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doi: 10.1242/10.1242/dev.00469

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Translational control of maternal glp-1 mRNA by POS-1 and its interacting protein SPN-4 in Caenorhabditis elegans

Ken-ichi Ogura1,*, Norihito Kishimoto1, Shohei Mitani2, Keiko Gengyo-Ando2 and Yuji Kohara1,{dagger}

1 Genome Biology Laboratory, Center for Genetic Resource Information, National Institute of Genetics, Mishima 411-8540, Japan
2 Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo 162-8666, Japan
* Present address: Department of Pharmacology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan



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  		Fig. 1. Expression of GLP-1 in pos-1 and spn-4 mutants. Embryos were stained with an anti-GLP-1 antibody. (A-F) Four-cell stage embryos. (G) One-cell stage embryo. Anterior is towards the upper left, posterior is toward the lower right, dorsal is towards the upper right and ventral is towards the lower left. Scale bar: 10 µm. (A) Wild type. GLP-1 was detected only in the ABa and ABp blastomeres, mainly at the membrane. (B) glp-1(RNAi) embryo. In this embryo, there was no expression of GLP-1. Thus, the staining shown here gives the level of the background signals. (C) pos-1(zu148) and (D) pos-1(ne51) embryos. GLP-1 was detected at the membrane in all four blastomeres. (E) spn-4(tm291). GLP-1 was not detectable. (F) pos-1(zu148); spn-4(tm291) double mutant embryo. GLP-1 was detected in all four blastomeres. (G) One-cell stage pos-1(zu148) embryo. GLP-1 is not detectable.

 


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  		Fig. 2. Interaction of POS-1 and SPN-4 with glp-1 3' UTR. (A) The glp-1 mRNA 3' UTR. The entire 3' UTR is 369 bases long from the stop codon UAA to the poly A addition site. The SCR was originally defined as lying between bases 180 and 240, and the TCR between bases 245 and 369 (Evans et al., 1994Go). The comparison of the 3' UTRs of closely related species (Rudel and Kimble, 2001Go) suggested some minor modifications on the regions as follows: modified SCR was between bases 172 and 237, and modified TCR was from base 242. Thus, the regions are as follows: the 294-base (bases 26 to 319), the 138-base (bases 26 to 163, SCR (172 to 237) and the TCR*(bases 242 to 319). TCR* indicates a subregion of TCR. (B) Outline of the structure of POS-1. Two CCCH zinc fingers are located in the middle of the 264 amino acid POS-1 protein. The allele ne51 is a missense mutation at codon 147, C(TGC) to Y(TAC), in the second zinc finger. (C) Yeast tri-hybrid analysis. Based on the reporter RNA study (Evans et al., 1994Go) and the comparative study (Rudel and Kimble, 2001Go), the following regions were taken from the 369-base glp-1 mRNA 3' UTR for the construction of hybrid RNA with RRE, a Rev responsive element recognized by HIV-1 RevM10 (Putz et al., 2000Go): row 1, bases 26-319 for 138-base SCR-TCR* (the entire region); row 2, bases 26-163 for 138-base (non SCR, non TCR region); row 3, bases 172-237 for the SCR; and row 4, bases 242-319 for the TCR* (TCR subregion). Row 5 is a negative control RNA. C indicates growth on a non-selection medium without 3AT; S indicates growth on a selection medium containing 50 mM (for POS-1) or 10 mM (for SPN-4) 3-AT. (D) Western blot analysis of the tri-hybrid strains by using an anti-POS-1 antibody: (1) GAL4 AD, (2) GAL4 AD::POS-1 and (3) GAL4 AD::POS-1(C147Y) were expressed in yeast. In all the yeast strains, RRE-138-base SCR-TCR* was co-expressed. POS-1(C147Y) was expressed at the same level as POS-1.

