Howard Beverley Osborne

Inactivation of CUG-BP1/CELF1 Causes Growth, Viability, and Spermatogenesis Defects in Mice

ABSTRACT CUG-BP1/CELF1 is a multifunctional RNA-binding protein involved in the regulation of alternative splicing and translation. To elucidate its role in mammalian development, we produced mice in which the Cugbp1 gene was inactivated by homologous recombination. These Cugbp1−/− mice were viable, although a significant portion of them did not survive after the first few days of life. They displayed growth retardation, and most Cugbp1−/− males and females exhibited impaired fertility. Male infertility was more thoroughly investigated. Histological examination of testes from Cugbp1−/− males showed an arrest of spermatogenesis that occurred at step 7 of spermiogenesis, before spermatid elongation begins, and an increased apoptosis. A quantitative reverse transcriptase PCR analysis showed a decrease of all the germ cell markers tested but not of Sertoli and Leydig markers, suggesting a general decrease in germ cell number. In wild-type testes, CUG-BP1 is expressed in germ cells from spermatogonia to round spermatids and also in Sertoli and Leydig cells. These findings demonstrate that CUG-BP1 is required for completion of spermatogenesis.

Embryo Deadenylation Element-Dependent Deadenylation Is Enhanced by a cis Element Containing AUU Repeats

ABSTRACT The deadenylation of maternal mRNAs in the Xenopusembryo is a sequence-specific process. One cis element that targets maternal mRNAs for deadenylation after fertilization is the embryo deadenylation element (EDEN). This element, composed of U/R repeats, is specifically bound by a protein, EDEN-BP. In the present study we show that the rate at which an RNA containing an EDEN is deadenylated can be increased by the presence of an additionalcis element composed of three AUU repeats. This effect was observed for a natural EDEN (c-mos) and two synthetic EDENs. Hence, the enhancement of EDEN-dependent deadenylation conferred by the (AUU)3 motif is not due to an interaction with a particular EDEN sequence. Mutation of the (AUU)3 motif abrogated the enhancement of EDEN-dependent deadenylation. These data indicate that the rate at which a specific maternal mRNA is deadenylated in Xenopus embryos is probably defined by a cross talk between multiple cis elements.

Regulation of EDEN-dependent deadenylation of Aurora A/Eg2-derived mRNA via phosphorylation and dephosphorylation in Xenopus laevis egg extracts.

Deadenylation is an intimate part of the post-transcriptional regulation of maternal mRNAs in embryos. EDEN-BP is so far the only known member of a complex regulating the deadenylation of maternal mRNA in Xenopus laevis embryos in a manner that is dependent on the 3′-untranslated region called EDEN (embryo deadenylation element). In this report, we show that calcium activation of cell-free extracts triggers EDEN binding protein (EDEN-BP) dephosphorylation and concomitant deadenylation of a chimeric RNA bearing Aurora A/Eg2 EDEN sequence. Deadenylation of mRNA deprived of EDEN sequence (default deadenylation) does not change with egg activation. Kinase and phosphatase inhibitors downregulate EDEN-dependent deadenylation but they do not substantially influence default deadenylation. Using indestructibleΔ 90 cyclin B to revert interphase extracts to the M-phase, we show that modulation of EDEN-dependent deadenylation is independent of M-phase promoting factor (MPF) activity. These results suggest that the increase in EDEN-dependent deadenylation following egg activation is achieved, at least partially, via dephosphorylation and/or phosphorylation of regulatory proteins, including EDEN-BP dephosphorylation. This regulation proceeds in a manner independent from MPF inactivation.

Post-transcriptional regulation in Xenopus embryos: role and targets of EDEN-BP.

EDEN (embryo deadenylation element)-dependent deadenylation is a regulatory process that was initially identified in Xenopus laevis early embryos and was subsequently shown to exist in Drosophila oocytes. Recent data showed that this regulatory process is required for somitic segmentation in Xenopus. Inactivation of EDEN-BP (EDEN-binding protein) causes severe segmentation defects, and the expression of segmentation markers in the Notch signalling pathway is disrupted. We showed that the mRNA encoding XSu(H) (Xenopus suppressor of hairless), a protein central to the Notch pathway, is regulated by EDEN-BP. Our data also indicate that other segmentation RNAs are targets for EDEN-BP. To identify new EDEN-BP targets, a microarray analysis has been undertaken.

Poly(A) metabolism in Xenopus laevis embryos: Substrate-specific and default poly(A) nuclease activities are mediated by two distinct complexes

The metabolism of the poly(A) tail is a process important for the translational regulation of maternal mRNAs in Xenopus laevis oocytes and early embryos. Two poly(A) nuclease (PAN) activities have been described in Xenopus embryo or activated egg extracts (Legagneux et al (1995) RNA 1, 1001-1008). These activities (default PAN and EgPAN) are distinguishable by their deadenylation kinetics and their substrate specificities. In this report, we show that these activities display different sensitivities to biochemical treatments. Urea and, to a lesser extent, spermidine, inhibit EgPAN at concentrations which have no effect on default PAN. Heparin activates default PAN but inhibits EgPAN. When extracts are fractionated by ultracentrifugation, the default activity is recovered in one unique fraction, whereas two fractions must be combined to reconstitute the EgPAN activity. Moreover, these two deadenylation activities are separable by size exclusion chromatography under native conditions. We conclude that these two deadenylation activities are mediated by two protein complexes.