Translational repression (TR) of mRNAs plays an important role in sexual differentiation and gametogenesis in multicellular eukaryotes. We show here that TR and mRNA turnover are key influences on stage specific gene expression in the protozoan Plasmodium. The DDX6 class RNA helicase, DOZI (development of zygote inhibited), is found in cytoplasmic bodies of female, blood stage gametocytes in a complex with mRNA species known to be translationally repressed. Genetic disruption of pbdozi inhibits the formation of translationally quiescent mRNPs and targets these and other (>350 different) transcripts to the degradation pathway rather than directing them to translating polysomes, preventing zygote development prior to meiosis in the mosquito. Thus TR is essential in malaria parasite development. A full catalogue of the proteins and processes associated with DOZI (the first described malaria parasite TR-effector protein) might lead to novel approaches to prevent parasite development.Translational repression (TR) of mRNAs in higher eukaryotes controls temporal expression of specific protein cascades or directs the location of translation within a cell, and is prominent after gamete fertilisation (zygote formation) in the early embryo when de novo transcription of mRNA is restricted (1-5). The hallmark of repression is the assembly of certain mRNAs together with proteins into quiescent messenger ribonucleoprotein particles (mRNPs) where these transcripts are stored for translation at a later time. The DDX6-family of DEAD-box RNA helicases is tightly linked both to storage of mRNAs encoding proteins associated with progression through meiosis into translationally silent mRNPs and with the * To whom correspondence should be addressed waters@lumc.nl. Post-fertilsation development of Plasmodium zygotes in the mosquito relys on the formation of translationally quiescent mRNPs in blood-stage, female sexual precursor cells Supporting Online Material www.sceincemag.org Materials and Methods Figs. S1 to S4 Tables S1 to S4 References

Vertebrate chromosomes terminate in variable numbers of T2AG3 nucleotide repeats. In order to study telomere repeats at individual chromosomes, we developed novel, quantitative fluorescence in situ hybridization procedures using labeled (C3TA2)3 peptide nucleic acid and digital imaging microscopy. Telomere fluorescence intensity values from metaphase chromosomes of cultured human hematopoietic cells decreased with the replication history of the cells, varied up to six-fold within a metaphase, and were similar between sister chromatid telomeres. Surprisingly, telomere fluorescence intensity values within normal adult bone marrow metaphases did not show a normal distribution, suggesting that a minimum number of repeats at each telomere is required and/or maintained during normal hematopoiesis.

A universal feature of metazoan sexual development is the generation of oocyte P granules that withhold certain mRNA species from translation to provide coding potential for proteins during early post-fertilization development. Stabilisation of translationally quiescent mRNA pools in female Plasmodium gametocytes depends on the RNA helicase DOZI, but the molecular machinery involved in the silencing of transcripts in these protozoans is unknown. Using affinity purification coupled with mass-spectrometric analysis we identify a messenger ribonucleoprotein (mRNP) from Plasmodium berghei gametocytes defined by DOZI and the Sm-like factor CITH (homolog of worm CAR-I and fly Trailer Hitch). This mRNP includes 16 major factors, including proteins with homologies to components of metazoan P granules and archaeal proteins. Containing translationally silent transcripts, this mRNP integrates eIF4E and poly(A)-binding protein but excludes P body RNA degradation factors and translation-initiation promoting eIF4G. Gene deletion mutants of 2 core components of this mRNP (DOZI and CITH) are fertilization-competent, but zygotes fail to develop into ookinetes in a female gametocyte-mutant fashion. Through RNA-immunoprecipitation and global expression profiling of CITH-KO mutants we highlight CITH as a crucial repressor of maternally supplied mRNAs. Our data define Plasmodium P granules as an ancient mRNP whose protein core has remained evolutionarily conserved from single-cell organisms to germ cells of multi-cellular animals and stores translationally silent mRNAs that are critical for early post-fertilization development during the initial stages of mosquito infection. Therefore, translational repression may offer avenues as a target for the generation of transmission blocking strategies and contribute to limiting the spread of malaria.

Chromosome ends are protected from degradation by the presence of the highly repetitive hexanucleotide sequence of TTAGGG and associated proteins. These so-called telomeric complexes are suggested to play an important role in establishing a functional nuclear chromatin organization. Using peptide nucleic acid (PNA) probes, we studied the dynamic behavior of telomeric DNA repeats in living human osteosarcoma U2OS cells. A¯uorescent cy3-labeled PNA probe was introduced in living cells by glass bead loading and was shown to speci®cally associate with telomeric DNA shortly afterwards. Telomere dynamics were imaged for several hours using digital¯uorescence microscopy. While the majority of telomeres revealed constrained diffusive movement, individual telomeres in a human cell nucleus showed signi®cant directional movements. Also, a subfraction of telomeres were shown to associate and dissociate, suggesting that in vivo telomere clusters are not stable but dynamic structures. Furthermore, telomeres were shown to associate with promyelocytic leukemia (PML) bodies in a dynamic manner.

The p53 tumor suppressor protein has a major role in protecting the integrity of the genome. In unstressed cells, p53 is maintained at low levels by the ubiquitin-proteasome pathway. A balance between ubiquitin ligase activity (Hdm2, COP1, and Pirh2) and the ubiquitin protease activity of the Herpes virus-associated ubiquitin-specific protease (HAUSP) determines the half-life of p53. HAUSP also modulates p53 stability indirectly by deubiquitination and stabilization of Hdm2. The Hdmx protein affects p53 stability as well through its interaction with and regulation of Hdm2. Vice versa, Hdmx is a target for Hdm2-mediated ubiquitination and degradation. Here, we show that HAUSP also interacts with Hdmx, resulting in its direct deubiquitination and stabilization. HAUSP activity is required to maintain normal Hdmx protein levels. Therefore, the balance between HAUSP and Hdm2 activity determines Hdmx protein stability. Importantly, impaired deubiquitination of Hdmx/Hdm2 by HAUSP contributes to the DNA damage-induced degradation of Hdmx and transient instability of Hdm2.