Analysis of Biological Development (K. Kalthoff)

Updates to Topic 18: Translational Control and Posttranslational Modifications


Answers to Questions in Text

Polyadenylation of c-mos mRNA in Xenopus oocytes (p. 460/461)

  1. The few oocytes that matured in spite of injection with antisense oligonucleotide contained full-length c-mos mRNA (Fig. 18.9e, lane 3). Why is this result significant? Answer: This result confirms that polyadenylated c-mos mRNA is necessary for oocyte maturation.
  2. The investigators also tested whether the polyadenylation elements near the 3' end of c-mos mRNA can confer polyadenylation and translational activation to other mRNAs. In these experiments, they also showed that the polyadenylation signals of c-mos mRNA stimulate translational activation before GVBD. How do you think they accomplished these goals? Answer: They constructed a fusion mRNA combining the translated region for a reporter protein (luciferase) with the 3' UTR containing the polyadenylation signals of c-mos mRNA. The latter conferred translatability to the reporter.

Stage-specific protein synthesis in oocytes and eggs (p. 464/465)

  1. Ascribing the synthesis of stage-specific proteins in Spisula as seen in Fig. 18.12 to selective recruitment of mRNAs into polysomes implies that oocytes, eggs, and embryos contain identical inventories of mRNAs. In other words, it is assumed that there is no selective synthesis or degradation of specific mRNA molecules between these stages. What would be a direct way of testing this implication? Answer: The investigators prepared total cytoplasmic mRNA from starfish oocytes, eggs, and embryos and purified the RNA by extraction with phenol to remove any associated proteins. Samples from each mRNA preparation were translated in a cell-free translation system. The results were striking: mRNAs from all stages directed the synthesis of the same proteins in vitro. In particular, those proteins that had shown major synthetic rate changes in vivo were synthesized at the same rate in vitro using mRNA from all stages (Rosenthal et al. (1982) Devel. Biol. 91: 215-220)
  2. Do the results shown in Figs. 18.12 and 18.13 show which of the translational control mechanisms discussed in Section 18.2 are involved in this case? Answer: Any control mechanism that affects specific mRNAs can account for these data. However, follow-up experiments indicate that the mRNAs for proteins A and C are masked, during the oocyte stage, by an 82 kDa protein bound to their trailer regions (Walker et al. 1996, Devel. Biol. 173: 292-305).

Translational Control during Spermiogenesis (p. 467/468)

  1. The first round of spermatogenesis in the mouse is synchronous. How does this fact facilitate collection of the data discussed above? Answer: Yes. It makes it easy to establish a time table of morphological and molecular events during the first round of spermiogenesis.
  2. A transgene introduced into a diploid cell, such as a primordial germ cell, is usually integrated only into one chromosome of a homologous pair. Thus, after meiosis, the transgene will be present only in half of the gametes. Is this a complication for the experiments reviewed here? Answer: No. Because the developing spermatids and spermatozoa are connected by cytoplasmic bridges (see Fig. 3.8), they will exchange mRNAs transcribed from resident genes as well as transgenes. Even if the mRNAs and proteins derived from the transgenes would be restricted to half of the developing germ cells they would still be detectable. The probes used to detect the human reporter gene products were designed not to cross-react with mouse gene products.
  3. How may the investigators proceed to delimit the exact sequence within the mP1 trailer region that is sufficient for delaying mP1 mRNA translation? Answer: By deletion mapping, that is, by deleting various portions of the mP1 trailer region of the transgene and testing the modified chimeric mRNA for conservation and delayed translation.

Comments

A class of small regulatory RNAs known as microRNAs (miRNAs) controls mRNA survival and/or translation similar to small interfering RNA (siRNA). Both siRNA and miRNA are cut by dicer from larger double-styranded RNA precursors (see Comments to Chapter 15). And both miRNAs and siRNAs act either by causing the destruction of their target RNAs or by preventing their transl;ation. However, while siRNA come from the same genes that they ultimately silence, miRNAs come from different genes whose sole function is to produce the small regulatory RNAs. One of the first miRNAs to be discovered is the one encoded by the lin-4 gene of C. elegans (see p. 680). Since then, dozens if not hundreds of miRNAs have been identified in C. elegans, Drosophila, plants, and the human (Carrington and Ambros, 2003).

