Instruct: Given a scientific claim, retrieve relevant documents that support or refute the claim.
Query: Overexpressing Cnp1 N-tail variants exacerbates the temperature-sensitive growth defect of scm3-139.
The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.
The Bacillus subtilis clpC operon is regulated by two stress induction pathways relying on either sigmaB or a class III stress induction mechanism acting at a sigmaA-like promoter. When the clpC operon was placed under the control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Pspac promoter, dramatic repression of the natural clpC promoters fused to a lacZ reporter gene was noticed after IPTG induction. This result strongly indicated negative regulation of the clpC operon by one of its gene products. Indeed, the negative regulator could be identified which is encoded by the first gene of the clpC operon, ctsR, containing a predicted helix-turn-helix DNA-binding motif. Deletion of ctsR abolished the negative regulation and resulted in high expression of both the clpC operon and the clpP gene under nonstressed conditions. Nevertheless, a further increase in clpC and clpP mRNA levels was observed after heat shock, even in the absence of sigmaB, suggesting a second induction mechanism at the vegetative promoter. Two-dimensional gel analysis and mRNA studies showed that the expression of other class III stress genes was at least partially influenced by the ctsR deletion. Studies with different clpC promoter fragments either fused to the reporter gene bgaB or used in gel mobility shift experiments with the purified CtsR protein revealed a possible target region where the repressor seemed to bind in vivo and in vitro. Our data demonstrate that the CtsR protein acts as a global repressor of the clpC operon, as well as other class III heat shock genes, by preventing unstressed transcription from either the sigmaB- or sigmaA-dependent promoter and might be inactivated or dissociate under inducing stress conditions.
In the yeast Saccharomyces cerevisiae that lacks lamins, the nuclear pore complex (NPC) has been proposed to serve a role in chromatin organization. Here, using fluorescence microscopy in living cells, we show that nuclear pore proteins of the Nup84 core complex, Nup84p, Nup145Cp, Nup120p, and Nup133p, serve to anchor telomere XI-L at the nuclear periphery. The integrity of this complex is shown to be required for repression of a URA3 gene inserted in the subtelomeric region of this chromosome end. Furthermore, altering the integrity of this complex decreases the efficiency of repair of a DNA double-strand break (DSB) only when it is generated in the subtelomeric region, even though the repair machinery is functional. These effects are specific to the Nup84 complex. Our observations thus confirm and extend the role played by the NPC, through the Nup84 complex, in the functional organization of chromatin. They also indicate that anchoring of telomeres is essential for efficient repair of DSBs occurring therein and is important for preserving genome integrity.
The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein-labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.
Expanded CGG repeats cause chromosomal fragility and hereditary neurological disorders in humans. Replication forks stall at CGG repeats in a length-dependent manner in primate cells and in yeast. Saccharomyces cerevisiae proteins Tof1 and Mrc1 facilitate replication fork progression through CGG repeats. Remarkably, the fork-stabilizing role of Mrc1 does not involve its checkpoint function. Thus, chromosomal fragility might occur when forks stalled at expanded CGG repeats escape the S-phase checkpoint.
Telomerase is a ribonucleoprotein enzyme complex that reverse-transcribes an integral RNA template to add short DNA repeats to the 3'-ends of telomeres. G-quadruplex structure in a DNA substrate can block its extension by telomerase. We have found that hnRNP A1--which was previously implicated in telomere length regulation--binds to both single-stranded and structured human telomeric repeats, and in the latter case, it disrupts their higher-order structure. Using an in vitro telomerase assay, we observed that depletion of hnRNP A/B proteins from 293 human embryonic kidney cell extracts dramatically reduced telomerase activity, which was fully recovered upon addition of purified recombinant hnRNP A1. This finding suggests that hnRNP A1 functions as an auxiliary, if not essential, factor of telomerase holoenzyme. We further show, using chromatin immunoprecipitation, that hnRNP A1 associates with human telomeres in vivo. We propose that hnRNP A1 stimulates telomere elongation through unwinding of a G-quadruplex or G-G hairpin structure formed at each translocation step.
