Abstract
During the early stages of Xenopus laevis development no transcription occurs. Many cell divisions take place in about 9 h, a process orchestrated by maternal mRNAs. When the embryo contains about thousand cells, zygotic transcription initiates during the so-called mid blastula transition or MBT. Before MBT, translational control is crucial
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for Xenopus embryonic development. This control includes regulation of the length of the poly (A) tail of the mRNA, localization and activation of mRNAs, binding of regulatory proteins to the 3' untranslated region (UTR), and modulation of mRNA half-life.
Several elements in the 5'UTR are known which affect the efficiency of translation: its length, CG-content, and secondary structure, protein binding sites, AUG triplets upstream of the open reading frame (uAUGs), upstream open reading frames (uORFs), and the sequence around each potential AUG initiation codon. In Chapter 1 several examples have been discussed of mRNAs controlled by uORFs. In general, uORFs down-regulate translation of the main ORF, although a few examples have been described in which uORFs enhance expression of the main ORF. Control of translation by uORFs is best explained by the ribosomal scanning model: ribosomes enter at the 5' cap-structure of the mRNA and scan the mRNA for an AUG initiation codon. Alternatively, ribosomes may enter via internal entry by the presence of an internal ribosome entry site (IRES), often located just upstream of the initiation codon.
Translation can be controlled by IRESes, uORFs, and protein binding sites in a tissue-specific way, in a cell cycle-dependent way, and can be dependent on the presence of particular amino acids or Fe-ions. How specificity is determined is not always clear but activation of kinases that phosphorylate and down-regulate translation initiation factor eIF2 is involved in control of uORF-dependent translation.
Connexins are proteins that form channels between cells, allowing the coordinated development of connected cells by sharing small molecules, such as cyclic AMP, inositol triphosphate, and Ca2+. In a database search for the occurrence of uAUGs, we noticed that connexin mRNAs contained uAUGs relatively frequently compared to non-connexin mRNAs. Therefore, we obtained the full length 5'UTRs for two of the four known Xenopus connexin mRNAs, Cx30 and Cx41. Chapters 2 and 3 described the cloning and sequencing of the Cx30 and Cx41 5'UTRs, respectively. The 5'UTRs were cloned upstream of a GFP reporter gene and the translation efficiency was compared to control constructs. The 3'UTR of Cx30 was also cloned. The first endogenous Cx30 transcripts are detected at embryonic stage 11 and are mainly present in endodermal cells. Later in development Cx30 transcripts are also present in ectodermal (hatching gland) and mesodermal (kidney) cells. Translation of reporter constructs occurred throughout the entire embryo, whether the construct contained ß-globin or Cx30 UTRs, suggesting that the 5'and 3'UTR do not restrict translation and that Cx30 expression is mainly controlled at the transcriptional level.
The Cx41 5'UTR contains 3 uORFs, each of which down-regulates translation, since mutation of the uAUG codons increased GFP synthesis considerably. The contribution of each of the three AUGs has been studied in Chapter 3, allowing the postulation of a model, which describes the flow of ribosomes along the Cx41 mRNA. This model was tested in Chapter 4, by mutating the termination codon of the most upstream uORF (uORF1), enabling the synthesis of an elongated form of GFP (designated #1 GFP). The model proposed in Chapter 3 predicted the synthesis of high amounts of #1 GFP. However, low levels of a #1 GFP were observed, implying low initiation efficiency at uAUG1. This indicated that the control of Cx41 translation is more complex than assumed in Chapter 3. The strong effect of mutation of uAUG1 together with the low initiation frequency at this uAUG, suggested a low translation rate of uORF1. As determined in Chapter 4, at least two elements contribute to the down-regulation of GFP synthesis: the rare leucine codon at position 3 of uORF1 as well as the termination rate at uORF1. All these factors have the effect that the low numbers of ribosomes that initiate at uORF1 physically block entry of 40S subunits on the mRNA.
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