Drosophila Yemanuclein associates with the cohesin and synaptonemal complexes.

Meiosis is characterized by two chromosome segregation rounds (meiosis I and II), which follow a single round of DNA replication, resulting in haploid genome formation. Chromosome reduction occurs at meiosis I. It relies on key structures, such as chiasmata, which are formed by repair of double-strand breaks (DSBs) between the homologous chromatids. In turn, to allow for segregation of homologs, chiasmata rely on the maintenance of sister chromatid cohesion. In most species, chiasma formation requires the prior synapsis of homologous chromosome axes, which is mediated by the synaptonemal complex, a tripartite proteinaceous structure specific to prophase I of meiosis. Yemanuclein (Yem) is a maternal factor that is crucial for sexual reproduction. It is required in the zygote for chromatin assembly of the male pronucleus, where it acts as a histone H3.3 chaperone in complex with Hira. We report here that Yem associates with the synaptonemal complex and the cohesin complex. A genetic interaction between yem(1) (V478E) and the Spo11 homolog mei-W68, modified a yem(1) dominant effect on crossover distribution, suggesting that Yem has an early role in meiotic recombination. This is further supported by the impact of yem mutations on DSB kinetics. A Hira mutation gave a similar effect, presumably through disruption of Hira-Yem complex.

Drosophila Yemanuclein and HIRA cooperate for de novo assembly of H3.3-containing nucleosomes in the male pronucleus.

The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI) de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM), the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male pronucleus. yem loss of function alleles affect male pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.

A single mutation results in diploid gamete formation and parthenogenesis in a Drosophila yemanuclein-alpha meiosis I defective mutant.

BACKGROUND: Sexual reproduction relies on two key events: formation of cells with a haploid genome (the gametes) and restoration of diploidy after fertilization. Therefore the underlying mechanisms must have been evolutionary linked and there is a need for evidence that could support such a model. RESULTS: We describe the identification and the characterization of yem1, the first yem-alpha mutant allele (V478E), which to some extent affects diploidy reduction and its restoration. Yem-alpha is a member of the Ubinuclein/HPC2 family of proteins that have recently been implicated in playing roles in chromatin remodeling in concert with HIRA histone chaperone. The yem1 mutant females exhibited disrupted chromosome behavior in the first meiotic division and produced very low numbers of viable progeny. Unexpectedly these progeny did not display paternal chromosome markers, suggesting that they developed from diploid gametes that underwent gynogenesis, a form of parthenogenesis that requires fertilization. CONCLUSIONS: We focus here on the analysis of the meiotic defects exhibited by yem1 oocytes that could account for the formation of diploid gametes. Our results suggest that yem1 affects chromosome segregation presumably by affecting kinetochores function in the first meiotic division. This work paves the way to further investigations on the evolution of the mechanisms that support sexual reproduction.

Repeat length and RNA expression level are not primary determinants in CUG expansion toxicity in Drosophila models.

Evidence for an RNA gain-of-function toxicity has now been provided for an increasing number of human pathologies. Myotonic dystrophies (DM) belong to a class of RNA-dominant diseases that result from RNA repeat expansion toxicity. Specifically, DM of type 1 (DM1), is caused by an expansion of CUG repeats in the 3'UTR of the DMPK protein kinase mRNA, while DM of type 2 (DM2) is linked to an expansion of CCUG repeats in an intron of the ZNF9 transcript (ZNF9 encodes a zinc finger protein). In both pathologies the mutant RNA forms nuclear foci. The mechanisms that underlie the RNA pathogenicity seem to be rather complex and not yet completely understood. Here, we describe Drosophila models that might help unravelling the molecular mechanisms of DM1-associated CUG expansion toxicity. We generated transgenic flies that express inducible repeats of different type (CUG or CAG) and length (16, 240, 480 repeats) and then analyzed transgene localization, RNA expression and toxicity as assessed by induced lethality and eye neurodegeneration. The only line that expressed a toxic RNA has a (CTG)(240) insertion. Moreover our analysis shows that its level of expression cannot account for its toxicity. In this line, (CTG)(240.4), the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene. Interestingly, CG9650 and (CUG)(240.4) expansion RNAs were found in the same nuclear foci. In conclusion, we suggest that the insertion context is the primary determinant for expansion toxicity in Drosophila models. This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.

The Drosophila Bruno paralogue Bru-3 specifically binds the EDEN translational repression element.

We reported in our previous work that the EDEN-dependent translational repression of maternal mRNAs was conserved between Drosophila and Xenopus. In Xenopus, this repression is achieved through the binding of EDEN to the Bruno-like factor, EDEN-BP. We show in the present work that the Drosophila Bruno paralogue, the 45 kDa Bru-3 protein (p45), binds specifically to the EDEN element and acts as a homodimer. We describe for the first time a previously undetected 67 amino acid domain, found in the divergent linker region, the lsm domain (lsm stands for linker-specific motif). We propose that the presence of this domain in a subset of the Bruno-like proteins, including Bru-3, EDEN-BP and CUG-BP but not Bruno nor its other paralogue Bru-2, might be responsible for specific RNA recognition. Interestingly, comparative structural analyses using threaders and molecular modelling suggest that the new domain might be distantly related to the first RNA recognition motif of the Drosophila sex-lethal protein (sxl). The phylogenetic analyses and the experimental data based on its specific binding to the EDEN element support the conclusion that Bru-3 is an EDEN-BP/CUG-BP orthologue.

EDEN-dependent translational repression of maternal mRNAs is conserved between Xenopus and Drosophila.

Translational control is a key level in regulating gene expression in oocytes and eggs because many mRNAs are synthesized and stored during oogenesis for latter use at various stages of oocyte maturation and embryonic development. Understanding the molecular mechanisms that underlie this translational control is therefore crucial. Another important issue is the evolutionary conservation of these mechanisms--in other words the determination of their universal and specific aspects. We report here a comparative analysis of a translational repression mechanism that depends on the EDEN (embryo deadenylation element) element. This small cis-acting element, localized in the 3' untranslated region of c-mos and Eg mRNAs, was shown to be involved in a deadenylation process. We demonstrate here that in Xenopus embryos, mRNAs that contain an EDEN are translationally repressed. Next, transgenic flies were used to study the effect of the EDEN motif on translation in Drosophila oocytes. We show that this element also causes the translational repression of a reporter gene in Drosophila demonstrating that the EDEN-dependent translational repression is functionally conserved between Xenopus and Drosophila.