The molecular chaperone CCT/TRiC plays a central role in maintaining cellular proteostasis as it mediates the folding of the major cytoskeletal proteins tubulins and actins. CCT/TRiC is also involved in the oncoprotein cyclin E, the Von Hippel-Lindau tumour suppressor protein, cyclin B and p21(ras) folding which strongly suggests that it is involved in cell proliferation and tumor genesis. To assess the involvement of CCT/TRiC in tumor genesis, we quantified its expression levels and activity in 18 cancer, one non-cancer human cell lines and a non-cancer human liver. We show that the expression levels of CCT/TRiC in cancer cell lines are higher than that in normal cells. However, CCT/TRiC activity does not always correlate with its expression levels. We therefore documented the expression levels of CCT/TRiC modulators and partners PhLP3, Hop/P60, prefoldin and Hsc/Hsp70. Our analysis reveals a functional interplay between molecular chaperones that might account for a precise modulation of CCT/TRiC activity in cell proliferation through changes in the cellular levels of prefoldin and/or Hsc/p70 and CCT/TRiC client protein availability. Our observation and approaches bring novel insights in the role of CCT/TRiC-mediated protein folding machinery in cancer cell development.
Glycosyl phosphatidylinositols (GPIs) anchor many proteins to the surface of eukaryotic cells and may also serve as sorting signals on proteins and participate in signal transduction. We have isolated a Saccharomyces cerevisiae GPI anchoring mutant, gpi1, using a colony screen for cells blocked in [3H]inositol incorporation into protein. The gpi1 mutant is defective in vitro in the synthesis of N-acetylglucosaminyl phosphatidylinositol, the first intermediate in GPI synthesis, and is also temperature-sensitive for growth. Completion of the first step in GPI assembly is therefore required for growth of the unicellular eukaryote S. cerevisiae. GPI synthesis could therefore be exploited as a target for antifungal or antiparasitic agents.
The yeast ribosomal protein gene RPL32 of Saccharomyces cerevisiae is of particular interest for two reasons: 1) it is adjacent to another ribosomal protein gene, RP29, whose divergent transcription may be driven from the same control sequences, and 2) it appears that the splicing of its transcript is regulated by the product of the gene, ribosomal protein in L32. RPL32 has been analyzed in detail. It is essential for cell growth. Its sequence predicts L32 to be a protein of 105 amino acids, somewhat basic near the NH2 terminus, rather acidic near the COOH terminus, and homologous to ribosomal protein L30 of mammals. The reading frame has been confirmed by partial NH2-terminal analysis of L32. The nucleotide sequence also predicts an intron of 230 nucleotides, which begins with the unusual sequence GTCAGT and ends 40 nucleotides downstream of the consensus sequence TAC-TAAC. The intron has been confirmed by determination of the sequence of a cDNA clone. Transcription initiates 58 nucleotides upstream of the AUG initiation codon, and the polyadenylation site occurs 100 nucleotides downstream of the termination codon. Regulation of the transcription of ribosomal protein genes has been linked to two related consensus sequences. Analysis of the intergenic region between RP29 and RPL32 reveals three copies of these sequences. A deletion removing all three sequences reduces synthesis of a L32-LacZ fusion protein by more than 90%. Some residual activity, however, remains.
FANCM is a component of the Fanconi anemia (FA) core complex and one FA patient (EUFA867) with biallelic mutations in FANCM has been described. Strikingly, we found that EUFA867 also carries biallelic mutations in FANCA. After correcting the FANCA defect in EUFA867 lymphoblasts, a "clean" FA-M cell line was generated. These cells were hypersensitive to mitomycin C, but unlike cells defective in other core complex members, FANCM(-/-) cells were proficient in monoubiquitinating FANCD2 and were sensitive to the topoisomerase inhibitor camptothecin, a feature shared only with the FA subtype D1 and N. In addition, FANCM(-/-) cells were sensitive to UV light. FANCM and a C-terminal deletion mutant rescued the cross-linker sensitivity of FANCM(-/-) cells, whereas a FANCM ATPase mutant did not. Because both mutants restored the formation of FANCD2 foci, we conclude that FANCM functions in an FA core complex-dependent and -independent manner.
