdc2/Cyclin B heterodimer was first characterized as maturation promoting factor, or MPF. Unfertilized frog eggs manifest cytoplasmic activity that can induce immature oocytes to undergo meiotic maturation. MPF activity drives the events of early mitosis such as nuclear breakdown, chromosome condensation and spindle formation by phosphorylating cellular substrates. While cdc2 (the catalytic subunit of the MPF heterodimer) is required to drive the events of early mitosis, it must be inactivated to allow the events of late mitosis to proceed. The metaphase-anaphase transition is a checkpoint during mitosis. At this juncture the cell is able to monitor the integrity of its spindle before proceeding to inactivate MPF and initiate chromosome separation (Murray, 1992 and references). What is the link between phosphorlyation and this checkpoint? A common mitotic error is the attachment of a chromosome to only one spindle pole rather than to both poles. Even a single such chromosomal error delays anaphase in cells. Recently it has been shown that when the tension associated with proper attachment is absent the kinetochore becomes phosphorylated and anaphase is delayed. It has been proposed that the kinetochore protein dephosphorylation caused by tension is the all-clear signal to the checkpoint. The involvement of Cyclin B/cdc2 in the events surrounding this mitotic checkpoint have yet to be documented, but the fact that Cyclin B/cdc2 is degraded at the metaphase-anaphase transition, suggests that MPF is the direct target of this checkpoint (Nicklas, 1995 and Dawson, 1995).
In the Drosophila embryo, Nanos and Pumilio collaborate to repress the translation of hunchback mRNA in the somatic cytoplasm. Both proteins are also required for repression of maternal Cyclin B mRNA in the germline; it has not been clear whether they act directly on Cyclin B mRNA, and if so, whether regulation in the presumptive somatic and germline cytoplasm proceeds by similar or fundamentally different mechanisms. This report shows that Pumilio and Nanos bind to an element in the 3' UTR to repress Cyclin B mRNA. Regulation of Cyclin B and hunchback differ in two significant respects. (1) Pumilio is dispensable for repression of Cyclin B (but not hunchback) if Nanos is tethered via an exogenous RNA-binding domain. Nanos probably acts, at least in part, by recruiting the CCR4-Pop2-NOT deadenylase complex, interacting directly with the NOT4 subunit. (2) Although Nanos is the sole spatially limiting factor for regulation of hunchback, regulation of Cyclin B requires another Oskar-dependent factor in addition to Nanos. Ectopic repression of Cyclin B in the presumptive somatic cytoplasm causes lethal nuclear division defects. It is suggested that a requirement for two spatially restricted factors is a mechanism for ensuring that Cyclin B regulation is strictly limited to the germline (Kadyrova, 2007).
Thus Nos and Pum directly regulate maternal CycB mRNA, binding to an NRE in its 3' UTR. Differences in the spacing and arrangement of protein-binding sites within the hb and CycB NREs appear to account for the regulation of hb but not CycB by Brat. For regulation of CycB, the main function of Pum is to recruit Nos, a role that can be bypassed by tethering Nos via an exogenous RNA-binding domain. CycB-bound Nos is then likely to act, at least in part, by recruiting a deadenylase complex, interacting with its NOT4 subunit. Regulation of CycB is limited to the PGCs to avoid the deleterious consequences of repression in the presumptive somatic cytoplasm. The requirement for both Nos plus at least one additional germline-restricted factor may be part of a mechanism to ensure that CycB regulation is strictly limited to the PGCs (Kadyrova, 2007).
The co-crystal structure of human Pum bound to a fragment of the hb NRE shows that a single Pum RBD directly contacts eight nucleotides of the RNA. However, Puf proteins bind with essentially wild-type affinity to many mutant sites, suggesting that all eight nucleotides are not rigidly specified. How, then, do Puf proteins recognize specific mRNA targets in vivo (Kadyrova, 2007)?
