N-terminal acetylation (N-Ac) is a highly abundant eukaryotic protein modification. Proteomics revealed a significant increase in the occurrence of N-Ac from lower to higher eukaryotes, but evidence explaining the underlying molecular mechanism(s) is currently lacking. We first analysed protein N-termini and their acetylation degrees, suggesting that evolution of substrates is not a major cause for the evolutionary shift in N-Ac. Further, we investigated the presence of putative N-terminal acetyltransferases (NATs) in higher eukaryotes. The purified recombinant human and Drosophila homologues of a novel NAT candidate was subjected to in vitro peptide library acetylation assays. This provided evidence for its NAT activity targeting Met-Lys- and other Met-starting protein N-termini, and the enzyme was termed Naa60p and its activity NatF. Its in vivo activity was investigated by ectopically expressing human Naa60p in yeast followed by N-terminal COFRADIC analyses. hNaa60p acetylated distinct Met-starting yeast protein N-termini and increased general acetylation levels, thereby altering yeast in vivo acetylation patterns towards those of higher eukaryotes. Further, its activity in human cells was verified by overexpression and knockdown of hNAA60 followed by N-terminal COFRADIC. NatF's cellular impact was demonstrated in Drosophila cells where NAA60 knockdown induced chromosomal segregation defects. In summary, our study revealed a novel major protein modifier contributing to the evolution of N-Ac, redundancy among NATs, and an essential regulator of normal chromosome segregation. With the characterization of NatF, the co-translational N-Ac machinery appears complete since all the major substrate groups in eukaryotes are accounted for.

Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G 2 , but not during M/G 1 or early S phase. We have also isolated and characterized a temperaturesensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses.

RNApolII-dependent transcription is repressed in primordial germ cells of many animals during early development and is thought to be important for maintenance of germline fate by preventing somatic differentiation. Germ cell transcriptional repression occurs concurrently with inhibition of phosphorylation in the carboxy-terminal domain (CTD) of RNApolII, as well as with chromatin remodeling. The precise mechanisms involved are unknown. Here, we present evidence that a noncoding RNA transcribed by the gene polar granule component (pgc) regulates transcriptional repression in Drosophila germ cells. Germ cells lacking pgc RNA express genes important for differentiation of nearby somatic cells and show premature phosphorylation of RNApolII. We further show that germ cells lacking pgc show increased levels of K4, but not K9 histone H3 methylation, and that the chromatin remodeling Swi/Snf complex is required for a second stage in germ cell transcriptional repression. We propose that a noncoding RNA controls transcription in early germ cells by blocking the transition from preinitiation to transcriptional elongation. We further show that repression of somatic differentiation signals mediated by the Torso receptor-tyrosine kinase is important for germline development.

In many species, germ cells form in a specialized germ plasm, which contains localized maternal RNAs. In the absence of active transcription in early germ cells, these maternal RNAs encode germ-cell components with critical functions in germ-cell specification, migration, and development. For several RNAs, localization has been correlated with release from translational repression, suggesting an important regulatory function linked to localization. To address the role of RNA localization and translational control more systematically, we assembled a comprehensive set of RNAs that are localized to polar granules, the characteristic germ-plasm organelles. We find that the 3'-untranslated regions (UTRs) of all RNAs tested control RNA localization and instruct distinct temporal patterns of translation of the localized RNAs. We demonstrate necessity for translational timing by swapping the 3'UTR of polar granule component (pgc), which controls translation in germ cells, with that of nanos, which is translated earlier. Translational activation of pgc is concurrent with extension of its poly(A) tail length but appears largely independent of the Drosophila CPEB homolog ORB. Our results demonstrate a role for 3'UTR mediated translational regulation in fine-tuning the temporal expression of localized RNA, and this may provide a paradigm for other RNAs that are found enriched at distinct cellular locations such as the leading edge of fibroblasts or the neuronal synapse.

Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I.

