The spindle apparatus dictates the plane of cell cleavage, which is critical in the choice between symmetric or asymmetric division. Spindle positioning is controlled by an evolutionarily conserved pathway, which involves LIN-5/GPR-1/2/Galpha in Caenorhabditis elegans, Mud/Pins/Galpha in Drosophila and NuMA/LGN/Galpha in humans. GPR-1/2 and Galpha localize LIN-5 to the cell cortex, which engages dynein and controls the cleavage plane during early mitotic divisions in C. elegans. Here we identify ASPM-1 (abnormal spindle-like, microcephaly-associated) as a novel LIN-5 binding partner. ASPM-1, together with calmodulin (CMD-1), promotes meiotic spindle organization and the accumulation of LIN-5 at meiotic and mitotic spindle poles. Spindle rotation during maternal meiosis is independent of GPR-1/2 and Galpha, yet requires LIN-5, ASPM-1, CMD-1 and dynein. Our data support the existence of two distinct LIN-5 complexes that determine localized dynein function: LIN-5/GPR-1/2/Galpha at the cortex, and LIN-5/ASPM-1/CMD-1 at spindle poles. These functional interactions may be conserved in mammals, with implications for primary microcephaly.

Meiosis is a cellular differentiation process in which hundreds of genes are temporally induced. Because the expression of meiotic genes during mitosis is detrimental to proliferation, meiotic genes must be negatively regulated in the mitotic cell cycle. Yet, little is known about mechanisms used by mitotic cells to repress meiosis-specific genes. Here we show that the poly(A)-binding protein Pab2, the fission yeast homolog of mammalian PABPN1, controls the expression of several meiotic transcripts during mitotic division. Our results from chromatin immunoprecipitation and promoter-swapping experiments indicate that Pab2 controls meiotic genes post-transcriptionally. Consistently, we show that the nuclear exosome complex cooperates with Pab2 in the negative regulation of meiotic genes. We also found that Pab2 plays a role in the RNA decay pathway orchestrated by Mmi1, a previously described factor that functions in the post-transcriptional elimination of meiotic transcripts. Our results support a model in which Mmi1 selectively targets meiotic transcripts for degradation via Pab2 and the exosome. Our findings have therefore uncovered a mode of gene regulation whereby a poly(A)-binding protein promotes RNA degradation in the nucleus to prevent untimely expression.Meiosis is a key differentiation process essential for the generation of genetically distinct individuals. During yeast meiosis, two cells of opposite mating types fuse and conjugate their DNA to form a diploid cell. This diploid cell undergoes DNA replication followed by two rounds of cell division to produce four haploid cells. Although many of the activities used to achieve cell division are common to both meiosis and mitosis, there are several features unique to meiosis. Importantly, the meiotic and mitotic cell cycles are mutually exclusive, and genes required for meiotic differentiation are solely expressed during meiosis. Such negative control of meiotic genes during mitosis suggests important regulatory systems to avoid inappropriate activation of meiosis. To date, however, the molecular mechanisms by which meiotic differentiation genes are suppressed during the mitotic cell cycle remain poorly understood.In the fission yeast Schizosaccharomyces pombe, meiotic differentiation involves the temporal activation of hundreds of genes (1). Although previous studies have established the importance of transcriptional regulation during fission yeast meiosis (1, 2), recent results implicate post-transcriptional mechanisms of gene regulation, including pre-mRNA splicing and mRNA degradation. Accordingly, several meiotic genes are specifically spliced during meiosis, but remain unspliced during the mitotic cell cycle (3, 4). Selective mRNA turnover is another mechanism used by fission yeast to ensure the absence of meiosis-specific transcripts during the mitotic cell cycle. The rapid elimination of specific meiotic transcripts in mitotic cells requires the YTH-family RNA-binding protein, Mmi1 (5). Mmi1 promotes the destruction of specific meiotic transcripts...

