Gene expression can be regulated at several different levels. While the primary control of gene expression is at the level of transcription (synthesis of specific mRNAs from a DNA template) in recent years it has become apparent that regulation at the level of translation (the synthesis of proteins from messenger RNA) is also very important.
Translational regulation linked to changes in mRNA poly(A) tail length is necessary for progression through meiosis, early development and localized translation at the neuronal synapse. This mechanism is called “polyadenylation induced translation”. Essentially, mRNAs containing a long poly A tail (50-300 nt) are translated, whereas those with a short poly A tail (<50 nt) are not. Molecular events that alter the length of the poly A tail can therefore directly influence the translation of mRNA.
C-mos mRNA in the prophase I oocyte is translationally repressed by interaction between CPEB, a repressing protein complex and the cap binding protein eIF4E. During progesterone stimulated meiotic resumption, XGef influences the phosphorylation of CPEB by Aurora A kinase. This activates CPEB, which then recruits a complex of proteins, cleavage and polyadenylation specificity factor and poly A polymerase (CPSF and PAP), to the mRNA, and the poly A tail is elongated. Multiple molecules of poly A binding protein (PABP) then associate with the poly A tail and recruit eIF4G to the mRNA. This assists in displacement of the repressing complex, and the 43 S ribosome is recruited to the mRNA to initiate the scanning phase of translation initiation.
Our research is currently focused on dissecting the molecular machinery of polyadenylation-induced translation and the signal transduction cascade that regulates this process during Xenopus oocyte meiosis.
Oocytes in most metazoans are frozen in prophase of meiosis I. Meiosis is reinitiated by the action of hormones on the oocyte membrane. In Xenopus oocytes, steroid hormones trigger a signal transduction cascade that activates at least two separate pathways that converge to activate maturation promoting factor (MPF), a powerful kinase that is the work horse of meiosis. In one pathway, Cdc25 phosphatase is activated, and it then removes an inhibitory phosphate from one of the subunits of MPF. In a parallel pathway (see figure below), the translation of mRNA encoding the Mos kinase is activated by polyadenylation-induced translation. This latter pathway is of particular interest to us because it links the signal transduction cascade with a specific mechanism of translational regulation.
The signal transduction cascade leading from activation of the progesterone receptor (PR) by progesterone, through the activation of Aurora A kinase, and the influence of Aurora A kinase and XGef on early CPEB activation. CPEB then participates in the polyadenylation induced translation of c-mos mRNA, which triggers the activation of mitogen activated protein kinase (MAPK). Activated MAPK, in conjunction with polyadenylation-induced translation of cyclin and Cdc25 activation (not shown) stimulates the timely activation of MPF (cyclin B: cdc2), which subsequently triggers resumption of meiosis. Arrows indicate positive feedback pathways that further stimulate c-mos mRNA translation.
We use Xenopus oocytes and eggs because we can obtain large amounts of material for examining the molecular machinery of polyadenylation-induced translation and we can induce the meiotic signal transduction cascade by adding progesterone to explanted oocytes. Microinjection allows us to explore the influence of various mRNAs and proteins on meiosis and the metabolism of components of meiosis and polyadenylation-induced translation.
Cytoplasmic polyadenylation element binding protein was the first component of the polyadenylation-induced translation mechanism to be identified and cloned. CPEB is involved in both the translational repression of stored maternal mRNAs and their translational activation. We are exploring how these two apects of CPEB function are regulated during meiosis in response to the signal transduction cascade using phosphopeptide analysis, site directed mutagenesis and overexpression assays, and perturbation of known signal transduction components. We have found that a subset of CPEB within the oocyte is targeted for degradation by the ubiquitin-proteasome pathway during meiosis. We are also learning about CPEB regulation through the identification and characterization of additional CPEB interacting proteins using the yeast two hybrid system and large scale immunoprecipitation.
XGef is a CPEB interacting protein that we identified using a yeast two hybrid screen. XGef is a Rho-family guanine nucleotide exchange factor. Extensive functional characterization has revealed that XGef interacts directly with CPEB in oocytes and participates in the activation of CPEB function in c-mos mRNA polyadenylation-induced translation. Through the creation of XGef deletion mutants, we have found that the influence of XGef on early CPEB phosphorylation requires that XGef interact with CPEB and that XGef retain exchange activity. The latter observation implies that the classical role of XGef, to activate a Rho-family G-protein, is also required for early signal transduction during meiosis. To date, small G-protein function has not been implicated in these early meiotic events, and we are currently attempting to identify the G-protein that is involved with XGef during early meiosis.