SLX-1 Is Required for Maintaining Genomic Integrity and Promoting Meiotic Noncrossovers in the Caenorhabditis elegans Germline

Although the SLX4 complex, which includes structure-specific nucleases such as XPF, MUS81, and SLX1, plays important roles in the repair of several kinds of DNA damage, the function of SLX1 in the germline remains unknown. Here we characterized the endonuclease activities of the Caenorhabditis elegans SLX-1-HIM-18/SLX-4 complex co-purified from human 293T cells and determined SLX-1 germline function via analysis of slx-1(tm2644) mutants. SLX-1 shows a HIM-18/SLX-4-dependent endonuclease activity toward replication forks, 5'-flaps, and Holliday junctions. slx-1 mutants exhibit hypersensitivity to UV, nitrogen mustard, and camptothecin, but not gamma irradiation. Consistent with a role in DNA repair, recombination intermediates accumulate in both mitotic and meiotic germ cells in slx-1 mutants. Importantly, meiotic crossover distribution, but not crossover frequency, is altered on chromosomes in slx-1 mutants compared to wild type. This alteration is not due to changes in either the levels or distribution of double-strand breaks (DSBs) along chromosomes. We propose that SLX-1 is required for repair at stalled or collapsed replication forks, interstrand crosslink repair, and nucleotide excision repair during mitosis. Moreover, we hypothesize that SLX-1 regulates the crossover landscape during meiosis by acting as a noncrossover-promoting factor in a subset of DSBs.     

Potential Roles for Centromere Pairing in Meiotic Chromosome Segregation

One of the key differences between mitosis and meiosis is the necessity for exchange between homologous chromosomes. Crossing-over between homologous chromosomes is essential for proper meiotic chromosome segregation in most organisms, serving the purpose of linking chromosomes to their homologous partners until they segregate from one another at anaphase I. In several organisms it has been shown that occasional pairs of chromosomes that have failed to experience exchange segregate with reduced fidelity compared to exchange chromosomes, but do not segregate randomly. Such observations support the notion that there are mechanisms, beyond exchange, that contribute to meiotic segregation fidelity. Recent findings indicate that active centromere pairing is important for proper kinetochore orientation and consequently, segregation of non-exchange chromosomes. Here we discuss the implications of these findings for the behavior of meiotic chromosomes.     

Meiotic chromosomes in motion: a perspective from Mus musculus and Caenorhabditis elegans

Vigorous chromosome movement during the extended prophase of the first meiotic division is conserved in most eukaryotes. The movement is crucial for the faithful segregation of homologous chromosomes into daughter cells, and thus for fertility. A prerequisite for meiotic chromosome movement is the stable and functional attachment of telomeres or chromosome ends to the nuclear envelope and their cytoplasmic coupling to the cytoskeletal forces responsible for generating movement. Important advances in understanding the components, mechanisms, and regulation of chromosome end attachment and movement have recently been made. This review focuses on insights gained from experiments into two major metazoan model organisms: the mouse, Mus musculus, and the nematode, Caenorhabditis elegans.

     

Pseudosynapsis and Decreased Stringency of Meiotic Repair Pathway Choice on the Hemizygous Sex Chromosome of Caenorhabditis elegans Males

During meiosis, accurate chromosome segregation relies on homology to mediate chromosome pairing, synapsis, and crossover recombination. Crossovers are dependent upon formation and repair of double-strand breaks (DSBs) by homologous recombination (HR). In males of many species, sex chromosomes are largely hemizygous, yet DSBs are induced along nonhomologous regions. Here we analyzed the genetic requirements for meiotic DSB repair on the completely hemizygous X chromosome of Caenorhabditis elegans males. Our data reveal that the kinetics of DSB formation, chromosome pairing, and synapsis are tightly linked in the male germ line. Moreover, DSB induction on the X is concomitant with a brief period of pseudosynapsis that may allow X sister chromatids to masquerade as homologs. Consistent with this, neither meiotic kleisins nor the SMC-5/6 complex are essential for DSB repair on the X. Furthermore, early processing of X DSBs is dependent on the CtIP/Sae2 homolog COM-1, suggesting that as with paired chromosomes, HR is the preferred pathway. In contrast, the X chromosome is refractory to feedback mechanisms that ensure crossover formation on autosomes. Surprisingly, neither RAD-54 nor BRC-2 are essential for DSB repair on the X, suggesting that unlike autosomes, the X is competent for repair in the absence of HR. When both RAD-54 and the structure-specific nuclease XPF-1 are abrogated, X DSBs persist, suggesting that single-strand annealing is engaged in the absence of HR. Our findings indicate that alteration in sister chromatid interactions and flexibility in DSB repair pathway choice accommodate hemizygosity on sex chromosomes.     

