| CONTENTS Main Page Dynamic Development
The Foundations of Developmental Biology
Gametogenesis
From Sperm and Egg to Embryo
Genetic Regulation of Development
Organizing the Multicellular Embryo
Generating Cell Diversity
Dynamic Development at a Glance
Learning Resources
Research Resources
The Developmental Biology Journal Club
Developmental Biology Tutorial |
Regulation of Protein Synthesis during Oogenesis, Oocyte
Maturation and Early Development
As you know, protein synthesis is required for oocyte maturation and early development,
but in many organisms initial protein synthesis utlilizes transcripts that are synthesized
during oogenesis and stored for later utilization. Furthermore, the utilization of
transcripts during development is very selective. What mechanisms select among transcripts
and determines which ones will be translated during oogenesis, which will not, and when
and where the stored transcripts will be utilized? This problem has been studied
extensively in Xenopus, and we shall use this species to focus on this problem.
One mechanism controlling translatability of some oogenic messengers is the level of
polyadenylation of those transcripts; the determinant of their polyadenylation is located
in their 3' UTR. One class of messengers is translationally inert during oogenesis and is
translationally activated during oocyte maturation (i.e., during the transition from
prophase I to metaphase II of meiosis). Two cis-acting elements are necessary for
this mode of regulation: the nuclear polyadenylation signal (AAUAAA; also called A2UA3)
and a U-rich cytoplasmic polyadenylation element (CPE). The sequence of the CPE and its
position relative to A2UA3 control the extent and timing of polyadenylation (Wormington,
1994; Vassalli and Stutz, 1995). Some transcripts respond to poly(A) tail length, whereas
others require the act of polyadenylation itself.
Translation of the transcripts encoding the cell-cycle regulatory proteins cyclin and c-mos
is controlled by polyadenylation; their translation - in turn - controls progression
through the meiotic cell cycle. You will recall that maturation of the oocyte is triggered
by progesterone, which is produced by the follicle cells surrounding the oocyte in
response to pituitary gonadotropins. Multiple cyclins (A1, A2, B1, and B2) have been
identified, which are synthesized during either oocyte maturation or after fertilization.
Although the exact roles of the various cyclins are unknown, translation of cyclin B is
necessary for the cleavage mitoses. Synthesis of c-mos is essential for maturation.
The synthesis of these various proteins is under very fine control. The messengers
encoding these proteins are present in the full-grown oocyte, during maturation, and after
fertilization, but there are dramatic differences in their translatability. Among the
cyclins, cyclin A1 accumulation is very low in oocytes and increases during maturation,
whereas both cyclin B1 and B2 accumulate to relatively high levels in oocytes (Kobayashi et
al., 1991, Figs. 9 and 3 ). Cyclin A2, on the other hand, does not accumulate in
significant amounts until after the mid-blastula transition (Howe et al., 1995,
Fig. 4).
Sheets et al. (1994) examined the possibilty that translational activation of
messengers encoding c-mos and cyclins A1, B1, and B2 during maturation is regulated
by cytoplasmic polyadenylation. The lengths of poly(A) tracts on the endogenous
transcripts were measured at different times after treatment of oocytes with progesterone.
To monitor poly(A) length, total RNA was isolated and annealed to a DNA oligo
complementary to a region 200 nucleotides upstream of the poly(A) tail. Half of each
sample was also annealed to oligo(dT). Both samples were then treated with RNAse H, and
the lengths of the 3' UTR were determined by Northern blotting, using a 3' UTR-specific
probe. The length of the poly(A) was the difference in length between the RNAs treated
with or without oligo(dT). Results are shown in Figs. 1 and 2 and summarized in Fig. 3
(Sheets et al., 1994). The data indicate a complex pattern of changes in poly(A)
tail length; different transcripts lengthen to different extents with different kinetics:
c-mos and cyclin B1 poly(A) increased in length by 70 and 220 nucleotides,
respectively within 4 hours of progesterone treatement, whereas cyclins B2 and A1
increased by 80 and 100 nucleotides, respectively 5 hours later. Whereas the elongated
poly(A) is retained on the cyclin messengers in 2-cell embryos, c-mos undergoes
poly(A) shortening late in maturation and becomes completely deadenylated after
fertilization.
Do the 3'UTRs of the transcripts possess the information that is sufficient to regulate
polyadenylation? To examine this possibility, Sheets et al. microinjected RNAs
containing only the terminal 100 nucleotides of these transcripts into oocytes and treated
them with progesterone. The RNA was recovered and the length of poly(A) added to each was
determined. The injected RNA fragments replicate the polyadenylation patterns of the
endogenous transcripts, indicating that the 3' UTR is sufficient to regulate this
behaviour (Sheets et al., 1994, Fig. 4).
