Endogenous small interfering RNAs (endo-siRNAs) regulate diverse gene expression programs in eukaryotes by either binding and cleaving mRNA targets or mediating heterochromatin formation; however, the mechanisms of endo-siRNA biogenesis, sorting, and target regulation remain poorly understood. Here we report the identification and function of a specific class of germline-generated endo-siRNAs in Caenorhabditis elegans that are 26 nt in length and contain a guanine at the first nucleotide position (i.e., 26G RNAs). 26G RNAs regulate gene expression during spermatogenesis and zygotic development, and their biogenesis requires the ERI-1 exonuclease and the RRF-3 RNA-dependent RNA polymerase (RdRP). Remarkably, we identified two nonoverlapping subclasses of 26G RNAs that sort into specific RNA-induced silencing complexes (RISCs) and differentially regulate distinct mRNA targets. Class I 26G RNAs target genes are expressed during spermatogenesis, whereas class II 26G RNAs are maternally inherited and silence gene expression during zygotic development. These findings implicate a class of endo-siRNAs in the global regulation of transcriptional programs required for fertility and development.endogenous siRNA ͉ germline ͉ RNA interference S mall RNAs bind Argonaute/Piwi proteins in the RNAinduced silencing complex (RISC) and, through base pairing, guide RISC to silence their cognate targets. While the taxonomy of small RNAs remains fluid, they can be defined in part by nucleotide length, 5Ј nucleotide composition, chemical modifications, genetic requirements for biogenesis, mode of silencing, and biological functions. For example, microRNAs are processed from double-stranded hairpin precursors by the RNase III-like enzyme Dicer to the Ϸ22-nt mature form containing a 5Ј-monophosphate nucleotide. The microRNAs associate with Argonaute (Ago) proteins in RISC and mediate translational repression and/or degradation of their target mRNAs (1). In contrast, Piwi-interacting RNAs (piRNAs) are typically longer than microRNAs, possess a uridine in the first nucleotide, and are generated by a Dicer-independent self-amplification pathway. The piRNAs bind to Piwi proteins in RISC and silence transposons (2).Endogenous small interfering RNAs (endo-siRNAs) represent an emerging class of small RNAs described and characterized in Caenorhabditis elegans by Ambros et al. (3). These endo-siRNAs are perfectly antisense to target transcripts and require the C. elegans Dicer, DCR-1, the RNA-dependent RNA polymerase (RdRP) RRF-3, and the exonuclease ERI-1 for expression (4,5). By large-scale pyrosequencing, Ruby et al. determined that other endo-siRNAs target transcripts associated with spermatogenesis and transposons (6). Therefore, C. elegans endo-siRNAs appear to be a diverse class of small RNAs, with distinct biological functions and genetic requirements for biogenesis. The recent discovery of endo-siRNAs derived from transposable elements, natural antisense transcripts, and hairpin RNAs in Drosophila melanogaster and Mus musculus (7-12) fur...

Male infertility is a long-standing enigma of significant medical concern. The integrity of sperm chromatin is a clinical indicator of male fertility and in vitro fertilization potential: chromosome aneuploidy and DNA decondensation or damage are correlated with reproductive failure. Identifying conserved proteins important for sperm chromatin structure and packaging can reveal universal causes of infertility. Here we combine proteomics, cytology and functional analysis in Caenorhabditis elegans to identify spermatogenic chromatin-associated proteins that are important for fertility. Our strategy employed multiple steps: purification of chromatin from comparable meiotic cell types, namely those undergoing spermatogenesis or oogenesis; proteomic analysis by multidimensional protein identification technology (MudPIT) of factors that co-purify with chromatin; prioritization of sperm proteins based on abundance; and subtraction of common proteins to eliminate general chromatin and meiotic factors. Our approach reduced 1,099 proteins co-purified with spermatogenic chromatin, currently the most extensive catalogue, to 132 proteins for functional analysis. Reduction of gene function through RNA interference coupled with protein localization studies revealed conserved spermatogenesis-specific proteins vital for DNA compaction, chromosome segregation, and fertility. Unexpected roles in spermatogenesis were also detected for factors involved in other processes. Our strategy to find fertility factors conserved from C. elegans to mammals achieved its goal: of mouse gene knockouts corresponding to nematode proteins, 37% (7/19) cause male sterility. Our list therefore provides significant opportunity to identify causes of male infertility and targets for male contraceptives.

In most sexually reproducing organisms, the fundamental process of meiosis is implemented concurrently with two differentiation programs that occur at different rates and generate distinct cell types, sperm and oocytes. However, little is known about how the meiotic program is influenced by such contrasting developmental programs. Here we present a detailed timeline of late meiotic prophase during spermatogenesis in Caenorhabditis elegans using cytological and molecular landmarks to interrelate changes in chromosome dynamics with germ cell cellularization, spindle formation, and cell cycle transitions. This analysis expands our understanding C. elegans spermatogenesis, as it identifies multiple spermatogenesis-specific features of the meiotic program and provides a framework for comparative studies. Post-pachytene chromatin of spermatocytes is distinct from that of oocytes in both composition and morphology. Strikingly, C. elegans spermatogenesis includes a previously undescribed karyosome stage, a common but poorly understood feature of meiosis in many organisms. We find that karyosome formation, in which chromosomes form a constricted mass within an intact nuclear envelope, follows desynapsis, involves a global down-regulation of transcription, and may support the sequential activation of multiple kinases that prepare spermatocytes for meiotic divisions. In spermatocytes, the presence of centrioles alters both the relative timing of meiotic spindle assembly and its ultimate structure. These microtubule differences are accompanied by differences in kinetochores, which connect microtubules to chromosomes. The sperm-specific features of meiosis revealed here illuminate how the underlying molecular machinery required for meiosis is differentially regulated in each sex.

