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Fig. 6. RNA binding and translational control by CPEB. (A) Extracts
from 3-week-old mice were incubated with CPEB antibody or IgG and subjected to
immunoprecipitation followed by RNA extraction and RT-PCR for specific mRNAs.
Two to 10% of the RNA was used for the loading control; RT refers to no
reverse transcription. The sequence of the putative CPE and its distance from
the AAUAAA is indicated for each RNA, as is the function of the encoded
protein. (B) The upper panel shows the scheme of the chromatography
procedure on poly (U) Sepharose followed by thermal elution. RNAs with shorter
poly(A) tails elute at lower temperatures than those mRNAs with longer tails,
and are detected by RT-PCR. The lower panel shows the distribution of the
RT-PCR products following chromatography. In wild-type ovaries, all the RNAs
eluted mostly at 45°C, but also at 60°C, indicating that the poly(A)
tails were heterogeneous in length and some were long enough to elute only at
the higher temperature. By contrast, all the RNAs from the TG ovaries except
-tubulin, Zp1 and Mater that lack a CPE eluted
completely at 45°C. These results suggest that the poly(A) tails of these
RNAs were short in the TG ovaries because they eluted at a lower temperature.
(C) Western blot shows that GDF9 (preprocessed) levels were
substantially reduced in all TG lines compared with wild type. NS, a
non-specific loading control.
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