Part of the answer appears to be that, within functional NREs, more than eight nucleotides are recognized, at least by Drosophila Pum. Mutations that simultaneously disrupt Pum binding in vitro and regulation in vivo are spread over 20 nts of the hb NRE and 18 nts of the CycB NRE. These extended Pum mutational 'footprints' are too large to be accounted for by binding of a single RBD; it is suggested that two or more Pum RBDs bind each NRE, an idea supported by the detection of two RNA-protein complexes in gel mobility shift experiments using both the CycB and hb NREs. This model disagrees with earlier experiments that suggested only a single Pum RBD binds to the hb NRE. Further biochemical and structural studies will be required to resolve the issue (Kadyrova, 2007).
The distribution of Pum- and Nos-binding sites within the CycB and hb NREs is different. In the former, the Nos binding site lies 5' to the Pum-binding site(s), whereas in the latter, the Nos-binding site is flanked by nucleotides recognized by Pum. It is assumed that the different arrangement of Nos- and Pum-binding sites is responsible for the assembly of Pum-NRE-Nos complexes with different topographies, such that Brat is recruited to hb but not to CycB. Further definition of each RNP structure will ultimately be required to understand the combinatorial assembly of different repressor complexes on each NRE (Kadyrova, 2007).
In addition to the NRE, Pum also binds with high affinity to at least two other sites in the CycB 3' UTR; however, binding to these sites does not mediate translational repression in the PGCs, perhaps because neither supports recruitment of Nos. These sites may simply bind Pum fortuitously, or they may mediate Nos-independent regulation at other stages of development. Pum has been suggested to destabilize bcd mRNA at the anterior of the embryo in a Nos-independent manner. Another Nos-independent function of Pum is the repression of CycB translation throughout the prospective somatic cytoplasm during the early syncitial nuclear cleavages. These processes might be mediated by elements in Fragments A and F of the 3' UTR, that bind Pum but not Nos (Kadyrova, 2007).
A general framework has been provided for understanding how Puf proteins act to control either the translation or stability of target mRNAs (Goldstrohm, 2006). The yeast Puf protein MPT5 interacts directly with Pop2, one of the catalytically active subunits of a large deadenylase complex. Subsequent deadenylation could either silence the mRNA or cause its degradation, depending on other signals in the transcript or the composition of the deadenylase complex (or both). The Puf-Pop2 interaction is conserved across species (including Drosophila), supporting the idea that the mechanism uncovered for MPT5 might generally be applicable to Puf proteins (Kadyrova, 2007).
In this context, it is surprising that Pum is dispensable if Nanos is tethered to CycB via MS2 CP. It is suggested that yeast Puf proteins both recognize target mRNAs and recruit the deadenylase, but that in the Drosophila germline these functions are partitioned, with Pum primarily responsible for target mRNA recognition and Nos primarily responsible for effector recruitment. This model has the attraction of attributing an important role to Nos, which is essential for Puf-mediated regulation in Drosophila, and probably other metazoans as well. What, then, might be the role of the conserved interaction between Pum and Pop2? One possibility is that it acts cooperatively with Nos to recruit the deadenylase; unlike CycB, other mRNA targets (e.g. hb) might require recruitment by both Nos and Pum to ensure efficient deadenylation. Another possibility is that it plays an essential role for mRNAs regulated by Pum but not Nos (Kadyrova, 2007).
Oscillations in CycB activity underlie normal cell cycle progression. During the early embryonic syncitial nuclear cleavages, degradation in the vicinity of the nuclei is thought to deplete CycB locally. Recent work has shown that Pum can inappropriately repress de novo translation of CycB mRNA during the initial nuclear cleavages if not antagonized by the PNG kinase, resulting in mitotic failure. This early Pum-dependent repression is thought to be Nos-independent, as it occurs efficiently in the anterior, where Nos activity is undetectable (Kadyrova, 2007).