 


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  		Fig. 3. Identification of POS-1-interacting proteins. (A) In vitro binding of recombinant proteins. (1) GST and MBP::POS-1, (2) GST::POS-1 and MBP::POS-1, (3) GST::MEX-3 and MBP::POS-1, (4) GST::SPN-4 and MBP::POS-1, (5) GST::LET-92 and MBP::POS-1, (6) GST and MBP::lacZ{alpha}, (7) GST::POS-1 and MBP::lacZ{alpha}, (8) GST::MEX-3 and MBP::lacZ{alpha}, (9) GST::SPN-4 and MBP::lacZ{alpha}, (10) GST::LET-92 and MBP::lacZ{alpha}. Positive signals were detected only in lanes 2~5. (B) The SPN-4 protein. The RNP-type RNA-binding domain (RNP) is shown in black. The cDNA sequence of spn-4 has been deposited with the DDBJ (DNA Data Bank of Japan) under the Accession Number AB052819. (C) Genomic structure of the spn-4 gene. Boxes represent exons. The ORF is shown in gray with the RNP region in black. The trans-splice leader SL1 was detected just one base upstream of the first ATG. The genomic region that was deleted in the tm291 mutant is shown.

 


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  		Fig. 4. Localization of spn-4 mRNA. Wild-type embryos were analyzed by in situ hybridization with a spn-4 cDNA probe. Anterior is leftwards, posterior is rightwards, dorsal is upwards and ventral is downwards. Scale bar: 10 µm. (A) One-cell embryo. (B) Two-cell embryo. (C) Four-cell embryo. (D) Eight-cell embryo. P3 and C are stained. (E) An embryo just before gastrulation. P4 and D are stained. (F) An embryo beginning gastrulation. Only P4 is stained. (G) An embryo during gastrulation. P4 has divided to produce Z2 and Z3, which are stained. (H) A mid-stage embryo. No staining is observed. The spn-4 mRNA was also detected in adult gonads and in oocytes (data not shown).

 


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  		Fig. 5. SPN-4 protein distribution. (A-J) SPN-4 and POS-1 distribution. Wild-type gonads and embryos were stained with an anti-SPN-4 antibody (red; A,B,E,H) and an anti-POS-1 antibody (green; C,F,I). (D,G,J) Merged images including DAPI-stained images (blue). Anterior is leftwards, posterior is rightwards, dorsal is upwards and ventral is downwards. Scale bars: 10 µm. (A) Oocytes. SPN-4 was detected in oocytes from adult animals. (B-D) One-cell embryo just before the first cleavage. (E-G) Two-cell stage embryo. (H-J) Four-cell stage embryo. SPN-4 was uniformly present in the cytoplasm up to the two-cell stage, then began to localize to the posterior blastomeres P2 and EMS. Co-localization of SPN-4 and POS-1 was mainly observed in the cytoplasm of the posterior blastomeres, including the germline (D,G,J; yellow regions) and P granules (yellow dots, see Fig. 6). (K) SPN-4 in spn-4 embryo at four-cell stage.

 


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  		Fig. 6. Co-localization of SPN-4 and the P granules. Wild-type embryos were stained with an anti-SPN-4 antibody (red, A) and mabK76, which recognizes P granules (green, B). (C) is a merged image and includes a DAPI-stained image (blue). The granular staining of the anti-SPN-4 antibody matches the mabK76 staining pattern of the P granules (C, yellow dots).

 


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  		Fig. 7. A model for the roles of POS-1 and SPN-4 in the glp-1 mRNA translation. (Top) SCR: the 66-base spatial control region within the glp-1 mRNA 3' UTR. TCR: the 125-base temporal control region within the glp-1 mRNA 3' UTR. In this model, POS-1 binds the SCR with its second CCCH finger. SPN-4 binds the TCR. POS-1 suppresses and SPN-4 activates the translation. SPN-4 may also inhibit the POS-1 function by direct binding. (Bottom) POS-1 is abundant in the posterior blastomere (right) but present at a much lower level in the anterior one (left), while SPN-4 is abundant in both. We propose that the translation of maternal glp-1 mRNA is suppressed in the posterior half of the embryo by the POS-1 repressor activity, while it is turned on in the anterior half, where the concentration of SPN-4 and its activating function predominates.

 

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