Gene-specific and chromosome-wide mechanisms of transcriptional regulation control development in multicellular organisms. SDC-2, the determinant of hermaphrodite fate in Caenorhabditis elegans, is a paradigm for both modes of regulation. SDC-2 represses transcription of X chromosomes to achieve dosage compensation, and it also represses the male sex-determination gene her-1 to elicit hermaphrodite differentiation. We show here that SDC-2 recruits the entire dosage compensation complex to her-1, directing this X-chromosome repression machinery to silence an individual, autosomal gene. Functional dissection of her-1 in vivo revealed DNA recognition elements required for SDC-2 binding, recruitment of the dosage compensation complex, and transcriptional repression. Elements within her-1 differed in location, sequence, and strength of repression, implying that the dosage compensation complex may regulate transcription along the X chromosome using diverse recognition elements that play distinct roles in repression. The complex patterns of gene expression that control development are established by multiple regulatory mechanisms that operate locally on individual genes or globally across entire chromosomes or subchromosomal domains. These gene-specific and chromosome-wide modes of regulation are generally controlled by different protein complexes; however, a small number of global repressors have also been shown to function as local silencers of individual genes (Dawes et al. 1999;Moazed 2001;Nielsen et al. 2001). For example, the Sir proteins of Saccharomyces cerevisiae repress transcription at individual silent mating-type loci as well as telomeric regions of chromosomes (Aparicio et al. 1991;Moretti et al. 1994). The SDC-2 (sex-determination and dosage compensation) chromatin-binding protein of Caenorhabditis elegans has the versatility to repress transcription of an autosomal sex-determination gene by 20-fold and the entire X chromosome by twofold (Nusbaum and Meyer 1989;Trent et al. 1991;Dawes et al. 1999;Meyer 2000). Such dual-function regulatory components provide unique opportunities to explore mechanisms of local and global regulation by analyzing the more tractable mechanisms of gene-specific regulation.SDC-2 coordinates all hermaphrodite-specific aspects of C. elegans development (Nusbaum and Meyer 1989). Acting as a gene-specific repressor, SDC-2 induces hermaphrodite sexual differentiation in XX animals by repressing transcription of the male (XO) sex-determining gene her-1 (Trent et al. 1991;Dawes et al. 1999). Acting simultaneously as a chromosome-wide repressor, SDC-2 activates dosage compensation by triggering the assembly of a specialized protein complex onto hermaphrodite X chromosomes to reduce X-linked gene expression by half (Dawes et al. 1999). The dosage compensation complex (Chuang et al. 1996;Lieb et al. 1996Lieb et al. , 1998 resembles the condensin complex, which drives mitotic chromosome compaction in vitro (Koshland and Strunnikov 1996;Hirano 2000). X-chromosome repression is essential an...

Clathrin‐associated protein (AP) complexes have been implicated in the assembly of clathrin coats and the selectivity of clathrin‐mediated protein transport processes. We have identified a yeast gene, APS1, encoding a homolog of the small (referred to herein as sigma) subunits of the mammalian AP‐1 complex. Sequence comparisons have shown that Aps1p is more similar to the sigma subunit of the Golgi‐localized mammalian AP‐1 complex than Aps2p, which is more related to the plasma membrane AP‐2 sigma subunit. Like their mammalian counterparts, Aps1p and Aps2p are components of distinct, large (> 200 kDa) complexes and a significant portion of the Aps proteins co‐fractionate with clathrin‐coated vesicles during gel filtration chromatography. Unexpectedly, even though the evolutionary conservation of AP small subunits is substantial (50% identity between mammalian and yeast proteins), disruptions of APS1 (aps1 delta) and APS2 (aps2 delta), individually or in combination, elicit no detectable mutant phenotypes. These data indicate that the Aps proteins are not absolutely required for clathrin‐mediated selective protein transport in cells expressing wild type clathrin. However, aps1 delta accentuated the slow growth and alpha‐factor pheromone maturation defect of cells carrying a temperature‐sensitive allele of clathrin heavy chain (Chc) (chc1‐ts). In contrast, aps1 delta did not influence the effects of chc1‐ts on vacuolar protein sorting or receptor‐mediated endocytosis. The aps2 delta mutation resulted in a slight effect on chc1‐ts cell growth but had no additional effects. The growth defect of cells completely lacking Chc was compounded by aps1 delta but not aps2 delta. These results comprise evidence that Aps1p is involved in a subset of clathrin functions at the Golgi apparatus. The effect of aps1 delta on cells devoid of clathrin function suggests that Aps1p also participates in clathrin‐independent processes.