The DAZL family proteins are PABP-binding proteins that regulate translation in germ cells

Abstract

DAZL proteins are germ-cell-specific RNA-binding proteins essential for gametogenesis. The precise molecular role of these proteins in germ-cell development remains enigmatic; however, they appear to function in the cytoplasm. In order to directly address the function of vertebrate DAZL proteins, we have used Xenopus laevis oocytes as a model system. Here we demonstrate that members of this family, including Xdazl, mouse Dazl, human DAZL, human DAZ and human BOULE, have the ability to stimulate translation and function at the level of translation initiation. We show that DAZL proteins interact with poly(A)-binding proteins (PABPs), which are critical for the initiation of translation. Mapping and tethered function experiments suggest that these interactions are physiologically important. This leads to an attractive hypothesis whereby DAZL proteins activate translationally silent mRNAs during germ cell development through the direct recruitment of PABPs.

Figure 1

Tethered Xdazl can stimulate translation. (A) The tethered function assay has two components: a luciferase reporter mRNA with binding sites for the MS2 coat protein within its 3′ UTR (Luc-MS2), which is unadenylated unless stated, and a fusion protein of MS2-coat protein and Xdazl. Binding of the coat protein tethers Xdazl to the mRNA. The effects of fusion proteins on translation are measured by luciferase assay, normalized to a β-Gal. (B) Oocytes expressing MS2, MS2-U1A, MS2-PABP or MS2-Xdazl were coinjected with Luc-MS2 and β-Gal mRNAs. (C) The stability of luciferase (upper panel) and β-Gal mRNAs (lower panel) is shown by Northern blotting. RNA was extracted at 0 h (T=0) and 16 h (T=16) after incubation in oocytes expressing fusion proteins. (D) Oocytes expressing MS2-PABP or MS2-Xdazl were coinjected with β-Gal mRNA and Luc-MS2 or Luc-ΔMS2 mRNAs.

Figure 2

Mammalian DAZL, DAZ and BOULE proteins can also stimulate translation. (A) A phylogenetic comparison of some vertebrate members of the DAZL family of proteins. The tree was created using Mega 2 and is rooted using Drosophila Boule. (B) Oocytes expressing MS2, MS2-Xdazl, MS2-mDazl or MS2-hDAZL or (D) MS2, MS2-mDazl, MS2-hDAZL, MS2-hDAZ or MS2-hBOULE were coinjected with Luc-MS2 and β-Gal mRNAs. (C) The overall identity between hDAZL and DAZ or BOULE. The majority of the conservation lies in the first 190 amino acids, including the RRM and DAZ motif.

Figure 3

Recruitment of multiple Dazl molecules further enhances translation in cis. (A) Cartoon of luciferase reporter mRNAs, which vary only in the number of MS2 sites within the 3′ UTR. (B) Oocytes expressing MS2, or MS2-mDazl were coinjected with β-Gal mRNA and Luc-MS2₁, Luc-MS2₃ or Luc-MS2₉. Normalized luciferase activity is plotted with MS2 set to an arbitrary level to allow direct comparison.

Figure 4

DAZL proteins enhance translation initiation and interact with a canonical translation initiation factor. (A) Sucrose gradient analysis of initiation intermediates showing the effects of mDazl (•) and U1A (▴) on the distribution of luciferase reporter mRNAs in the presence of cycloheximide. The positions of 80S ribosomes, ribosomal subunits and mRNPs are indicated. The A254 profile is represented by a curved black line. (B) Yeast-two-hybrid analysis with Xdazl and the C-terminus of Xenopus PABP1 (PABP-Ct) using qualitative β-Gal filter assays. IRP-1, an RNA-binding protein that mediates translational control, and MS2 are used as specificity controls. The interaction between Xenopus PABP-Ct and Paip-1 (Gray et al, 2000) acts as a positive control. (C) Oocytes injected with MS2-Xdazl and Xenopus PABP1 were immunoprecipitated with an anti-PABP1 antibody co-precipitating MS2-Xdazl. MS2-Xdazl was detected by Western (upper panel) blotting or by ³⁵S-methionine labeling of total oocyte proteins (middle panel). Endogenous Xdazl was detected by Western blot after IP with anti-PABP1 antibody from X. laevis testes (lower panel). Input: 1 μl of testis extract (D). Yeast two-hybrid analysis with PABP-Ct and DAZL family members using qualitative β-Gal filter assays. (E) Yeast two-hybrid analysis with ePABP C-terminus and Xdazl using qualitative β-Gal filter assays. (F) Endogenous Xdazl was detected by Western blot after IP with anti-ePABP antibody from X. laevis oocytes. Input: 5 μl of stage VI oocyte extract.

Figure 5

Mapping of the minimal PABP–mDazl interaction domain and analysis of the minimal activation domain required for mDazl stimulation. (A) On the left, mDazl deletion constructs are represented. Black lines indicate sequences that are present in construct. Brackets denote internal deletions. Qualitative β-Gal filter assays generated by yeast-two-hybrid analysis are shown on the right. Where shown, IRP-1 is used as a specificity control. (B) Representation of mDazl deletion constructs. Black lines indicate sequences that are present in constructs. Brackets denote internal deletions. The data shown in panel C are summarized on the right. (C) Oocytes expressing MS2, MS2-mDazl or the deletion constructs of mDazl shown in (B) were coinjected with Luc-MS2 and β-Gal mRNAs, and relative stimulation is shown. (D) Upper panel, coimmunoprecipitation of ePABP from oocytes expressing MS2-mDazl, or the deletion constructs of mDazl shown in (C). Lower panel, quantitation of ePABP binding to MS2-mDazl deletions is shown.

Figure 6

Model for the role of PABPs in DAZL-mediated stimulation of target mRNAs. During oogenesis and spermatogenesis, many mRNAs are stored with short poly(A) tails. (A) Many of these mRNAs are activated at specific times in response to cytoplasmic polyadenylation (shown as green A's). This is proposed to recruit additional PABP molecules (shown in green), which facilitates additional interactions with factors bound to the 5′ end (depicted as arrows), increasing end-to-end complex formation. (B) In our working model, DAZL family members directly recruit additional molecules of PABP (shown in green), increasing end-to-end complex formation, leading to enhanced ribosomal subunit recruitment (not shown). Multiple DAZL proteins may recruit multiple PABPs. This does not require changes in polyadenylation.

Figure 7

DAZL proteins appear to functionally substitute for poly(A) tails. (A) Cartoon of reporter mRNAs used. The reporters vary only with respect to their adenylation status. Oocytes expressing MS2-U1A or MS2-mDazl were coinjected with Luc-MS2 or Luc-MS2-pA alongside β-Gal mRNA. (B) Normalized luciferase activity is plotted. (C) The relative fold stimulation of MS2-mDazl compared to MS2-U1A is shown for Luc-MS2 or Luc-MS2-pA. MS2-U1A has been set to 1.