Two Types of Sites Required for Meiotic Chromosome Pairing in Caenorhabditis Elegans

Previous studies have shown that isolated portions of Caenorhabditis elegans chromosomes are not equally capable of meiotic exchange. These results led to the proposal that a homolog recognition region (HRR), defined as the region containing those sequences enabling homologous chromosomes to pair and recombine, is localized near one end of each chromosome. Using translocations and duplications we have localized the chromosome I HRR to the right end. Whereas the other half of chromosome I did not confer any ability for homologs to pair and recombine, deficiencies in this region dominantly suppressed recombination to the middle of the chromosome. These deletions may have disrupted pairing mechanisms that are secondary to and require an HRR. Thus, the processes of pairing and recombination appear to utilize at least two chromosomal elements, the HRR and other pairing sites. For example, terminal sequences from other chromosomes increase the ability of free duplications to recombine with their normal homologs, suggesting that telomere-associated sequences, homologous or nonhomologous, play a role in facilitating meiotic exchange. Recombination can also initiate at internal sites separated from the HRR by chromosome rearrangement, such as deletions of the unc-54 region of chromosome I. When crossing over was suppressed in a region of chromosome I, compensatory increases were observed in other regions. Thus, the presence of the HRR enabled recombination to occur but did not determine the distribution of the crossover events. It seems most likely that there are multiple initiation sites for recombination once homolog recognition has been achieved.     

The Multiple Roles of Cohesin in Meiotic Chromosome Morphogenesis and Pairing

Sister chromatid cohesion, mediated by cohesin complexes, is laid down during DNA replication and is essential for the accurate segregation of chromosomes. Previous studies indicated that, in addition to their cohesion function, cohesins are essential for completion of recombination, pairing, meiotic chromosome axis formation, and assembly of the synaptonemal complex (SC). Using mutants in the cohesin subunit Rec8, in which phosphorylated residues were mutated to alanines, we show that cohesin phosphorylation is not only important for cohesin removal, but that cohesin's meiotic prophase functions are distinct from each other. We find pairing and SC formation to be dependent on Rec8, but independent of the presence of a sister chromatid and hence sister chromatid cohesion. We identified mutations in REC8 that differentially affect Rec8's cohesion, pairing, recombination, chromosome axis and SC assembly function. These findings define Rec8 as a key determinant of meiotic chromosome morphogenesis and a central player in multiple meiotic events.     

An Aneuploidy-Free and Structurally Defined Balancer Chromosome Toolkit for Caenorhabditis elegans

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Roles of Hop1 and Mek1 in Meiotic Chromosome Pairing and Recombination Partner Choice in Schizosaccharomyces pombe

