Multiple maternal proteins coordinate to restrict the translation of C. elegans nanos-2 to primordial germ cells - PubMed (original) (raw)

. 2008 May;135(10):1803-12.

doi: 10.1242/dev.013656. Epub 2008 Apr 16.

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Multiple maternal proteins coordinate to restrict the translation of C. elegans nanos-2 to primordial germ cells

Shreyas Jadhav et al. Development. 2008 May.

Abstract

Although germ cell formation has been relatively well understood in worms and insects, how germ cell-specific developmental programs are initiated is not clear. In Caenorhabditis elegans, translational activation of maternal nos-2 mRNA is the earliest known molecular event specific to the germline founder cell P(4). Cis-elements in nos-2 3'UTR have been shown to mediate translational control; however, the trans-acting proteins are not known. Here, we provide evidence that four maternal RNA-binding proteins, OMA-1, OMA-2, MEX-3 and SPN-4, bind nos-2 3'UTR to suppress its translation, and POS-1, another maternal RNA-binding protein, relieves this suppression in P(4). The POS-1: SPN-4 ratio in P(4) increases significantly over its precursor, P(3); and POS-1 competes with SPN-4 for binding to nos-2 RNA in vitro. We propose temporal changes in the relative concentrations of POS-1 and SPN-4, through their effect on the translational status of maternal mRNAs such as nos-2, initiate germ cell-specific developmental programs in C. elegans.

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Figures

Fig. 1

Fig. 1

A line diagram showing abbreviated embryonic lineage (Sulston et al., 1983). The P lineage is shown in red.

Fig. 2

Fig. 2

A and 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.

Fig. 3

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; and 5×, 10× and 50× – 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).

Fig. 4

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 and 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 and 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.

Fig. 5

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., 2006). 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 and 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). Note that 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).

Fig. 6

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 Δ27 mutation.

Fig. 7

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.

References

    1. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. - PMC - PubMed
    1. Barton MK, Schedl TB, Kimble JE. Gain-of-function mutations of fem-3, a sex-determination gene in Caenorhabditis elegans. Genetics. 1987;115:107–119. - PMC - PubMed
    1. Ciosk R, DePalma M, Priess JR. ATX-2, the C. elegans ortholog of ataxin 2, functions in translational regulation in the germline. Development. 2004;131:4831–41. - PubMed
    1. D'Agostino I, Merritt C, Chen PL, Seydoux G, Subramaniam K. Translational repression restricts expression of the C. elegans Nanos homolog NOS-2 to the embryonic germline. Dev. Biol. 2006;292:244–252. - PubMed
    1. Dahanukar A, Walker JA, Wharton RP. Smaug, a novel RNA-binding protein that operates a translational switch in Drosophila. Mol. Cell. 1999;4:209–18. - PubMed

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