Temporal control of cell division is critical for proper animal development. To identify mechanisms involved in developmental arrest of cell division, we screened for cell-cycle mutants that disrupt the reproducible pattern of somatic divisions in the nematode C. elegans. Here, we show that the cdc-14 phosphatase is required for the quiescent state of specific precursor cells. Whereas budding yeast Cdc14p is essential for mitotic exit, inactivation of C. elegans cdc-14 resulted in extra divisions in multiple lineages, with no apparent defects in mitosis or cell-fate determination. CDC-14 fused to the green fluorescent protein (GFP-CDC-14) localized dynamically and accumulated in the cytoplasm during G1 phase. Genetic interaction and transgene expression studies suggest that cdc-14 functions upstream of the cki-1 Cip/Kip inhibitor to promote accumulation of CKI-1 in the nucleus. Our data support a model in which CDC-14 promotes a hypophosphorylated and stable form of CKI-1 required for developmentally programmed cell-cycle arrest.

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Schizosaccharomyces pombe Rmt3 is a member of the proteinarginine methyltransferase (PRMT) family and is the homolog of human PRMT3. We previously characterized Rmt3 as a ribosomal protein methyltransferase based on the identification of the 40 S Rps2 (ribosomal protein S2) as a substrate of Rmt3. RMT3-null cells produce nonmethylated Rps2 and show misregulation of the 40 S/60 S ribosomal subunit ratio due to a small subunit deficit. For this study, we have generated a series of RMT3 alleles that express various amino acid substitutions to characterize the functional domains of Rmt3 in Rps2 binding, Rps2 arginine methylation, and small ribosomal subunit production. Notably, catalytically inactive versions of Rmt3 restored the ribosomal subunit imbalance detected in RMT3-null cells. Consistent with a methyltransferase-independent function for Rmt3 in small ribosomal subunit production, the expression of an Rps2 variant in which the identified methylarginine residues were substituted with lysines showed normal levels of 40 S subunit. Importantly, substitutions within the zinc finger domain of Rmt3 that abolished Rps2 binding did not rescue the 40 S ribosomal subunit deficit of RMT3-null cells. Our findings suggest that the Rmt3-Rps2 interaction, rather than Rps2 methylation, is important for the function of Rmt3 in the regulation of small ribosomal subunit production.Protein arginine methylation is a posttranslational modification catalyzed by a family of enzymes known as protein-arginine methyltransferases (PRMTs).2 Although protein-arginine methyltransferase activity has never been demonstrated in prokaryotic organisms, genes encoding PRMTs have been identified in a variety of unicellular and multicellular eukaryotes (1, 2). In humans, 10 PRMTs have so far been identified (3). Most PRMTs are divided in two major classes, depending of the type of dimethylarginine they produce. Whereas both type I and II PRMTs use S-adenosyl-L-methionine as a cofactor for the monomethylation of specific arginines within substrate proteins, type I and type II enzymes can also produce asymmetricarginine, respectively (1). Interestingly, protein arginine methylation is often found within arginine-glycine (RG)-rich regions of nucleic acid-binding proteins (4). The functional role of PRMTs is likely to be mediated by the modification of substrate proteins. Accordingly, proteins involved in specific steps of gene expression, including transcription (5, 6), splicing (7), polyadenylation (8, 9), mRNA export (10), and translation (11-13), are modified by arginine methylation. Methylation of specific arginine residues within the N-terminal tails of nucleosomal histones is also important for gene regulation and chromatin remodeling (14, 15), thereby influencing biological processes, such as cell fate determination (16) and oncogenesis (17). As yet, however, the biological role of most PRMTs remains poorly understood.The ribosome is the macromolecular complex responsible for protein synthesis in all living cells. In eukaryotes, the 80 S ribos...

