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First published online 14 March 2007
doi: 10.1242/dev.002212

Development 134, 1519-1527 (2007)
Published by The Company of Biologists 2007
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Translational control of maternal Cyclin B mRNA by Nanos in the Drosophila germline

Lyudmila Y. Kadyrova1, Yasuaki Habara1,*, Tammy H. Lee1 and Robin P. Wharton1,2,{dagger}

1 Howard Hughes Medical Institute, Department of Molecular Genetics and Microbiology.
2 Department of Cell Biology, Box 3657, Duke University Medical Center, Durham, NC 27710, USA.


Figure 1
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  		Fig. 1. Identification of a Nos Response Element in the CycB 3' UTR. (A) Drawing to scale of the CycB 3' UTR and the fragments used in the experiments below. The critical regulatory region (fragment E in red) is present in the maternal mRNA isoform but spliced out of the zygotic isoform (dashed line). Numbers identify the 3' UTR nucleotides present in each fragment. Note that fragment F contains 25 nt encoded by genomic DNA downstream of the polyadenylation and cleavage site. (B) Gel mobility shift experiments with RNA bearing the hb NRE or various fragments of the CycB 3' UTR. On the left, RNA was incubated with embryonic extract prepared as described (Murata and Wharton, 1995Go). On the right, the Pum RBD was incubated at concentrations of 0, 0.14, 0.42 and 1.3 µM in lanes 1-4 of each titration. The figure is a composite of two different gels. (C) Drawing of the region spanning fragments D and E, where each dot represents a potential Pum-binding site (UGU trinucleotide). The functionally defined 50 nt CycB NRE (black box, nts 594-643 of the 3' UTR) contains two such UGU trinculeotides (red dots, see the sequence in E). Repression in the PGCs at stage 4 for various derivatives is indicated to the right. (D) Accumulation of CycB (green) in stage 4 embryos in which the PGCs are marked by accumulation of Vasa (red), detected by immunofluorescence and confocal microscopy. In these and all subsequent images, repression of CycB mRNA causes the pole cells to appear red, whereas de-repression results in colocalization of CycB and Vasa, causing the pole cells to appear yellow-orange. Embryos are either from w1118 (wt) nosBN, or pumET3/pumMsc females, or CycB2/+ females that also bear the indicated CycB transgene. (E) Sequence of the 50 nt CycB NRE (nts 594-643 of the 3' UTR). Binding sites for Nos and Pum are inferred from experiments with purified proteins and a collection of mutant RNAs. Supporting data is presented in Fig. S3 of the supplementary material.

 

Figure 2
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  		Fig. 2. The CycB NRE confers regulation on hb mRNA exclusively in the PGCs. Accumulation of Hb in embryos from wild-type (wt) females or females carrying the indicated hb transgene, with high magnification views (of other embryos) shown below to allow visualization of the pole cells (marked with arrowheads). Accumulation in the anterior ~50% of the embryo derives from both unrepressed maternal mRNA and zygotically transcribed mRNA; accumulation in the posterior ~50% of the embryo derives primarily from unrepressed maternal mRNA (in the transgenic embryos), with a minor contribution at the stage shown from zygotic transcription under the control of the terminal system (most easily seen in the wild-type embryo).

 

Figure 3
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  		Fig. 3. Interaction of NOT4 with the N-terminal region of Nos. (A) CycB (green) and Vasa (red) accumulation in embryos from wild-type (wt) and CCR4 KG877/Df(3R)crb-F89-4 females. (B) GST-pulldown experiments show that Hs Pum1 and Dm Pum interact in a similar fashion with three different Pop2 homologs; each GST fusion was incubated with Hs CNOT7 (lane 1), Hs CNOT8 (lane 2) and Dm Pop2 (lane 3). Note that a truncated fragment of CNOT8 generated during in vitro synthesis binds preferentially to both Pum RBDs. (C) Four different fragments of NOT4 interact with full-length Nos in yeast, as does Cup [supporting growth -His + 1 mM 3-aminotriazole (3-AT)]. At the bottom, interaction of NOT4 residues 597-1051 with various portions of Nos is summarized on the left, based on growth -His + 5 mM 3-AT, shown on the right. Inclusion of residues 1-42 of Nos is necessary for efficient expression of the C-terminal zinc finger domain (ZF) in yeast. The AD-Cup fusion provides a positive control and the AD encoded by the empty pGAD424 vector provides a negative control.

 

Figure 4
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  		Fig. 4. Tethered Nos can repress CycB but not hb. (A) Accumulation of CycB (green) monitored in the primordial germ cells, marked by accumulation of Vasa (red). The relevant maternal genotypes are: (1) CycB+/CycB+ (wild type, wt), (2) CycB3/CycB+; CycB(2x MS2hp), (3) CycB3/CycB+, Nos-CP; CycB(2x MS2hp), (4) CycB3/CycB2, Nos-CP; pumET3 / CycB(2x MS2hp), pumMsc (all transgenes single-copy). (B) Accumulation of Hb (green) in embryos from wild-type females or females bearing the indicated transgenes, with pole cells marked by arrowheads and nuclei labeled with TOPRO3 (red). Note that the accumulation of Hb in somatic nuclei at the posterior of the transgenic embryos derives from both torso-dependent zygotic transcription and unrestrained translation of the maternal hb(2x MS2hp) mRNA, whereas wild-type embryos contain only the former source of Hb (as in Fig. 2).

 

Figure 5
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  		Fig. 5. Deleterious consequences of Nos-dependent repression of CycB mRNA outside the PGCs. (A) Darkfield photomicrographs of cuticle secreted by embryos from wild-type (wt) or CycB2/CycB3; CycB(2x hbNRE) females. Note the reduction of abdominal segments, each marked by a band of large denticles (e.g. black arrowhead) and the rudimentary posterior terminalia (filzkörper, white arrowhead); both features are absent in the limit CycB (2x hb NRE) phenotype (above). (B) Surface views of nuclei in two different cycle 10-13 embryos from females of the indicated genotype. The wild-type embryos are from CycB2/+ females. Note that many nuclei in the posterior of the embryo at the lower right have fallen into the center and are not visible in the focal plane shown. (C) Distributions of CycB (green) and Vasa (red) in mid-line views of embryos from females of the indicated genotype. The posterior region that contains lower levels of CycB is bracketed. To the right are high magnification views of post-cycle 9 embryos, showing repression of CycB (2x hbNRE) in the PGCs. The white arrowhead highlights nuclei that have fallen into the interior. The embryo on the left is from a CycB2/+ female and the embryo in the middle from a CycB2/+ female bearing the CycB ({Delta}I+II) transgene.

 

Figure 6
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  		Fig. 6. Ectopic Nos not sufficient to repress wild-type CycB mRNA outside the pole plasm. Distributions of Nos (left) and nuclei (right) in embryos from various females, genotypes indicated at the right. Note the ectopic pole cells at the anterior of the osk-bcd embryo.

 

Figure 7
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  		Fig. 7. Puf proteins nucleate the assembly of different repressor complexes in Drosophila and budding yeast. Models of the mechanism by which Pum and MPT5 repress CycB and HO mRNAs in Drosophila and S. cerevisiae, respectively. The data of Figs 2 and 4 are consistent with the idea that a germline-limited co-factor (magenta) binds directly to the CycB NRE; however, other roles for the co-factor are possible.

 

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