Does the polyadenylation of these transcripts influence their translation? To examine this
possibility, Sheets et al. fused the cyclin A1, B1, and c-mos UTRs to the
luciferase coding region, synthesized RNA in vitro, injected the RNA into oocytes,
and determined both the polyadenylation status and the level of luciferase activity in the
presence and absence of progesterone (Sheets et al., 1994, Fig. 6). The ratio of
luciferase activity in the matured vs. nonmatured oocytes was used as an index of
translational stimulation. Mutations in the regions that control polyadenylation prevent
translational activation. Sheets et al. concluded that each of the 3' UTRs
stimulated translation during maturation as a result of their polyadenylation. In
addition, the extent of translational stimulation can be correlated with the length of
added poly(A). As shown in Figure 3 (Sheets et al., 1994), the chronology of
poly(A) addition is quite different for some of these transcripts. Cyclins A1 and B1
represent two extremes; the former is polyadenylated several hours after the latter. Do
the 3' UTRs exert this temporal control? The data in Fig. 7 (Sheets et al., 1994)
suggest that they do.
The effects of poly(A) on translation are intriguing, because protein synthesis is
initiated at the opposite, or 5', end of RNA. One hypothesis for the mechanism underlying
this effect is that 3' polyadenylation induces 5' cap ribose methylation (Kuge and
Richter, 1995). There is considerable evidence that not only poly(A) but also other 3'
elements can exert profound influences on translation. It is clear that long-range
interactions occur in RNA such that 3' elements can influence events occuring at the 5'
end. One possibility is that the 3' end of RNA can influence the effectiveness of
translation initiation factors in protein synthesis.
The messenger encoding the FGF receptor-1 (XFGFR-1) provides further evidence for
long-range interactions in regulation of translation of oogenic mRNA. This receptor is
involved in mesoderm induction, as demonstrated by mesoderm insufficiency and repression
of mesoderm-specific gene expression when FGF signaling is inhibited in Xenopus
embryos (Amaya et al., 1991, Cell 66: 257). FGF signaling is also required for
activin-mediated induction of mesoderm (Cornell and Kimelman, 1994, Development 120: 453;
LaBonne and Whitman, 1994, Development 120: 463). Thus, the appropriate blastomeres must
display the appropriate surface receptors at the right time to be able to respond to the
specific signals. Because mesoderm induction occurs before the onset of zygotic
transcription, the FGF receptor must be encoded by oogenic messengers, and translation of
those messengers must be regulated to ensure that the receptor is present in the right
cells at the right time and in sufficient amounts.
The XFGFR-1 messenger is present in oocytes and early embryos. The messenger is not
translated in immature oocytes; translation is initiated at oocyte maturation, coincident
with a lengthening of the poly(A) tail (Fig. 1; Robbie et al., 1995). Robbie et
al. examined the possibility that a cis-acting signal in the 3' UTR represses
translation in the immature oocyte. Their results (Fig. 2) show that the 3' UTR is
sufficient to repress translation and that the repression is specific to oocytes. To
examine whether the inhibition by the 3' UTR is intrinsic to the UTR or whether an
interaction between it and the coding sequence is necessary, chimeric constructs
incorporating 3' UTR elements and a truncated human EGF receptor coding sequence (HED)
were produced. As shown in Figure 3, the XFGFR-1 UTR was sufficient to inhibit
translation.
Deletion analysis was conducted to determine which portion(s) of the UTR are responsible
for translation inhibition. The results indicate that a 180 nucleotide proximal region was
sufficient to inhibit translation. This portion of the UTR was coined the translation
inhibitory element (TIE; Fig. 4, Robbie et al., 1995). Is polyadenylation involved
in either translation inhibition or release of inhibition at oocyte maturation? To examine
the possible involvement of polyadenylation in translation in immature oocytes,
truncations to remove the synthetic poly(A) and the A2UA3 polyadenylation elements were
made. As shown in Fig. 5 (Robbie et al., 1995), inhibition is not dependent upon
downstream elements. RNA recovered from injected oocytes is the same length as the
uninjected RNA, indicating that no polyadenylation had occurred.