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Ribosome biogenesis is an evolutionarily conserved pathway that requires ribosomal and nonribosomal proteins. Here, we investigated the role of the ribosomal protein S2 (Rps2) in fission yeast ribosome synthesis. As for many budding yeast ribosomal proteins, Rps2 was essential for cell viability in fission yeast and the genetic depletion of Rps2 caused a complete inhibition of 40S ribosomal subunit production. The pattern of pre-rRNA processing upon depletion of Rps2 revealed a reduction of 27SA2 pre-rRNAs and the concomitant production of 21S rRNA precursors, consistent with a role for Rps2 in efficient cleavage at site A2 within the 32S pre-rRNA. Importantly, kinetics of pre-rRNA accumulation as determined by rRNA pulse-chases assays indicated that a small fraction of 35S precursors matured into 20S-containing particles, suggesting that most 40S precursors were rapidly degraded in the absence of Rps2. Analysis of steady-state RNA levels revealed that some pre-40S particles were produced in Rps2-depleted cells, but that these precursors were retained in the nucleolus. Our findings suggest a role for Rps2 in a mechanism that monitors pre-40S export competence.

The initiation of eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at chromosomal origins of DNA replication. Pre-RC assembly requires the essential DNA replication proteins ORC, Cdc6, and Cdt1 to load the MCM DNA helicase onto chromatin. Saccharomyces cerevisiae Noc3 (ScNoc3), an evolutionarily conserved protein originally implicated in 60S ribosomal subunit trafficking, has been proposed to be an essential regulator of DNA replication that plays a direct role during pre-RC formation in budding yeast. We have cloned Schizosaccharomyces pombe noc3 ؉ (Spnoc3 ؉ ), the S. pombe homolog of the budding yeast ScNOC3 gene, and functionally characterized the requirement for the SpNoc3 protein during ribosome biogenesis, cell cycle progression, and DNA replication in fission yeast. We showed that fission yeast SpNoc3 is a functional homolog of budding yeast ScNoc3 that is essential for cell viability and ribosome biogenesis. We also showed that SpNoc3 is required for the normal completion of cell division in fission yeast. However, in contrast to the proposal that ScNoc3 plays an essential role during DNA replication in budding yeast, we demonstrated that fission yeast cells do enter and complete S phase in the absence of SpNoc3, suggesting that SpNoc3 is not essential for DNA replication in fission yeast.The timely and faithful duplication of large and discontinuous eukaryotic genomes is achieved by the coordinated initiation of DNA replication from hundreds to thousands of chromosomal origins of DNA replication during S phase. The initiation of DNA replication is a stepwise process involving (i) the assembly of prereplication complexes (pre-RCs) at replication origins during G 1 phase, (ii) pre-RC activation by conserved S-phase-promoting kinases, and (iii) the establishment of bidirectional DNA replication forks (the replisome) (reviewed in references 6, 24, 34, and 47). Genetic and biochemical approaches have revealed many, if not all, of the factors required for pre-RC assembly (ORC, Cdc6, Cdt1, MCM, and Mcm10), pre-RC activation (CDK and DDK), and replisome assembly (Cdc45, Sld2, Sld3, GINS, RPA, and DNA polymerases). However, the precise functions of many of these proteins await elucidation.Although the size and sequence composition of chromosomal origins vary widely among eukaryotes (reviewed in reference 12), ORC is universally required to recruit Cdc6 and Cdt1 to chromosomal replication origins prior to S phase (10, 11, 28). In turn, Cdc6 and Cdt1 cooperate with ORC to mediate the ATP-dependent loading of the heterohexameric MCM complex (Mcm2-7) onto chromatin to establish a functional pre-RC (16,19,22,23,28,35,44,48,49). The periodic expression and chromatin association of both Cdc6 and Cdt1 serve to restrict MCM loading and pre-RC formation to the G 1 phase and ensure that DNA replication occurs only once per cell cycle (2,20,21,25,35,36,39).Results from experiments in budding yeast suggest that Saccharomyces cerevisiae Noc3 (ScNoc3), a protein essential for large (...