The results show that if CycB is inappropriately subjected to Pum+Nos-dependent repression via the hb NRE, CycB is locally depleted, resulting in mitotic failure during nuclear division cycles 10-13. Since it is thought to be the case during the early cycles (1-7), de novo synthesis of CycB apparently is required to counteract the local degradation that probably occurs during M phase of each cycle. The CycB NRE must therefore be precisely tuned to repress translation only in the PGCs and not in the presumptive somatic cytoplasm (Kadyrova, 2007).
Osk is the limiting factor for assembly of pole plasm in the embryo; the results suggest that it stimulates the accumulation or activity of at least one factor in addition to Nos that is required for repression of CycB in the PGCs. The existence of a co-factor is inferred from the finding that ectopic Nos can repress CycB in the somatic cytoplasm only in the presence of ectopic Osk. Regulation of CycB may depend on more than one germline-restricted factor to ensure that potentially deleterious repression does not occur in the somatic cytoplasm (Kadyrova, 2007).
A germline Nos co-factor might act in a variety of ways. It could bind to the CycB NRE adjacent to Pum and Nos, substituting functionally for Brat, which is recruited to the Pum-hb NRE-Nos complex. The 50 nt CycB NRE is inactivated by a truncation at both ends that leaves the Pum- and Nos-binding sites intact, consistent with the idea that another factor binds to the element. Another possibility is that the co-factor is a germline-specific component of the adenylation/deadenylation machinery, as is the case for the GLD-2 cytoplasmic poly(A)-polymerase in C. elegans. Distinguishing among these ideas awaits identification of the cofactor (Kadyrova, 2007).
The Drosophila embryo is a promising model for isolating gene products that coordinate S phase and mitosis. Increasing maternal Cyclin B dosage to up to six copies (six cycB) increases Cdk1-Cyclin B (CycB) levels and activity in the embryo, delays nuclear migration at cycle 10, and produces abnormal nuclei at cycle 14. The level of CycB in the embryo inversely correlates with the ability to lengthen interphase as the embryo transits from preblastoderm to blastoderm stages and defines the onset of a checkpoint that regulates mitosis when DNA replication is blocked with aphidicolin. A screen for modifiers of the six cycB phenotypes identified 10 new suppressor deficiencies. In addition, heterozygote dRPA2 (a DNA replication gene) mutants suppressed only the abnormal nuclear phenotype at cycle 14. Reduction of dRPA2 also restored interphase duration and checkpoint efficacy to control levels. It is proposed that lowered dRPA2 levels activate Grp/Chk1 to counteract excess Cdk1-CycB activity and restore interphase duration and the ability to block mitosis in response to aphidicolin. These results suggest an antagonistic interaction between DNA replication checkpoint activation and Cdk1-CycB activity during the transition from preblastoderm to blastoderm cycles (Crest, 2007).
It has been proposed (model 1) that the ability of the DNA replication checkpoint to regulate the entry into M-phase is not active before cycle 11 in wild-type (two cycB) embryos. This model is based on observations that the earliest defects in interphase duration can be detected only after cycle 10 in grp (dChk1) or mei-41 (dATR) mutant embryos. In this view, before cycle 10 in two cycB embryos, nuclei have normal morphology because the DNA replication machinery is abundant. The exponentially increasing numbers of nuclei, however, titrate components of this machinery to a critical level after cycle 10, thereby inducing the checkpoint activation, interphase extension, and delay of M-phase in a Grp- and Mei-41-dependent manner (Crest, 2007).
An alternative (model 2) is proposed: Checkpoint function is active all the time, but before cycle 10 checkpoint activity is too low and is overridden by the high level of maternal Cdk1-CycB. The difference between the two models is that in model 1, checkpoint is activated by a critical amount of the replication machinery. In model 2, at a critical concentration of Cdk1-CycB, this kinase (Cdk1) can no longer override checkpoint activity. Several observations are not compatible with model 1, but are with model 2. First, here this study shows that checkpoint activity does not depend on a specific number of nuclei, number of rounds of divisions, or time after fertilization, but on the amount of CycB-Cdk1. It occurs earlier in one cycB embryos with fewer nuclei and later in six cycB embryos with more nuclei (Crest, 2007).
