Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development.

The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented. This section will be followed by a review of translational mechanisms operating in oocytes, eggs, and early cleavage-stage embryos and conclude with a discussion of how the regulation of key maternal cell-fate determinants at the level of translation functions in Xenopus embryogenesis. A key theme is that the molecular asymmetries critical for forming the body axes are established and further elaborated upon by the selective temporal and spatial regulation of maternal mRNA translation.

A gradient of maternal Bicaudal-C controls vertebrate embryogenesis via translational repression of mRNAs encoding cell fate regulators.

Vertebrate Bicaudal-C (Bicc1) has important biological roles in the formation and homeostasis of multiple organs, but direct experiments to address the role of maternal Bicc1 in early vertebrate embryogenesis have not been reported. Here, we use antisense phosphorothioate-modified oligonucleotides and the host-transfer technique to eliminate specifically maternal stores of both bicc1 mRNA and Bicc1 protein from Xenopus laevis eggs. Fertilization of these Bicc1-depleted eggs produced embryos with an excess of dorsal-anterior structures and overexpressed organizer-specific genes, indicating that maternal Bicc1 is crucial for normal embryonic patterning of the vertebrate embryo. Bicc1 is an RNA-binding protein with robust translational repression function. Here, we show that the maternal mRNA encoding the cell-fate regulatory protein Wnt11b is a direct target of Bicc1-mediated repression. It is well established that the Wnt signaling pathway is crucial to vertebrate embryogenesis. Thus, the work presented here links the molecular function of Bicc1 in mRNA target-specific translation repression to its biological role in the maternally controlled stages of vertebrate embryogenesis.

Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

Children with congenital hypothyroidism have similar neuroradiological abnormal findings as healthy ones.

OBJECTIVE: To assess the neuroradiological findings of children with congenital hypothyroidism (CHT) compared to healthy controls (HC). PATIENTS AND METHODS: Thirty children with CHT, mean age 12.5 ± 1.6 years, 14 (44.8%) males, were compared with 38 HC mean age 11.7 ± 1.7 years, 16 (45.7%) males. Clinical data were collected from medical charts and questionnaires seeking information on family history, birth and perinatal period events, medications, and overall health history. Neurocognitive function was assessed for global intelligence, visual and verbal memory, and executive functioning using standardized tests. Neuroimaging was performed using 1.5 T magnetic resonance imaging and assessed by two pediatric radiologists. RESULTS: Children with CHT had a similar proportion of incidental findings as did the children in the HC group, at 43.3% and 39.5%, respectively, P = 0.69. Abnormalities of the sellar region were reported in 13.3% of CHT group and 7.9% of HC group, P = 0.46. Other incidental findings included cerebellar ectopia, choroidal fissure and pineal cysts, and multiple increased signal intensity foci. Neuroradiological findings were not associated with clinical and neurocognitive abnormalities. CONCLUSION: Neuroimaging of children with CHT demonstrated a similar incidence of structural abnormalities as in the healthy population. There is no association between those findings and neurocognitive function.

Characterization of human septin interactions.

Septins constitute a group of GTP binding proteins that assemble into homo- and hetero-oligomeric complexes and filaments. These higher order septin structures are thought to function like scaffolds and/or diffusion barriers serving as spatial localizers for many proteins with key roles in cell polarity and cell cycle progression. In this study, we extensively characterized septin interaction partners using yeast two-hybrid and three-hybrid systems in addition to precipitation analyses in platelets. As a result, we identified human hetero-trimeric septin complexes on a large scale, which had been only postulated in the past. In addition, we illustrated roles of SEPT9 that might contribute to hetero-trimeric septin complex formation. SEPT9 can substitute for septins of the SEPT2 group and partially for SEPT7. Mutagenic analyses revealed that mutation of a potential phosphorylation site in SEPT7 (Y318) regulates the interaction with other septins. We identified several septin-septin interactions in platelets suggesting a regulatory role of diverse septin complexes in platelet function.

Human endothelial and platelet septin SEPT11: cloning of novel variants and characterisation of interaction partners.

Septins are cytoskeletal GTPases forming heteropolymeric complexes involved in processes characterised by active membrane movement such as cytokinesis, vesicle trafficking, and exocytosis. Septins are expressed in non-mitotic cells such as neurons and platelets. SEPT11 belongs to the SEPT6 group and was identified as interaction partner of SEPT5. We cloned and characterised novel SEPT11 variants and investigated interaction partners of SEPT11 in platelets and human umbilical vein endothelial cells. An endothelial cell library was used for cloning novel SEPT11 variants. Using Northern analysis the different SEPT11 transcripts were illustrated. Interaction studies were performed using yeast two-hybrid system, precipitation, FRET, and immunofluorescence microscopy. We demonstrate that SEPT11 partners with SEPT2, SEPT4 and SEPT7 using yeast two-hybrid system and precipitation. The interaction of SEPT11 with SEPT7 is also demonstrated by FRET. In addition to the known SEPT11 transcript (SEPT11_v1) we identified a novel SEPT11 variant (SEPT11_v2) as interaction partner of SEPT4 and SEPT7. Library screening of an endothelial cell library also revealed the presence of this novel SEPT11_v2 transcript. In addition, a third SEPT11 variant (SEPT11_v3) was identified. Expression of SEPT11_v1 and of SEPT11_v2 and SEPT11_v3 in human brain regions was investigated by Northern analysis. Further interaction partners of SEPT11 are characterised using immunofluorescence. Co-localisation of SEPT2, SEPT4, SEPT7 and SEPT11 with tubulin and transferrin receptor (endocytotic marker) is demonstrated. In addition, co-localisation of SEPT4 and SEPT11 with the vesicle-associated protein synaptobrevin 1 (VAMP1), but not clearly with actin, was shown. Only SEPT2 and SEPT7 definitely co-localised with actin, but not clearly with VAMP1.