Charged multivesicular body protein 2B (CHMP2B) - a component of the endosomal complex required for transport-III (ESCRT-III) - is responsible for the vital membrane deformation functions in autophagy and endolysosomal trafficking. A dominant mutation in CHMP2B (CHMP2BIntron5) is associated with a subset of heritable frontotemporal dementia - frontotemporal dementia linked to chromosome 3 (FTD-3). ESCRT-III recruits Vps4, an AAA-ATPase that abscises the membrane during various cellular processes including autophagy and intraluminal vesicle formation. CHMP2BIntron5 results in a C-terminus truncation removing an important Vps4 binding site as well as eliminating the normal autoinhibitory resting state of CHMP2B. CHMP2B is expressed in most cell types but seems to be especially vital for proper neuronal function. CHMP2BIntron5-mediated phenotypes include misregulation of transmembrane receptors, accumulation of multilamellar structures, abnormal lysosomal morphology, down regulation of a brain-specific micro RNA (miRNA-124), abnormal dendritic spine morphology, decrease in dendritic arborization, and cell death. Currently, transgenic-fly,-mouse, and -human cell lines are being used to better understand the diverse phenotypes and develop therapeutic approaches for the CHMP2BIntron5-induced FTD-3. This article is part of a Special Issue entitled SI:Autophagy.
Cornelia de Lange syndrome (CdLS) is a multiple malformation disorder characterized by dysmorphic facial features, mental retardation, growth delay and limb reduction defects. We indentified and characterized a new gene, NIPBL, that is mutated in individuals with CdLS and determined its structure and the structures of mouse, rat and zebrafish homologs. We named its protein product delangin. Vertebrate delangins have substantial homology to orthologs in flies, worms, plants and fungi, including Scc2-type sister chromatid cohesion proteins, and D. melanogaster Nipped-B. We propose that perturbed delangin function may inappropriately activate DLX genes, thereby contributing to the proximodistal limb patterning defects in CdLS. Genome analyses typically identify individual delangin or Nipped-B-like orthologs in diploid animal and plant genomes. The evolution of an ancestral sister chromatid cohesion protein to acquire an additional role in developmental gene regulation suggests that there are parallels between CdLS and Roberts syndrome.
Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40 °C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142-5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40 °C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142-5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142-5p and miR-143.
Post-translational modifications of Notch3 and their functional role with respect to Notch3 overexpression in T-cell leukemia are still poorly understood. We identify here a specific novel property of Notch3 that is acetylated and deacetylated at lysines 1692 and 1731 by p300 and HDAC1, respectively, a balance impaired by HDAC inhibitors (HDACi) that favor hyperacetylation. By using HDACi and a non-acetylatable Notch3 mutant carrying K/R1692−1731 mutations in the intracellular domain, we show that Notch3 acetylation primes ubiquitination and proteasomal-mediated degradation of the protein. As a consequence, Notch3 protein expression and its transcriptional activity are decreased both in vitro and in vivo in Notch3 transgenic (tg) mice, thus impairing downstream signaling upon target genes. Consistently, Notch3-induced T-cell proliferation is inhibited by HDACi, whereas it is enhanced by the non-acetylatable Notch3-K/R1692−1731 mutant. Finally, HDACi-induced Notch3 hyperacetylation prevents in vivo growth of T-cell leukemia/lymphoma in Notch3 tg mice. Together, our findings suggest a novel level of Notch signaling control in which Notch3 acetylation/deacetylation process represents a key regulatory switch, thus representing a suitable druggable target for Notch3-sustained T-cell acute lymphoblastic leukemia therapy.