Second, model 1 does not explain interphase extension before cycle 10 and the slight increase of interphase even in grp mutant embryos after cycle 10 (Crest, 2007).
Third, Grp protein is required for the degradation of cyclin A in the presence of cycloheximide as early as cycle 4. Although the effect of Grp on CycA degradation may be different from its effect on replication checkpoint activation, this observation suggests that Grp is present and functional before cycle 10. How then do nuclei enter M-phase at the normal time when aph. is applied before cycle 10, i.e., show no DNA replication checkpoint? It is reasoned that successful execution of the checkpoint requires the inhibitory effect of Grp to overcome the M-phase-promoting effect of Cdk1-CycB. Early embryos might have too few nuclei, thus limited numbers of replication forks, to trigger sufficient Grp/Chk1 activity necessary to overcome the relatively high levels of Cdk1-CycB. Despite this situation, nuclear morphology is normal because replication machinery is not limited before cycle 10, S-phase is rapidly completed, and nuclei can successfully undergo a normal mitosis (Crest, 2007).
Fourth, during normal S-phase of either yeast or mammalian cells, a low level of replication checkpoint activity is observed. This low level of checkpoint activity can be detected as phosphorylation on Chk1 in S-phase cells without any replication stress or DNA damage. Physiological regulation of Chk1 is under the control of similar factors of the DNA replication checkpoint machinery, and thus it was proposed that DNA replication per se generates lesions that activated the checkpoint pathway. Alternatively, but not mutually exclusively, this constitutively low level of replication checkpoint activation may be due to transient signaling from the replication forks, which does not lead to cell cycle arrest, but serves as a mechanism to coordinate the firing of replication origins, thereby moderating the rate of S-phase (Crest, 2007).
Model 2 not only accounts for observations with the aph. experiments, but also accounts for observation of the CycB effects on the replication checkpoint effect. In one cycB embryos the Grp/Chk1 effect is observed earlier, presumably because levels of Cdk1-CycB become limiting earlier, whereas in six cycB embryos, these occur later. Using PH3 staining on anaphase chromosomes as a measurement for Chk1-dependent Cyclin A degradation indicates that Chk1 is not functioning in six cycB embryos before cycle 11, possibly because it is overridden by the abundance of Cdk1-CycB in these embryos. It is proposed that in six cycB embryos at cycle 11 or later, Cdk1-CycB activity is still too high and forces the nuclei into mitosis at a time when the DNA replication machinery is limited, resulting in precocious M-phase and abnormal nuclei (Crest, 2007).
This study addresses how dRPA2 might suppress the six cycB phenotype at cycle 14. dRPA2 is a subunit of a highly conserved heterotrimeric complex of proteins that make up the RPA complex. All three subunits contain DNA-binding domains, which stabilize ssDNA as it is unwound at the replication fork. This stabilized DNA allows for Cdc45 and DNA polymerase-α to initiate DNA replication. Additional roles for RPA have been implicated in DNA repair and recombination (Crest, 2007).
In six cycB embryos, during the blastoderm cycles, elevated levels of Cdk1-CycB override Chk1 and the nuclei divide before DNA replication is completed, leading to abnormal nuclei. It is speculated that reducing dRPA2 is likely to slow DNA replication because RPA can facilitate DNA replication by unwinding dsDNA and by modulating the activities of several enzymes, such as DNA helicases, DNA polymerases, and primases. This would result in less RPA coated, primed DNA and ssDNA. Such a DNA structure may potentiate the TopBP1-mediated ATR-ATRIP kinase activation, leading to stronger Chk1 activation. Thus in dRPA2/six cycB embryos, a stronger Chk1 activation would have a stronger inhibitory effect on Cdk1-CycB activity that cancels out the effect caused by extra Cdk1-CycB. This interpretation for the suppressive effect of RPA2 on the six cycB phenotype suggests an antagonistic relationship between the DNA replication checkpoint activation and Cdk1-CycB activity in regulating the transition from the preblastoderm cycles to the blastoderm cycles (Crest, 2007).