The S-M checkpoint delays mitosis until DNA replication is complete; cells defective in this checkpoint lose viability when DNA replication is inhibited. This inviability can be suppressed in fission yeast by overexpression of Cid1 or the related protein Cid13. Fission yeast contain six cid1͞cid13-like genes, whereas budding yeast has just two, TRF4 and TRF5. Trf4 and Trf5 were recently reported to comprise an essential DNA polymerase activity required for the establishment of sister chromatid cohesion. In contrast, we find that Cid1 is not a DNA polymerase but instead uses RNA substrates and has poly(A) polymerase activity. Unlike the previously characterized yeast poly(A) polymerase, which is a nuclear enzyme, Cid1 and Cid13 are constitutively cytoplasmic. Cid1 has a degree of substrate specificity in vitro, consistent with the notion that it targets a subset of cytoplasmic mRNAs for polyadenylation in vivo, hence increasing their stability and͞or efficiency of translation. Preferred Cid1 targets presumably include mRNAs encoding components of the S-M checkpoint, whereas Cid13 targets are likely to be involved in dNTP metabolism. Cytoplasmic polyadenylation is known to be an important regulatory mechanism during early development in animals. Our findings in yeast suggest that this level of gene regulation is of more general significance in eukaryotic cells.A fter exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase, eukaryotic cells arrest in S phase and do not enter mitosis, owing to the activation of the DNA replication (S-M) checkpoint. In the fission yeast Schizosaccharomyces pombe, loss of function of S-M checkpoint genes such as rad3 does not impair normal growth but results in rapid loss of viability when cells are exposed to HU (1). Rad3 is a large protein kinase related to the vertebrate proteins ATR and ATM that, like Rad3, are required for S-M and DNA damage checkpoint responses (2). These checkpoints arrest the cell cycle to allow time for repair of damaged DNA or completion of DNA replication.During DNA replication, cohesion is established between newly replicated sister chromatids, which are held together by the multiprotein complex cohesin (3). Maintenance of sister chromatid cohesion from S phase through G 2 and until the onset of anaphase is necessary for accurate chromosome segregation. Cohesion is also important for efficient DNA repair, as it ensures that the undamaged sister chromatid is positioned to serve as a template for repair by homologous recombination. The mechanism by which cohesion is established during S phase is unknown. Recent data suggest that Trf4 and Trf5, two closely related proteins in Saccharomyces cerevisiae, act as a DNA polymerase to couple cohesion to replication, possibly directly by replicating sites of cohesion (4). The S. pombe genome contains six TRF4͞5 related genes: cid1, cid11, cid12, cid13, cid14, and cid16.S. pombe cid1 is required for S-M checkpoint integrity when DNA polymerase (Pol) ␦ or is inhibited (5). Overexpression of cid1 confers ...

Drosophila syncytial nuclear divisions limit transcription unit size of early zygotic genes. As mitosis inhibits not only transcription, but also pre-mRNA splicing, we reasoned that constraints on splicing were likely to exist in the early embryo, being splicing avoidance a possible explanation why most early zygotic genes are intronless. We isolated two mutant alleles for a subunit of the NTC/Prp19 complexes, which specifically impaired pre-mRNA splicing of early zygotic but not maternally encoded transcripts. We hypothesized that the requirements for pre-mRNA splicing efficiency were likely to vary during development. Ectopic maternal expression of an early zygotic pre-mRNA was sufficient to suppress its splicing defects in the mutant background. Furthermore, a small early zygotic transcript with multiple introns was poorly spliced in wild-type embryos. Our findings demonstrate for the first time the existence of a developmental pre-requisite for highly efficient splicing during Drosophila early embryonic development and suggest in highly proliferative tissues a need for coordination between cell cycle and gene architecture to ensure correct gene expression and avoid abnormally processed transcripts.DOI: http://dx.doi.org/10.7554/eLife.02181.001

Two paradigms exist for maintaining order during cell-cycle progression: intrinsic controls, where passage through one part of the cell cycle directly affects the ability to execute another, and checkpoint controls, where external pathways impose order in response to aberrant structures. By studying the mitotic inhibitor Mik1, we have identified evidence for an intrinsic link between unperturbed S phase and mitosis. We propose a model in which S/M linkage can be generated by the production and stabilization of Mik1 protein during S phase. The production of Mik1 during unperturbed S phase is independent of the Rad3-and Cds1-dependent checkpoint controls. In response to perturbed S phase, Rad3-Cds1 checkpoint controls are required to maintain high levels of Mik1, probably indirectly by extending the S phase period, where Mik1 is stable. In addition, we find that Mik1 protein can be moderately induced in response to irradiation of G 2 cells in a Chk1-dependent manner.