SummaryThe centriole, and the related basal body, is an ancient organelle characterized by a universal 9-fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are incompletely understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles.

Argonaute (Ago) proteins mediate silencing of nucleic acid targets by small RNAs. In fission yeast, Ago1, Tas3 and Chp1 assemble into a RITS complex, which silences transcription near centromeres. Here we describe a repetitive motif within Tas3, termed the 'Argonaute hook', that is conserved from yeast to humans and binds Ago proteins through their PIWI domains in vitro and in vivo. Site-directed mutation of key residues in the motif disrupts Ago binding and heterochromatic silencing in vivo. Unexpectedly, a PIWI domain pocket that binds the 5' end of the short interfering RNA guide strand is required for direct binding of the Ago hook. Moreover, wild-type but not mutant Ago hook peptides derepress microRNA-mediated translational silencing of a target messenger RNA. Proteins containing the conserved Ago hook may thus be important regulatory components of effector complexes in RNA interference.

The FACT complex is a conserved cofactor for RNA polymerase II elongation through nucleosomes. FACT bears histone chaperone activity and contributes to chromatin integrity. However, the molecular mechanisms behind FACT function remain elusive. Here we report biochemical, structural, and mutational analyses that identify the peptidase homology domain of the Schizosaccharomyces pombe FACT large subunit Spt16 (Spt16-N) as a binding module for histones H3 and H4. The 2.1-Å crystal structure of Spt16-N reveals an aminopeptidase P fold whose enzymatic activity has been lost. Instead, the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails. Mutations within a conserved surface pocket in Spt16-N or posttranslational modification of the histone H4 tail reduce interaction in vitro, whereas the globular domains of H3-H4 and the H3 tail bind distinct Spt16-N surfaces. Our analysis suggests that the N-terminal domain of Spt16 may add to the known H2A-H2B chaperone activity of FACT by including a H3-H4 tail and H3-H4 core binding function mediated by the N terminus of Spt16. We suggest that these interactions may aid FACT-mediated nucleosome reorganization events.histone chaperone ͉ histone modifications ͉ protein evolution ͉ site-directed mutagenesis ͉ transcription N ucleosomes create a natural barrier to RNA polymerase II (Pol II) progression. Transcription of histone-wrapped DNA thus requires factors that promote nucleosome remodeling, such as the histone chaperone FACT (facilitates chromatin transcription), which in human cells purifies as a heterodimer of Spt16 and SSRP1 (1, 2).FACT is a highly conserved complex (2-5). In fungi, SSRP1 is largely encoded by POB3, whereas NHP6 encodes the archetypal HMG-box of SSRP1. The gene for the Spt16 subunit is essential in Saccharomyces cerevisiae and Schizosaccharomyces pombe, probably reflecting important chromatin-related functions in transcription, replication, and DNA repair (4, 6-9). Genetic screens identified Spt16 as a factor whose mutation restores the expression of a Ty1 transposon-silenced reporter gene by promoting its cryptic transcription, known as a suppressor of Ty1 phenotype, [Spt Ϫ ] (4, 7, 10, 11). Several POB3 alleles genes display [Spt Ϫ ] phenotypes (12), indicating that the maintenance of correct chromatin structure involves Pol II cofactors such as FACT (13). Indeed, FACT acts as a coactivator of transcriptional initiation and elongation (14), and many Spt16 alleles display genetic interactions with basal transcription factors. Furthermore, FACT subunits biochemically interact with the Pol II elongation complex Paf1 (15), bind the coding region of transcribed Pol II genes, and are recruited to inducible genes upon activation (16-19).FACT's biological roles in transcription (and replication) may stem from its histone chaperone activity (20,21). Histone chaperones stimulate reactions involving the transfer of histones (22), thereby mediating chromatin reorganiz...

Centromeres exert vital cellular functions in mitosis and meiosis. A specialized histone and other chromatin-bound factors nucleate a dynamic protein assembly that is required for the proper segregation of sister chromatids. In several organisms, including the fission yeast, Schizosaccharomyces pombe, the RNAi pathway contributes to the formation of silent chromatin in pericentromeric regions. Little is known about how chromatin-remodeling factors contribute to heterochromatic integrity and centromere function. Here we show that the histone chaperone and remodeling complex FACT is required for centromeric-heterochromatin integrity and accurate chromosome segregation. We show that Spt16 and Pob3 are two subunits of the S. pombe FACT complex. Surprisingly, yeast strains deleted for pob3+ are viable and alleviate gene silencing at centromeric repeats and at the silent mating-type locus. Importantly, like heterochromatin and RNAi pathway mutants, Pob3 null strains exhibit lagging chromosomes on anaphase spindles. Whereas the processing of centromeric RNA transcripts into siRNAs is maintained in Pob3 mutants, Swi6-association with the centromere is reduced. Our studies provide the first experimental evidence for a role of the RNA polymerase II cofactor FACT in heterochromatin integrity and in centromere function.

The Ustilago maydis mrb1 gene specifies a mitochondrial matrix protein with significant similarity to mitochondrial p32 family proteins known from human and many other eukaryotic species. Compatible mrb1 mutant strains were able to mate and form dikaryotic hyphae; however, proliferation within infected tissue and the ability to induce tumor development of infected maize (Zea mays) plants were drastically impaired. Surprisingly, manifestation of the mrb1 mutant phenotype selectively depended on the a2 mating type locus. The a2 locus contains, in addition to pheromone signaling components, the genes lga2 and rga2 of unknown function. Deletion of lga2 in an a2Dmrb1 strain fully restored pathogenicity, whereas pathogenicity was partially regained in an a2Dmrb1Drga2 strain, implicating a concerted action between Lga2 and Rga2 in compromising pathogenicity in Dmrb1 strains. Lga2 and Rga2 localized to mitochondria and Mrb1 interacted with Rga2 in the yeast two-hybrid system. Conditional expression of lga2 in haploid cells reduced vegetative growth, conferred mitochondrial fragmentation and mitochondrial DNA degradation, and interfered with respiratory activity. The consequences of lga2 overexpression depended on the expression strength and were greatly exacerbated in Dmrb1 mutants. We propose that Lga2 interferes with mitochondrial fusion and that Mrb1 controls this activity, emphasizing a critical link between mitochondrial morphology and pathogenicity.