Down syndrome cell adhesion molecule (Dscam) seems likely to play a key role in the "alternative adaptive immunity" that has been reported in invertebrates. Dscam consists of a cytoplasmic tail that is involved in signal transduction and a hypervariable extracellular region that might use a pathogen recognition mechanism similar to that used by the vertebrate antibodies. In our previous paper, we isolated a unique tail-less form of Dscam from Litopenaeus vannamei. In this study, we report the first membrane-bound form of shrimp Dscam: PmDscam was isolated from Penaeus monodon, and it occurred in both membrane-bound and tail-less forms. Phylogenetic analysis showed that while the crustacean Dscams from shrimp and water flea did not share a single subclade, they were distinct from the invertebrate Dscam-like molecules and from the insecta Dscams. In the extracellular region, the variable regions of PmDscam were located in N-terminal Ig2, N-terminal Ig3 and the entire Ig7 domain. The PmDscam extracellular variants and transmembrane domain variants were produced by mutually exclusive alternative splicing events. The cytoplasmic tail variants were produced by exon inclusion/exclusion. Based on the genomic organization of Daphnia Dscam's cytoplasmic tail, we propose a model of how the shrimp Dscam genomic locus might use Type III polyadenylation to generate both the tail-less and membrane-bound forms.
Maternally expressed gene 3 (MEG3) is a maternally expressed imprinted gene representing a large noncoding RNA in which microRNAs (miRNAs) and small nucleolar RNAs are also hosted. It is capable of interacting with cyclic AMP, p53, murine double minute 2 (MDM2) and growth differentiation factor 15 (GDF15) playing a role in cell proliferation control. MEG3 expression is under epigenetic control, and aberrant CpG methylation has been observed in several types of cancer. Moreover, gene copy number loss has been reported as additional mechanism associated with tumorigenesis. MEG3 deletion seems to upregulate the paternally expressed genes and on the other hand downregulate the expression of downstream maternally expressed genes and tumor suppressor miRNAs, although there are conflicting data on the topic. MEG3 could represent a tumor suppressor gene located in chromosome 14q32 and its association with tumorigenesis is growing every day.
In the yeast Saccharomyces cerevisiae, genetic studies suggest that the RIM1 gene encodes a positive regulator of meiosis. rim1 mutations cause reduced expression of IME1, which is required for expression of many meiotic genes, and thus lead to a partial defect in meiosis and spore formation. We report the sequence of RIM1 and functional analysis of its coding region. The RIM1 gene product (RIM1) contains three regions similar to C2H2 zinc fingers. Serine substitutions for cysteine in each of the putative zinc fingers abolish RIM1 function. The carboxyl-terminus of RIM1 is enriched in acidic amino acids and is required for full RIM1 activity. RIM1 also contains two putative cAMP-dependent protein kinase (cAPK) phosphorylation sites. At one site, substitution of alanine for serine does not affect RIM1 activity; at the other site, this substitution impairs activity. This analysis of RIM1 suggests that the protein may function as a transcriptional activator. We have used the cloned RIM1 gene to create a complete rim1 deletion. This null allele, like previously isolated rim1 mutations, causes a partial meiotic defect. In addition to RIM1, maximum IME1 expression requires the MCK1 and IME4 gene products. Defects associated with rim1, mck1, and ime4 mutations in expression of a meiotic reporter gene (ime2-lacZ) and in sporulation are additive. These findings suggest that RIM1 acts independently of MCK1 and IME4 to stimulate IME1 expression.
A 3' overhang is critical for the protection and maintenance of mammalian telomeres, but its synthesis must be regulated to avoid excessive resection of the 5' end, which could cause telomere shortening. How this balance is achieved in mammals has not been resolved. Here, we determine the mechanism for 3' overhang synthesis in mouse cells by evaluating changes in telomeric overhangs throughout the cell cycle and at leading- and lagging-end telomeres. Apollo, a nuclease bound to the shelterin subunit TRF2, initiates formation of the 3' overhang at leading-, but not lagging-end telomeres. Hyperresection by Apollo is blocked at both ends by the shelterin protein POT1b. Exo1 extensively resects both telomere ends, generating transient long 3' overhangs in S/G2. CST/AAF, a DNA polα.primase accessory factor, binds POT1b and shortens the extended overhangs produced by Exo1, likely through fill-in synthesis. 3' overhang formation is thus a multistep, shelterin-controlled process, ensuring functional telomeric overhangs at chromosome ends.
