Generation of Anti-DAZL1 Antibodies
A polyclonal antiserum was generated against the last 76 amino acid residues of the human DAZL1 protein, which is 86% identical to the corresponding region of mouse DAZL1. On Western blots, the anti-DAZL1 antiserum detected a major band in mouse testicular extracts that comigrated with in vitro-synthesized DAZL1 and that was absent in the liver and the brain (Fig. 3a). The apparent molecular size of the putative DAZL1 was larger than the 33 kDa predicted from the cDNA sequence. A discrepancy between predicted molecular size and mobility in the SDS-PAGE gel was also reported for the Drosophila Boule protein. Preincubation of the antiserum with increasing amounts of in vitro-synthesized DAZL1 reduced the signal of the putative DAZL1 but not of other minor bands, further supporting the authenticity of the protein (Fig. 3b).
Detection of DAZL1 in Mouse Testicular Fractions
The anti-DAZL1 antiserum was used to trace DAZL1 during fractionation of mouse testicular extracts. When the extracts were separated into nuclear, mitochondrial, and postmitochondrial fractions by differential centrifugation, over 75% of DAZL1 was detected in the PMS fraction (data not shown). Further fractionation of PMS on 15-45% sucrose gradients showed that most DAZL1 comigrated with the polysomes (Fig. 4a), similar to FMRP, which was shown previously to be associated with polysomes. Other proteins that cross-reacted with the antiserum stayed at the top of the gradients (data not shown). Inclusion of 30 mM EDTA in the testicular extracts dissociated polysomes into 60S and 40S subunits, and caused a shift of DAZL1 to the top fractions of the gradients (Fig. 4b). On a 5-30% sucrose gradient, the DAZL1 protein in the EDTA-treated extracts showed a more heterogeneous distribution than that of FMRP (Fig. 4e). Addition of 0.5 M KCl, which is known to remove translation factors and ami-noacyl-tRNA synthetases, but not most intrinsic ribosomal proteins, dissociated most of DAZL1 from the polysomes (Fig. 4c). RNase A treatment of PMS separated the polysomes into monosomes and caused a significant reduction in the sedimentation of DAZL1 (Fig. 4d). The sedimentation profiles of DAZL1 under the various conditions indicate that most DAZL1 in mouse testes is associated with polysomes.
Capture of DAZL1-Associated mRNP
To investigate whether DAZL1 is associated with polysomes through its binding to mRNA molecules, poly(A) containing mRNP particles in PMS were captured on oli-go(dT) beads, and the presence of DAZL1 in the captured mRNP fractions was analyzed by SDS-PAGE followed by Western blotting. DAZL1, but not other cytoplasmic proteins that cross-reacted with anti-DAZLl, was captured on oligo(dT) beads (Fig. 5a). FMRP was also captured on oli-go(dT) beads, as previously reported. Capture of dAzLI on oligo(dT) was not affected by 30 mM EDTA but was reduced by preincubation with RNase and abolished by NaOH treatment, further supporting the binding of DAZL1 to poly(A) RNA (Fig. 5b).
FIG. 3. Identification of DAZL1 in mouse tissue extracts. a) Detection of DAZL1 by Western blotting with anti-DAZL1 antibody. Extracts of mouse liver, brain, and testis, and in vitro-synthesized DAZL1 were fractionated by SDS-PAGE and Western blotted with anti-DAZL1 antiserum. The arrowhead points to the putative DAZL1 protein. b) Inhibition of immunodetection of DAZL1 by in vitro-synthesized DAZL1. Anti-DAZL1 antiserum was preincubated with the indicated volume ofTNT protein synthesis reaction before incubation with membranes containing equal amounts of mouse liver (L) and testis (T) extracts. T3 and T7 RNA polymerases transcribed the Dazl1 cDNA clone in the sense and antisense directions, respectively.
FIG. 4. Sucrose gradient analyses of DAZL1 in mouse testes. Mouse testis PMS in the presence of 5 mM MgCl2 (a), 30 mM EDTA (b, e), or 0.5 M KCl (c), or after RNase A treatment (d), were fractionated by centrifugation through 15-45% sucrose gradients (except 5-30% in e). Aliquots of the fractions were analyzed on 10% SDS-PAGE gels and then Western blotted with antibodies against DAZL1, FMRP, and a human autoantibody against ribosomal P antigens (P-Ag).
FIG. 5. Association of DAZL1 with poly(A) mRNA. a) Poly(A) containing RNP particles in mouse PMS were captured on oligo(dT) beads, and the presence of DAZL1 and FMRP in the captured RNP particles was analyzed by 10% SDS-PAGE followed by Western blotting. L, An amount equivalent to 8% of the initial load to the beads; W, the wash; and B, the bound fraction. b) Mouse testis PMS was pretreated with EDTA, RNase, or NaOH before oligo(dT) capture, and the presence of DAZL1 in the captured fraction was analyzed.