Clarifications and Corrections

p. 458, Fig. 18.6. The two yellow proteins should be labeled "4E-BP" insead of "F4-BP".

New Review Articles

Carrington J.C. and Ambros V. (2003) Role of microRNAs in plant and animal development. Science 301: 336-338.

Cooperstock R.L., Lipshitz H.D. (2001) RNA localization and translational regulation during axis specification in the Drosophila oocyte. Int. Rev. Cytol. 203: 541-566

Leatherman J.L. and Jongens T.A. (2003) Transcriptional silencing and translational control: key features of early germ line development. BioEssays 25: 326-335

Mendez R., Richter J.D. (2001) Translational control by CPEB: a means to the end. Nature Rev. Mol. Cell Biol. 2: 521-529

Preiss T. and Hentze M.W. (2003) Starting the protein synthesis machine: eukaryotic translation initiation. BioEssays 25: 1201-1211

New Research Articles

Hodgman R., Tay J., Mendez R., Richter J.D. (2001) CPEB phosphorylation and cytoplasmic polyadenylation are catalyzed by the kinase IAK1/Eg2 in maturing mouse oocytes. Development 128: 2815-2822.

Mendez R., Hake L.E., Andresson T., Littlepage L.E., Ruderman J.V., Richter J.D. (2000) Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA. Nature 404: 302-307.

Mendez R., Murthy K.G., Ryan K., Manley J.L., Richter J.D. (2000) Phosphorylation of CPEB by Eg2 mediates the recruitment of CPSF into an active cytoplasmic polyadenylation complex. Mol Cell 6: 1253-1259.

Stebbins-Boaz B., Cao Q., de Moor C.H., Mendez R., Richter J.D. (1999) Maskin is a CPEB-associated factor that transiently interacts with elF-4E. Mol Cell. 4: 1017-1027.

These studies address the mechanism that connects the cytoplasmic polyadenylation of mRNAs with their translational activation in the oocytes of frogs and mice. Cytoplasmic polyadenylation depends on two regulatory sequences in the 3' UTR: the AAUAAA polyadenylation signal and an adjacent cytoplasmic polyadenylation element (CPE). The CPE is bound by CPEB, a sequence-specific RNA binding protein. In the dormant oocyte, CPEB is associated with the protein Maskin, which also interacts with the cap-associated initiation factor eIF4E. This interaction prevents eIF4G from binding to eIF4E and recruiting the ribosomal initiation complex to the cap site. Polyadenylation is initiated by the phosphorylation of CPEB by a kinase known as Eg2 in Xenopus and IAK1 in mice. The phosphorylated CPEB recruits a group of proteins collectively called the cleavage and polyadenylation-specific factor (CPSF) and poly(A) polymerase to the AAUAAA sequence, with poly(A) tail formation ensuing. At the same time, eIF4E dissociates from Maskin and allows eIF4G to bind, so that translation is initiated.

Groisman I., Jung M.Y., Sarkissian M., Cao Q., Richter J.D. (2002) Translational control of the embryonic cell cycle. Cell 109: 473-483.

Cell cycle progression is regulated at the level of cyclin B synthesis and destruction. The investigators prepared cycling extracts from Xenopus embryos and found that progression into M phase requires the polyadenylation-induced translation of cyclin B1 mRNA. Polyadenylation requires the binding of cytoplasmic polyadenylation elements (CPEs) by a CPE-binding protein (CPEB). This step is mediated by the phosphorylation of CPEB by Aurora, a kinase whose activity oscillates with the cell cycle. Exit from M phase seems to require deadenylation and subsequent translational silencing of cyclin B1 mRNA by Maskin, a CPEB and eIF4E binding factor, whose expression is cell cycle regulated. These observations suggest that regulated cyclin B1 mRNA translation is essential for the embryonic cell cycle. Mammalian cells also display a cell cycle-dependent cytoplasmic polyadenylation, suggesting that translational control by polyadenylation might be a general feature of mitosis in animal cells.


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