Transfer RNAs specific for Gln, Lys, and Glu from all organisms (except Mycoplasma) and organelles have a 2-thiouridine derivative (xm(5)s(2)U) as wobble nucleoside. These tRNAs read the A- and G-ending codons in the split codon boxes His/Gln, Asn/Lys, and Asp/Glu. In eukaryotic cytoplasmic tRNAs the conserved constituent (xm(5)-) in position 5 of uridine is 5-methoxycarbonylmethyl (mcm(5)). A protein (Tuc1p) from yeast resembling the bacterial protein TtcA, which is required for the synthesis of 2-thiocytidine in position 32 of the tRNA, was shown instead to be required for the synthesis of 2-thiouridine in the wobble position (position 34). Apparently, an ancient member of the TtcA family has evolved to thiolate U34 in tRNAs of organisms from the domains Eukarya and Archaea. Deletion of the TUC1 gene together with a deletion of the ELP3 gene, which results in the lack of the mcm(5) side chain, removes all modifications from the wobble uridine derivatives of the cytoplasmic tRNAs specific for Gln, Lys, and Glu, and is lethal to the cell. Since excess of the unmodified form of these three tRNAs rescued the double mutant elp3 tuc1, the primary function of mcm(5)s(2)U34 seems to be to improve the efficiency to read the cognate codons rather than to prevent mis-sense errors. Surprisingly, overexpression of the mcm(5)s(2)U-lacking tRNA(Lys) alone was sufficient to restore viability of the double mutant.
In the yeast S. cerevisiae, ribosome assembly is linked to environmental conditions by the coordinate transcriptional regulation of genes required for ribosome biogenesis. In this study we show that two nonessential stress-responsive genes, YAR1 and LTV1, function in 40S subunit production. We provide genetic and biochemical evidence that Yar1, a small ankyrin-repeat protein, physically interacts with RpS3, a component of the 40S subunit, and with Ltv1, a protein recently identified as a substoichiometric component of a 43S preribosomal particle. We demonstrate that cells lacking YAR1 or LTV1 are hypersensitive to particular protein synthesis inhibitors and exhibit aberrant polysome profiles, with a reduced absolute number of 40S subunits and an excess of free 60S subunits. Surprisingly, both mutants are also hypersensitive to a variety of environmental stress conditions. Overexpression of RPS3 suppresses both the stress sensitivity and the ribosome biogenesis defect of Deltayar1 mutants, but does not suppress either defect in Deltaltv1 mutants. We propose that YAR1 and LTV1 play distinct, nonessential roles in 40S subunit production. The stress-sensitive phenotypes of strains lacking these genes reveal a hitherto unknown link between ribosome biogenesis factors and environmental stress sensitivity.
The nuclear matrix antigen recognized by the monoclonal antibody (mAb) B1C8 is a novel serine (S) and arginine (R)-rich protein associated with splicing complexes and is named here SRm160 (SR-related matrix protein of 160 kD). SRm160 contains multiple SR repeats, but unlike proteins of the SR family of splicing factors, lacks an RNA recognition motif. SRm160 and a related protein SRm300 (the 300-kD nuclear matrix antigen recognized by mAb B4A11) form a complex that is required for the splicing of specific pre-mRNAs. The SRm160/300 complex associates with splicing complexes and promotes splicing through interactions with SR family proteins. Binding of SRm160/300 to pre-mRNA is normally also dependent on U1 snRNP and is stabilized by U2 snRNP. Thus, SRm160/300 forms multiple interactions with components bound directly to important sites within pre-mRNA. The results suggest that a complex of the nuclear matrix proteins SRm160 and SRm300 functions as a coactivator of pre-mRNA splicing.