The earliest divisions in the embryo (with the exception of mammals) are maternally regulated until the zygotic genome takes over. On the basis of many observations with different animals such as Xenopus and Drosophila, a simple concept developed: Early cell cycles are invariant, synchronous, and lack both gap phases until the transition to zygotic control occurs. The time point at which this change happens has been called the midblastula transition (MBT). It is assigned to the specific cycle when zygotic transcription is activated. Furthermore, G2 is induced in Drosophila and G1 and G2 in Xenopus into the abbreviated cell cycles and cell division is patterned (Crest, 2007).
This concept is attractive; however, as with many simple concepts, the more that is learned the harder it is to accept the concept at face value. For example, synchronous divisions have never been observed in Caenorhabditis elegans. Even in Drosophila, the simplification that early cell cycles are synchronous and equal in length is incorrect since interphase durations steadily increase after cycle 7 and metasynchronous mitoses are observed as early as cycle 4. These changes occur long before cycle 14, the time that has been designated by many as the MBT. The data presented in this study clearly demonstrate a change in the maternal program as the embryo develops: A DNA replication checkpoint is first detectable after cycle 10, but becomes increasingly robust in the subsequent cycles, indicating that the ability to regulate M-phase by checkpoints is not completely'off' or 'on'. In addition, it was found that the DNA replication checkpoint is detectable earlier in one cycB and later in six cycB embryos, clearly indicating that changes do not have to occur at a specific stage. A gradual attainment of full checkpoint function is also supported by the fact that aph. injections before cycle 11 can stall/delay the nuclear cycle, but not the centrosomal cycle. These results are not compatible with the idea of an invariant maternal program for pre-MBT cycles (Crest, 2007).
Another simplification is that changes that define the MBT are events that envelope the entire embryo. In sea urchins the deceleration of the cell cycle in the micromeres occurs long before these changes have been observed in the macromeres. Thus an MBT has not been proposed for this animal. But differences are also observed among the blastoderm cycles in Drosophila, where divisions in the middle of the embryo are slower than at the poles, correlating with their nuclear densities (Crest, 2007).
The initiation of the zygotic program also does not occur suddenly from off to on. fushi tarazu (ftz) transcripts are first observed at cycle 9 in one or the other embryo and in one or the other nucleus. Transcription gradually increases over the next three cycles (cycles 9-12). This gradual increase is a consequence of the dose-dependent repressor Tramtrack (TTK), where with one gene dose of maternal ttk, initial transcription of ftz occurs one cycle earlier, and conversely extra copies of ttk result in initial transcription of ftz one cycle later. These data are interpreted as a decline of TTK during cycles 8-10 to a threshold level where TTK repression is insufficient, enabling low-level transcription of ftz (Crest, 2007).
Despite the many observations that do not fit the MBT concept, textbooks, reviews, research articles, and grant proposals still hold on to it, hindering progress in the understanding of how maternally controlled development declines and terminates and zygotic programming gradually takes over (Crest, 2007).
The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin Bactivity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. This study shows that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, it was shown that Fest is required for proper execution of meiosis I (Baker, 2015).
This study shows that the developmental program of male gametogenesis imposes several levels of cell type- and stage-specific post-transcriptional control on expression of the key G2/M cell cycle regulatory component CycB during meiotic prophase and identifies two key developmentally regulated trans-acting factors involved. First, the cycB RNA expressed in spermatocytes has a short 3' UTR, only 130 nt long and missing previously identified translational regulatory sequences used in other cell types. Second, the RNA-binding protein Rbp4, expressed starting early in meiotic prophase soon after completion of pre-meiotic DNA synthesis, binds the short 3' UTR and blocks translation of cycB in immature spermatocytes. Third, the Rbp4-interacting protein Fest, also upregulated early in the spermatocyte period, is also required for blocking CycB expression in immature spermatocytes (Baker, 2015).