Because the translation of endogenous XFGFR-1 mRNA is triggered at oocyte maturation, one
would predict that the inhibitory effects of the TIE are reversed at maturation. Oocytes
were injected with transcripts containing the TIE, treated with progesterone, and assayed
for protein. As shown in Figure 6 (Robbie et al., 1995), the inhibition caused by
TIE is relieved by progesterone treatment.
As shown in Figure 1 (Robbie et al., 1995), the endogenous RNA lengthened at oocyte
maturation. Based upon the apparent effects of polyadenylation on recruitment of many
kinds of RNA onto polysomes at oocyte maturation, it would be reasonable to assume that
polyadenylation triggers the translation of XFGFR-1 in response to progesterone. However,
as shown in Figure 6, the injected RNA showed no significant increase in size. (Keep in
mind that no cytoplasmic polyadenylation elements are present in these transcripts. This
result suggests that activation of XFGFR-1 translation at maturation does not require
polyadenylation.
Once again, the question of how a 3' UTR element can regulate translation becomes
relevant. Is repression of translation in oocytes dependent upon the binding of protein to
TIE? A gel retardation assay was conducted to detect RNA-protein binding (Robbie et al.,
1995, Fig. 7). Radiolabeled RNA was incubated with oocyte protein and subjected to
electrophoresis. Proteins in the extract caused the RNA to shift; the shift pattern was
distinct from that of the coding region. To examine the binding of protein to TIE, the
authors conducted UV cross-linking of protein with radioactive TIE. The predominant
labeled protein runs at 43kDa. Unlike the control probe, binding of the protein to TIE is
competed out by excess cold probe, indicating the specificity of the binding of p43 to
TIE. Further work will be necessary to determine how derepression of translation occurs.
If p43 is responsible for repression, there is likely a mechanism triggered by
progesterone that eliminates its repressive effects, probably by dissociating the TIE/p43
association. One common way to alter protein function is by changing its phosphorylation
status. Because we know that a number of changes in protein phosphorylation occur at
maturation, this is one possibility that should be examined.
Digging Deeper:
deMoor, C.H. and Richter, J.D. 1997. The Mos pathway regulates cytoplasmic
polyadenylation in Xenopus oocytes. Mol. Cell. Biol. 17: 6419-6426.
Gebauer, F., Xu, W., Cooper, G.M. and Richter, J. D. 1994. Translational control by
cytoplasmic polyadenylation of c-mos mRNA is necessary for oocyte maturation in the mouse.
EMBO J 13: 5712.
Lieberfarb, M. E., Chu, T., Wreden, C., Theurkauf, W., Gergen, J. P. and Strickland, S.
1996 .Mutations that perturb poly (A) - dependent maternal mRNA activation block the
initiaion of development. Development 122: 579-588.
Weinstein, D. C., Honore,
E. and Hemmati-Brivanlou, A. Epidermal induction and inhibition of neural fate by
translation initiation factor 4AIII.
Wu, L., Good, P.J. and Richter, J.D. 1997. The 36-kilodalton embryonic-type cytoplasmic
polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the
ELAV family of RNA-binding proteins. Mol. Cell. Biol. 17: 6402-6409.
References
Howe, J.A., Howell, M., Hunt, T. and Newport, J.W. Identification of a developmental timer
regulating the stability of embryonic cyclin A and a new somatic A-type cuclin at
gastrulation. Genes & Dev. 9: 1164-1176.
Kobayashi, H., Minshull, J., Ford, C., Golsteyn, R., Poon, R. and Hunt, T. 1991. On the
synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation
in Xenopus laevis. J. Cell Biology114: 755-765.
Kuge, H. and J.D. Richter. 1995. Cytoplasmic 3' poly(A) addition induces 5' cap ribose
methylation: implications for translational control of maternal mRNA. EMBO J. 14:
6301-6310.
Robbie, E. P., Peterson, M., Amaya, E. and Musci, T. J. 1995 Temporal regulation of the
Xenopus FGF receptor in development: a translation inhibitory element in the 3'
untranslated region Development 121: 1775-1785
Sheets, M. D., Fox, C. A., Hunt, T., Vande Woude, G. and Wickens, M. 1994. The
3'-untranslated regions of c-mos and cyclin mRNAs stimulate translation by
regulating cytoplasmic polyadenylation. Genes & Development 8: 926-938.
Vassalli, J.-D. and Stutz, A. 1995. Awakening dormant mRNAs. Current Biology 5 476-479.
Wormington, M. 1994. Unmasking the role of the 3' UTR in the cytoplasmic polyadenylation
and translational regulation of maternal mRNAs. Bioessays 16: 533-535.
|