In wild-type budding yeast strains, the proteins encoded by SIR3, SIR4 and RAP1 co-localize with telomeric DNA in a limited number of foci in interphase nuclei. Immunostaining of Sir2p shows that in addition to a punctate staining that coincides with Rap1 foci, Sir2p localizes to a subdomain of the nucleolus. The presence of Sir2p at both the spacer of the rDNA repeat and at telomeres is confirmed by formaldehyde cross-linking and immunoprecipitation with anti-Sir2p antibodies. In strains lacking Sir4p, Sir3p becomes concentrated in the nucleolus, by a pathway requiring SIR2 and UTH4, a gene that regulates life span in yeast. The unexpected nucleolar localization of Sir2p and Sir3p correlates with observed effects of sir mutations on rDNA stability and yeast longevity, defining a new site of action for silent information regulatory factors.
Mutations in the PARN gene (encoding poly(A)-specific ribonuclease) cause telomere diseases including familial idiopathic pulmonary fibrosis (IPF) and dyskeratosis congenita, but how PARN deficiency impairs telomere maintenance is unclear. Here, using somatic cells and induced pluripotent stem cells (iPSCs) from patients with dyskeratosis congenita with PARN mutations, we show that PARN is required for the 3′-end maturation of the telomerase RNA component (TERC). Patient-derived cells as well as immortalized cells in which PARN is disrupted show decreased levels of TERC. Deep sequencing of TERC RNA 3′ termini shows that PARN is required for removal of post-transcriptionally acquired oligo(A) tails that target nuclear RNAs for degradation. Diminished TERC levels and the increased proportion of oligo(A) forms of TERC are normalized by restoring PARN, which is limiting for TERC maturation in cells. Our results demonstrate a new role for PARN in the biogenesis of TERC and provide a mechanism linking PARN mutations to telomere diseases.
We isolated a mutant allele of dnaX, encoding the tau and gamma subunits of the DNA polymerase III holoenzyme, that causes extreme cell filamentation but does not affect either cell growth or DNA replication. This phenotype results from a defect in daughter chromosome decatenation during rapid growth. In these cells, ParC, one subunit of topoisomerase IV, no longer associated with the replication factory, as occurs in wild-type cells, and was instead distributed uniformly on the nucleoid; the distribution of ParE, the other subunit of topoisomerase IV, was unaffected. In addition, the majority of topoisomerase IV activity in synchronized cell populations was restricted to late in the cell cycle, when replication was essentially complete. These observations suggest that topoisomerase IV activity in vivo might be dependent on release of ParC from the replication factory.
Small regulatory RNAs are key regulators of gene expression. One class of small regulatory RNAs, termed the endogenous small interfering RNAs (endo siRNAs), is thought to negatively regulate cellular transcripts via an RNA interference (RNAi)-like mechanism termed endogenous RNAi (endo RNAi). A complex of proteins composed of ERI-1/3/5, RRF-3, and DICER (the ERI/DICER complex) mediates endo RNAi processes in Caenorhabditis elegans. We conducted a genetic screen to identify additional components of the endo RNAi machinery. Our screen recovered alleles of eri-9, which encodes a novel DICER-interacting protein, and a missense mutation within the helicase domain of DICER [DCR-1(G492R)]. ERI-9(-) and DCR-1(G492) animals exhibit defects in endo siRNA expression and a concomitant failure to regulate mRNAs that exhibit sequence homology to these endo siRNAs, indicating that ERI-9 and the DCR-1 helicase domain function in the C. elegans endo RNAi pathway. We define a subset of Eri mutant animals (including eri-1, rrf-3, eri-3, and dcr-1, but not eri-9 or ergo-1) that exhibit temperature-sensitive, sperm-specific sterility and defects in X chromosome segregation. Among these mutants we find multiple aberrations in sperm development beginning with cytokinesis and extending through terminal differentiation. These results identify novel components of the endo RNAi machinery, demonstrate differential requirements for the Eri factors in the sperm-producing germline, and begin to delineate the functional requirement for the ERI/DICER complex in sperm development.