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Fig. 5. XSu(H) mRNA is regulated by EDEN-BP. (A) Radiolabelled
GbORF-mosEDEN (lanes 1-4), ESR5 3'UTR (lanes 5-8), X-Delta2 3'UTR
(lanes 9-12) and XSu(H) 3'UTR (lanes 13-16) transcripts were incubated
in Xenopus egg extracts in the absence or the presence of the
indicated molar excess of unlabelled RNA with an EDEN (+E, GbORF-mosEDEN) or
with the EDEN deleted (–E, GbORF). After UV-cross-linking and RNase
treatment, radiolabelled proteins were resolved by denaturing electrophoresis
and revealed by autoradiography. The position of the 51 kDa molecular weight
marker is indicated on the left. The asterisks indicate the position of the
EDEN-BP signal. (B) Embryo extracts were treated for immunoprecipitation with
anti-EDEN-BP antibodies (lanes 7-8), or pseudo-immunoprecipitations with beads
alone (lanes 3-4) or non-immune serum (lanes 5-6). RNA present in the
supernatant (S) or pellet (P) was identified by RT-PCR using primers specific
for XSu(H) (upper panel) or EF1
(lower panel). Lanes 1 and 2 show
products from PCR performed on the input fraction, either with (lane 2) or
without (lane 1) previous reverse transcription. Positions of molecular weight
markers are indicated on the left. (C) A capped, radiolabelled, polyadenylated
transcript corresponding to GbXSu(H) was injected into two-cell embryos in the
presence of 150 ng of the control (C-Ab, lanes 6-10) or anti-EDEN-BP (E-Ab,
lanes 1-5) immunoglobulins, and incubated for the indicated times. RNA was
extracted, and analyzed by electrophoresis and autoradiography. Positions of
A+ (A65) and A– (A0) RNAs are indicated. (D)
Quantification of C. The amount of deadenylated GbXSu(H) transcript, expressed
as a percentage of the total signal, is plotted against time. (E) Relative
quantities of XSu(H) and EF1 mRNA were quantified by real-time RT-PCR
in stage 25 embryos that had been previously injected in both blastomeres at
the 2-cell stage with EDEN-BP-Mo (E-Mo), EDEN-BP-Mo and 2 fmol of EDEN-BP mRNA
(E-Mo+R), or that had been left uninjected (NI). Results, expressed as the
ratio between XSu(H) and EF1 mRNAs, are shown for five independent
embryos. Statistical analysis showed that the ratios are significantly
different between EDEN-BP-Mo-injected embryos, and non-injected or morpholino
and RNA-injected embryos (Student's t-test, P<0.05).
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