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First published online 16 April 2008
doi: 10.1242/dev.013656

Development 135, 1803-1812 (2008)
Published by The Company of Biologists 2008
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Multiple maternal proteins coordinate to restrict the translation of C. elegans nanos-2 to primordial germ cells

Shreyas Jadhav, Mainpal Rana and Kuppuswamy Subramaniam*

Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India.


Figure 1
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  		Fig. 1. A line diagram showing abbreviated embryonic lineage. The P lineage is shown in red. See Sulston et. al.Go

 

Figure 2
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  		Fig. 2. Identification of proteins that control nos-2 translation. (A,B) OMA-1, OMA-2, MEX-3 and SPN-4 are essential for the translation suppression of nos-2 mRNA. Distribution pattern of GFP:H2B expressed under the control of nos-2 3'UTR in oocytes (A; a single oocyte in each panel is outlined) and embryos (B) is shown. Genes disrupted by RNAi treatment are indicated in each panel; WT, non-RNAi control. To facilitate visualization, we expressed GFP as a fusion protein with the histone H2B, which concentrates fluorescence signal in nuclei. (C) POS-1 acts as a de-repressor of nos-2 translation. Epistasis analysis of GFP:H2B expression among mex-3(-), spn-4(-) and pos-1(-) shown here reveals that POS-1 is not required for nos-2 translation in the absence of repressors such as MEX-3 and SPN-4.

 

Figure 3
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  		Fig. 3. MEX-3, SPN-4, OMA-1, OMA-2 and POS-1 physically interact with nos-2 3'UTR. (A) Electrophoretic mobility patterns of radiolabeled 200 bp nos-2 3'UTR RNA in the presence of MBP:MEX-3 (M), GST:SPN-4 (S), GST:OMA-1 (O1) and GST:OMA-2 (O2). L nos-2, radiolabeled 200 bp nos-2 3'UTR; UL nos-2, unlabeled nos-2 3'UTR; NS RNA, unlabeled non-specific RNA; 5x, 10x and 50x, number of times molar excess over L nos-2. (B) Electrophoretic mobility shift with GST:POS-1. Three different concentrations of GST-POS-1 were used: 75, 350 and 200 ng/µl. Comparison of lanes 2-4 indicates multimerization of this protein-RNA complex at higher protein concentrations. (C) Binding of radiolabeled nos-2 3'UTR RNA to solid matrix in presence of the indicated components (see Materials and methods for details).

 

Figure 4
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  		Fig. 4. Determination of nos-2 3'UTR regions that are critical for interaction with the various proteins. (A) Schematic illustration of the five regions of nos-2 3'UTR that were mutated by substitution. SubA begins immediately downstream of the stop codon. (B-E) Electrophoretic mobility shifts of various mutant versions of radiolabeled nos-2 3'UTR by MBP:MEX-3 (B), GST:SPN-4 (C) and GST:OMA-2 (D,E). The first lane in each set is the mobility of RNA in the absence of protein. Radiolabeled RNA used in D contained the wild-type version of the following regions only: 1, SubA-C; 2, SubB-E; 3, SubA-D; 4, SubD-E, whereas those in other panels contained the 200 bp nos-2 3'UTR with the indicated regions substituted with (TG)15. WT in all panels indicate the wild-type version of the 200 bp nos-2 3'UTR. (F) Binding of radiolabeled WT and mutant nos-2 3'UTR RNA to solid matrix in presence of the indicated components (see Materials and methods for details). L RNA, radiolabeled RNA; UL RNA, unlabeled RNA.

 

Figure 5
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  		Fig. 5. Binding to nos-2 3'UTR is essential for the translation suppression activity of MEX-3 and SPN-4. Two 8 bp direct repeats present in nos-2 3'UTR are critical for the binding of MEX-3 and SPN-4. (A) Alignment of the nos-2 3'UTR of the indicated species (D'Agostino et al., 2006Go). Only the region with two 8 bp direct repeats (DR1 and DR2; boxed) is shown. Stars indicate bases conserved in all three species. Sequences of mutations used in B,C are shown in red. (B) Electrophoretic mobility shifts by MBP:MEX-3 (top) and GST:SPN-4 (bottom) of the various mutant versions of radiolabeled nos-2 3'UTR. (C) Expression pattern of GFP:H2B in embryos of transgenic worms carrying the GFP:H2B:nos-2 3'UTR transgene bearing the indicated mutations, or following spn-4(RNAi) or mex-3(RNAi). The GFP:H2B distribution pattern in DR1 and DR2 is similar to that of spn-4(RNAi) and the pattern in DR1+DR2 is similar to that of mex-3(RNAi).

 

Figure 6
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  		Fig. 6. Interaction with nos-2 3'UTR is essential for the translation suppression activity of OMA-2. (A) Sequence of the SubE region. Sequences targeted by substitution analysis in EMSA are boxed and named. (B) Electrophoretic mobility shift by OMA-2 of radiolabeled nos-2 3'UTR bearing the indicated mutations. The first lane in each set is the mobility of RNA in the absence of protein. (C) Expression pattern of GFP:H2B in embryos of transgenic worms carrying the GFP:H2B:nos-2 3'UTR transgene with wild-type sequence (WT) or bearing {Delta}27 mutation.

 

Figure 7
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  		Fig. 7. POS-1 competes with SPN-4 for binding to nos-2 3'UTR. (A) Electrophoretic mobility shift of the radiolabeled 200 bp nos-2 3'UTR incubated with GST:SPN-4 alone (lane 1), GST:POS-1 alone (lane 2) or with increasing concentration of GST:POS-1 at a constant concentration of GST:SPN-4 (lanes 3-7). No protein was added to RNA in lane 8. Lanes 1 and 3-7 contain 2 µl of GST:SPN-4 per lane. Amounts of GST:POS-1 in lane 2 are 4 µl and in lanes 3-7 are 2, 4, 6, 8 and 10 µl, respectively. (B) Representative examples of 16- and 28-cell embryos immunostained with anti-SPN-4 and anti-POS-1 antibodies. (C) Bar graph showing average POS-1: SPN-4 ratios obtained by quantitation of immunofluorescence signals from 12 embryos for the indicated stages.

 

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