Immunoreactivity of the septins SEPT4, SEPT5, and SEPT8 in the human eye.

We aimed to examine the distribution of SEPT4, SEPT5, and SEPT8 in the human eye. For each septin, five to six normal human eyes were examined by immunohistochemical staining of paraffin sections using polyclonal antibodies against SEPT4, SEPT5, and SEPT8 and an avidin biotin complex immunodetection system. SEPT4 immunoreactivity (IR) was detected primarily in the epithelium of cornea, lens, and nonpigmented ciliary epithelium; in the endothelium of cornea and vessels of iris and retina; and in the retinal nerve fiber layer, the outer plexiform layer, the outer segments of the photoreceptor cells, the inner limiting membrane of the optic nerve head, and optic nerve axons. SEPT5-IR was present in corneal endothelial cells, iris tissue, nonpigmented ciliary epithelium, and epithelial cells of the lens. SEPT8-IR almost paralleled that of SEPT4, except for a lower SEPT8-IR of the outer photoreceptor segments and a positive staining of the meningothelial cell nests in the subarachnoidal space of the bulbar segment of the orbital optic nerve. In conclusion, SEPT4, SEPT5, and SEPT8 are expressed in various ocular tissues, each revealing a distinct expression pattern. Both physiological and potential pathophysiological role of septins in the human eye deserve further investigation.

The novel human platelet septin SEPT8 is an interaction partner of SEPT4.

Septins are a family of GTP-binding proteins, which are essential for active membrane movement such as cytokinesis and vesicle trafficking. In non-dividing cells (such as platelets and neurons) septins are implicated in exocytosis. Platelets from a SEPT5 knockout mouse showed an altered serotonin secretion and platelet aggregation, suggesting that SEPT5 is involved in secretion in platelets. Septins form complexes consisting of multiple septin polypeptides. Using the yeast two-hybrid system we had demonstrated that SEPT5 partners with SEPT8. The aim of this study was to identify other interaction partners of the human platelet septin SEPT8. Using the yeast two-hybrid system with SEPT8 as bait protein we identified the human septin SEPT4 as an interaction partner of SEPT8. The interaction between SEPT4 and SEPT8 was confirmed by immunoprecipitation. Expression analysis revealed that SEPT4 is also expressed in human platelets. Thus, SEPT4 is the third described platelet septin besides SEPT5 and SEPT8. Transmission electron microscopy of platelets revealed that SEPT8 and SEPT4 are localized surrounding alpha-granules (as it had been shown for the septin SEPT5) suggesting that the three septins may be components of the septin complex in platelets and contribute in such a way to platelet biology. Activation of platelets by agonists resulted in the translocation of SEPT4 and SEPT8 to the platelet surface indicating a possible functional role of these proteins in platelet granular secretion.

Isolation of new splice isoforms, characterization and expression analysis of the human septin SEPT8 (KIAA0202).

SEPT8 (KIAA0202) is a member of the highly conserved septin family. Septins are membrane-associated GTPases which are involved in cytokinesis and cellular morphogenesis. Using the yeast two-hybrid system and the glutathione-S-transferase pull-down assay we previously had identified the SEPT8 (KIAA0202) as interaction partner of the human septin SEPT5 (cell division cycle related-1, CDCrel-1). Since the complete cDNA sequence of the human septin SEPT8 (KIAA0202) was not known at that time, we isolated new 5' and 3' cDNA sequence of SEPT8 (KIAA0202) by screening three different cDNA libraries. In addition, we performed the characterization of SEPT8 (KIAA0202) and identified new splice variants of SEPT8 (KIAA0202). The expression pattern of SEPT8 (KIAA0202) and its interaction partner SEPT5 (CDCrel-1) is illustrated.

Human septin-septin interaction: CDCrel-1 partners with KIAA0202.

Septins are evolutionary conserved cytoskeletal GTPases forming heteropolymer complexes involved in cytokinesis and other cellular processes. CDCrel-1 (cell division cycle related-1) is a recently cloned and characterized human septin which is highly expressed in non-dividing cells, such as neurons. Using a yeast two-hybrid system we demonstrate that CDCrel-1 partners with another uncharacterized human septin, KIAA0202. The interaction of CDCrel-1 and KIAA0202 was confirmed in the human leukemia cell line K-562 using pull-down assays with a KIAA0202-glutathione S-transferase fusion protein and by immunoprecipitation of the CDCrel-1-KIAA0202 complex with an anti-KIAA0202 antibody. Expression studies of the two human septins revealed a concomitant expression of both proteins in certain cells.