25-07-2012, 02:30 PM
Selective degradation of cyclin B1 mRNA in rat oocytes by RNA interference (RNAi)
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Abstract
Cyclic adenosine monophosphate (cAMP) keeps oocytes in meiotic arrest, thereby preventing activation
of the key regulators of meiosis, p34cdc2/cyclin B1, (known as maturation-promoting factor (MPF)) and
Erk 1 and 2, members of the mitogen-activated protein kinase (MAPK) family. The activity of MAPK in
oocytes is upregulated by Mos. We previously demonstrated that Mos translation in rat oocytes is
negatively regulated by a PKA-mediated cAMP action, which inhibits c-mos mRNA polyadenylation and
is associated with the suppression of p34 cdc2 kinase. The goal of the present study was to provide
definitive evidence that Mos translation is subjected to MPF regulation. In order to inhibit MPF activity, we
employed the double-stranded (ds) RNA interference (RNAi) of gene expression. We demonstrated that
the introduction of cyclin B1 dsRNA into rat oocytes selectively depleted the corresponding mRNA, further
ablating its protein product. These oocytes, which exhibit low MPF activity, failed to elongate the c-mos
mRNA poly(A) tail, did not accumulate Mos and were unable to activate MAPK. We conclude that an
active MPF in rat oocytes is necessary for c-mos mRNA polyadenylation and Mos translation.
Journal of Molecular Endocrinology (2004) 33, 73–85
Introduction
Meiosis in oocytes of all animals is arrested at the
prophase that corresponds to the late G2-phase of
the cell-division cycle. The G2-arrested oocytes
are characterized by a distinct nuclear structure
known as germinal vesicle (GV). Oocytes that
escape G2-arrest undergo GV breakdown (GVB)
and chromosome condensation. These oocytes
proceed to the first metaphase (MI), emit the first
polar body (PBI) and are arrested again at the
second metaphase (MII) of meiosis. This process,
defined as oocyte maturation, is subjected to
regulation by the maturation-promoting factor
(MPF), which comprises a catalytic 34 kDa
Ser/Thr kinase, p34 cdc2, and a regulatory
45 kDa cyclin B1 (reviewed by Dekel 1996).
Materials and methods
Reagents and antibodies
Leibovitz’s L-15 tissue culture medium and
Lipofectamine were purchased from Gibco BRL
(Paisley, UK). Antibiotics were purchased from
Bio-Lab Ltd (Jerusalem, Israel). PKI, isobutylmethylxantine
(IBMX), N6, 2-o-dibutyryladenosine
3:5-cyclic monophosphate (dibutyryl cyclic AMP,
dbcAMP), Nonident P-40, -glycerophosphate,
PMSF, leupeptine, aprotonin, DTT, ethidium
bromide, agarose and fetal bovine serum were
purchased from Sigma. Affinity-purified polyclonal
goat anti-Mos antibody, mouse anticyclin B1 and
mouse anti-p34 cdc2 were purchased from Santa
Cruz, Inc. (Santa-Cruz, CA, USA).
Ribonuclease protection assay
Volumes of 350 μl digestion buffer (300 mM NaAc,
10 mM Tris, 5 mM EDTA), 4 μg RNase A and
4 U RNase T1 were added to each sample. After
1-h incubation at 30 C, 10 μl of 20% SDS and
2·5 μl of 10 mg/ml proteinase K were added, and
the samples were incubated for 20 min at 37 C.
The products were extracted with 400 μl of
phenol/chloroform/isoamyl alcohol (25:24:1). The
supernatant was transferred into 1 ml 100% EtOH
and 10 μg of glycogen and incubated at 70 C
for 30 min. Samples were centrifuged for 15 min,
EtOH was aspirated and pellets were allowed to
dry.