Rbp4 and Fest RNA and protein are expressed in very early spermatocytes prior to onset of transcription of cycB, which depends on action of the tMAC complex (White-Cooper, 1998; Beall 2007). As a result, when the cycB RNA is expressed, it arrives in a cytoplasm already primed for its proper cell type- and stage-specific translational repression. Expression of Cyclin B3 (Clb3) protein in budding yeast has been shown to be restricted to meiosis II via sequences in the CLB3 3' UTR that block translation during meiosis I. Although translational repression of CLB3 in meiosis I was important to prevent premature separation of sister chromatids, an event appropriate for meiosis II rather than meiosis I, trans-acting factors responsible for the stage-specific translational repression have yet to be identified. The current data reveal that translational repression of a cyclin (in this case CycB) is also a key feature of meiotic prophase during spermatogenesis in a metazoan animal. Surprisingly, expression of CycB in immature spermatocytes -- either in rbp4 or fest mutants or by a mutated CycB-eYFP reporter -- was insufficient to drive those cells immediately into meiotic division. This might be because action of the Cdc25 cell cycle phosphatase encoded by twine, which is also translationally repressed in immature spermatocytes and becomes translationally activated by the RNA-binding protein Boule only in mature spermatocytes, is also required to generate active Cdk1/CycB. (Baker, 2015)
One general model for Fest function invokes the possibility that Rbp4 recruits Fest to the cycB 3' UTR, where Fest is able to interfere with cycB translation. However, no compelling evidence was found of specific binding of Fest to the cycB 3' UTR in biotin pull-down experiments from testis extracts from flies expressing eYFP-Fest either with or without functional Rbp4, which suggests that either Fest is not recruited to the cycB 3' UTR, or that the biotin pull-down assay has limitations in detecting indirect RNA-protein interactions. As a result, it is important to consider other mechanisms for Fest function, including the possibility that binding of Fest to Rbp4 is needed only briefly to enact a post-translational modification of or conformational change within Rbp4 to promote its ability to recruit partners and/or repress translation. It is also technically possible that Fest and Rbp4 act in parallel pathways to regulate CycB. Finally, as the fest germ cell phenotype is dramatically stronger than that of rbp4, it is likely that Fest regulates other proteins in addition to Rbp4 (Baker, 2015)
It is not yet known how information about spermatocyte maturation is communicated to Rbp4 or Fest to allow translation of cycB in mature spermatocytes. One or more proteins could respond to input regarding cell size, given that spermatocytes grow 25-fold in volume during meiotic G2. Alternatively, given that translation of cycB in mature spermatocytes requires function of the testis TAF proteins, signals indicating the completion of the spermatocyte transcription program (not merely its onset) could trigger the reprieve from translational repression. Another possibility might be a meiotic arrest checkpoint mechanism triggered by transcriptional activity from unpaired chromatin, as seen in mammalian spermatocytes. Whatever the stimulus, it is clear that through stage-specific expression of the translational regulators Rbp4 and Fest in very early spermatocytes, the developmental program of male germ cell differentiation exerts additional layers of control over the core cell cycle machinery (Baker, 2015)
Bases in 5' UTR -123 or more
Bases in 3' UTR - 776
Within a central region spanning 206 amino acid residues, the Drosophila Cyclins A and B share 35% identity, whereas clam Cyclin A and Drosophila Cyclin A share 53% identity as do clam Cyclin B and Drosophila Cyclin B. In particular, the Drosophila Cyclin B sequence contains a consensus cAMP-dependent protein kinase site, a feature common to all other Cyclin B sequences (Whitfield, 1990).
date revised: 21 APR 97
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