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Dev Growth Differ
2009 Aug 01;516:595-605. doi: 10.1111/j.1440-169X.2009.01121.x.
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Xtr, a plural tudor domain-containing protein, coexists with FRGY2 both in cytoplasmic mRNP particle and germ plasm in Xenopus embryo: its possible role in translational regulation of maternal mRNAs.
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Xtr is present exclusively in early embryonic and germline cells. We have previously shown that loss-of-function of the Xtr in embryos causes arrest of karyokinesis progression. Since Xtr contains plural tudor domains, which are known to associate with target proteins directly, we examined Xtr-interacting proteins by immunoprecipitation with an anti-Xtr monoclonal antibody and detected a few RNA-binding proteins such as FRGY2, a component of messenger ribonucleoprotein (mRNP) particle. The coexistence of Xtr with FRGY2 by constituting an mRNP particle was further confirmed by gel filtration assay. Search of mRNAs in the immunoprecipitate with Xtr suggested that the Xtr-associated molecules included several mRNAs, of which translational products were known to play crucial roles in karyokinesis progression (RCC1, XRHAMM, and so on) and in germ cell development (XDead end). Immunohistochemical observation clearly showed the co-localization of Xtr with FRGY2 also in germ plasm, in which XDead end mRNA has been shown to be localized specifically. Taken together, we proposed the possible role of Xtr in translational activation of the maternal mRNAs repressed in mRNP particle.
Fig. 2. Reverse transcription–polymerase chain reaction (RT–PCR)
of normal mouse IgG (NMI)- or anti-Xtr-coimmunoprecipitated
mRNAs from the supernatant of centrifuged eggs using oligo dT
primer for RT and gene-specific primers (RCC1, XRHAMM,
XL-INCENP, XMad2, XDead end, and SP4) for PCR. W: Total RNA
(1 lg) from fertilized eggs (30 min after insemination) was used
for RT–PCR. Spermatogenic cell-specific SP4 mRNA was
added to the immunoprecipitates with NMI (NMI) and anti-Xtr
mAb (a Xtr) for comparing the recovery of RNA during
purification process and efficiency of RT–PCR between the
immunoprecipitates.
Fig. 3. (A) Gel filtration of the supernatant of ultracentrifuged eggs with Sephacryl S-300HR column with HB. Two milliliters of the
supernatant was applied onto the column (diameter 1.6 cm, height 55 cm) and 3 mL fractions were collected. Arrowheads show the
fractions in which thyroglobuline (T; M.W. approximately 670 kDa) and catalase (C; M.W. approximately 250 kDa) were eluted,
respectively. (B) Western blot analysis of gel-filtrated fractions using anti-Xtr (upper panel) and anti-FRGY2 (lower panel) rabbit antisera.
The numbers and SM above the upper panel represent the fraction numbers and start material (the supernatant; 5 lg), respectively. The
elute (fractions 16–19 as indicated with a bidirectional arrow in A) was pooled. (C) Western blot analysis of immunoprecipitates from
pooled fractions with normal mouse IgG (NMI) (lane d) and anti-Xtr mAb (lane e). Detection of Xtr and FRGY2 was carried out by using
anti-Xtr (upper panel) and anti-FRGY2 (lower panel) rabbit antisera, respectively. Xtr and FRGY2 in supernatant of centrifuged eggs
(10 lg protein each) (at 15 000 g in lane a and 150 000 g in lane b) and the pooled fractions (1 lg protein) (lane c) were also detected.
Note that FRGY2 was coimmunoprecipitated with Xtr. (D) Reverse transcription–polymerase chain reaction (RT–PCR) of NMI- or anti-Xtr
mAb-coimmunoprecipitated mRNAs from the pooled fractions 16–19 from gel filtration using oligo dT primer for RT and gene-specific
primers (RCC1, XRHAMM, XL-INCENP, XMad2, XDead end, and SP4) for PCR. W indicates total RNA (1 lg) from fertilized eggs
(30 min after insemination). Spermatogenic cell-specific SP4 mRNA was added to the immunoprecipitates with NMI (NMI) and anti-Xtr
mAb (a Xtr) for comparing the recovery of RNA during purification process and efficiency of RT–PCR between the immunoprecipitates.
Fig. 4. Effect of RNase A treatment of egg homogenate on
interaction between Xtr and FRGY2. Supernatant of egg
homogenate was treated with (+) or without ()) RNase A.
Western blot analyses of immunoprecipitates with normal mouse
IgG (NMI) and anti-Xtr mAb (a Xtr) were carried out using anti-Xtr
(top panel) and anti-FRGY2 (middle panel) rabbit antisera as
probes. The efficacy of RNase A treatment was assessed by
analyzing total RNA from the supernatant (bottom panel).
Fig. 5. (A) Subcellular localization of Xtr and FRGY2 in a 32 cell-stage embryo. Three serial sections were treated with anti-Xtr (a, a’, b,
and b’) or anti-FRGY2 (c and c’) rabbit antiserum or normal rabbit serum (d and d’) and then with fluorescein isothiocyanate (FITC)-
conjugated anti-rabbit IgG antibody. The upper panels (a–d) represent the bright-field images, while the lower panels (a’–d’) show the
corresponding fluorescence images of the same sections. b and b’ show higher magnification views of the boxed areas in a and a’,
respectively. Scale bars represent 200 lm (a and a’) and 20 lm (b, b’, c, c’, d, and d’). ap and vp in a represent the position of animal
pole and vegetal pole of the embryo, respectively. (B) Photographs of fluorescent images at vegetal hemisphere of activated eggs that
had been injected with either enhanced green fluorescent protein (EGFP)-Xtr mRNA (a and a’) or EGFP mRNA (b and b’) at fully-grown
oocyte stage, induced the maturation by progesterone treatment, and activated by pricking. Two hours after egg activation, the eggs
were stained with MitoTracker and accumulation of EGFP-tagged Xtr (a), EGFP (b), and mitochondria (a’ and b’) at the vegetal pole
were observed. Arrowheads represent the areas of germ plasm. Scale bar represents 500 lm. (C) Western blot analyses of
immunoprecipitates with anti-Xtr mAb from supernatant of animal half (a) and vegetal half (b) of early blastula using anti-Xtr (upper panel)
and anti-FRGY2 (lower panel) antisera.