Synaptonemal complex (SC) proteins Hop1 and Mek1 have been proposed to promote homologous recombination in meiosis of Saccharomyces cerevisiae by establishment of a barrier against sister chromatid recombination. Therefore, it is interesting to know whether the homologous proteins play a similar role in Schizosaccharomyces pombe. Unequal sister chromatid recombination (USCR) was found to be increased in hop1 and mek1 single and double deletion mutants in assays for intrachromosomal recombination (ICR). Meiotic intergenic (crossover) and intragenic (conversion) recombination between homologous chromosomes was reduced. Double-strand break (DSB) levels were also lowered. Notably, deletion of hop1 restored DSB repair in rad50S meiosis. This may indicate altered DSB repair kinetics in hop1 and mek1 deletion strains. A hypothesis is advanced proposing transient inhibition of DSB processing by Hop1 and Mek1 and thus providing more time for repair by interaction with the homologous chromosome. Loss of Hop1 and Mek1 would then result in faster repair and more interaction with the sister chromatid. Thus, in S. pombe meiosis, where an excess of sister Holliday junction over homologous Holliday junction formation has been demonstrated, Hop1 and Mek1 possibly enhance homolog interactions to ensure wild-type level of crossover formation rather than inhibiting sister chromatid interactions.Sexual reproduction in eukaryotes involves formation of haploid gametes from diploid cells by one round of DNA replication, pairing of the homologous chromosomes, and recombination and then by the two meiotic divisions (53). In fungi the gametes differentiate into haploid spores, which germinate to form vegetative cells. Crossover (CO) formation between homologous chromosomes and DNA repair processes between sister chromatids are required for spore viability (10, 55, 58).In vegetative cells homologous recombination (HR) is important for repair of DNA damage and stalled replication forks, with the sister chromatid as the preferred partner (28). Many of the enzymes involved in mitotic HR also contribute to meiotic recombination. In addition, meiosis-specific cytological structures and enzymes enhance recombination frequency (meiotic induction) and shift partner preference from sister chromatids to homologous chromosomes (3, 47, 64, 74). In detail the steps of HR vary between different types of sequence organization (allelic versus sister versus ectopic), between different types of DNA damage, between meiotic and mitotic cells, and between species (10, 55, 58).Meiotic recombination, including CO formation, is initiated by DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae and other eukaryotes, DSBs are formed by Spo11. Many cofactors are required (29). The Schizosaccharomyces pombe homolog is Rec12, also requiring auxiliary factors whose elimination leads to loss of meiotic DSB formation (12). The 5′ single-strand ends at DSBs are processed by nucleases. In S. cerevisiae the MRX complex made up by the proteins Rad50, Mre11, and Xrs2 is required for this resection, as well as for DSB formation. The corresponding MRN complex of S. pombe (Rad50, Rad32, and Nbs1) is not required for DSB formation but is essential for DSB repair (43, 72). Deletion of rad50, rad32, or ctp1 (homologous to SAE2/COM1 in S. cerevisiae and CtIP in humans) leads to very low spore viability. These proteins are also essential for DSB processing (23, 24, 32, 43, 60, 62).Free DNA 3′ ends at DSBs are recruited for invasion of a sister or homologous chromatid by the strand transfer proteins Rad51 and Dmc1, again involving many accessory proteins (16). This results in the central intermediates of HR: heteroduplex DNA consisting of single strands originating from different chromatids and Holliday junctions (HJs). In S. cerevisiae HJs form preferably between homologs with a two- to sixfold excess over intersister HJs (64). Surprisingly, meiotic HJs form with about a fourfold excess between sisters in S. pombe (11). Eventually the intermediates are resolved into crossover (CO) and noncrossover (NCO) events. COs show exchange of the flanking sequences of the two chromatids involved and usually carry a patch of conversion (unilateral transfer of DNA sequences from one chromatid to its interacting partner) near the DSB site. NCOs are conversion events without associated COs (22). In S. pombe loss of core HR functions leads to very low spore viability: deletion of rad51 but not of dmc1 (20), double mutation of rad54 and rdh54 (7), inactivation of the endonuclease activity encoded by mus81 and eme1 (5, 52), and combined deletion of rad22 and rti1 (homologs of RAD52 of S. cerevisiae). But, differently from the other core functions, Rad22 and Rti1 are not required for CO and NCO (50).Early in meiotic prophase of many eukaryotes, axial elements (called lateral elements in later stages) form along sister chromatids, and pairing of homologous chromosomes is initiated, leading to juxtaposition of the homologous chromosomes along their whole length in the synaptonemal complex (SC) (54). In S. pombe no SC is formed, but linear elements (LEs), resembling axial elements of other eukaryotes, are formed. LEs do not form continuously along the chromosomes (1) but load the proteins Rec10, Hop1, and Mek1 (36, 44, 57), which are homologs of, or at least related, to the S. cerevisiae proteins Red1, Hop1, and Mek1, respectively, localizing to axial/lateral elements (2, 67). Hop1 carries a HORMA domain, also present in proteins associating with axial elements and regulating the progress of recombination in higher eukaryotes: Arabidopsis thaliana (61), Caenorhabditis elegans (9, 41), and mammals (18).In S. cerevisiae localization of Hop1 and Mek1 (meiosis-specific protein kinase) to axial elements is dependent on Red1 (2, 67). Mutation of the three S. cerevisiae genes results in reduction of DSB formation, CO and conversion frequencies, and spore viability (26, 31, 59). Direct comparison of unequal sister chromatid recombination (USCR) frequencies in an assay excluding the scoring of intrachromatid recombination (ICR) revealed no increase in the hop1 null mutant but about fourfold increases in the red1 and mek1 null mutants (69). The S. cerevisiae Hop1, Red1, and Mek1 proteins are involved in biasing meiotic DSB repair to occur between homologous chromosomes rather than between sister chromatids (47). Activated Mek1 kinase is required for the inhibition of sister chromatid-mediated DSB repair by Rad51, when the DMC1 gene is deleted and the meiotic recombination checkpoint is activated (4, 27, 38, 47). For Mek1 activation, phosphorylation of Hop1 by the Mec1/Tel1 kinases is also required (6).Less is known about the S. pombe proteins. Hop1 of S. pombe was identified as a nonsignificant hit by sequence comparison with full-length S. cerevisiae Hop1 and contains an N-terminal HORMA domain and a central zinc finger motif like Hop1 in S. cerevisiae. In addition they share a short homology block toward the C terminus (36). The Mek1 protein of S. pombe shares 34% identity and 54% similarity with its S. cerevisiae counterpart along the whole sequence. It contains an FHA domain in the N-terminal part like the other members of its family of checkpoint kinases and is involved in regulation of the meiotic cell cycle (57). Hop1 and Mek1 are strongly expressed in meiosis but not expressed or only slightly expressed in vegetative cells (42, 57). In prophase both proteins localize to LEs as defined by colocalization with the LE component Rec10 (36). Deletion of the distant RED1 homolog rec10 abolishes LE formation (36, 44) and strongly reduces meiotic recombination (17, 70). Rec10, but not Hop1 and Mek1, is required for localization of Rec7 (a distant homolog of S. cerevisiae Rec114) to meiotic chromosomes (34). Rec7 and Rec10 are required for Rec12 activity (12, 29).Obtaining information on the functions of Hop1 and Mek1 in S. pombe was the aim of the work presented here, especially on their possible roles in homolog versus sister discrimination for DSB repair. Deletion mutants have been studied with respect to spore viability and the frequencies of CO and conversion. They have also been assessed for genetic recombination events between sister chromatids in the known PS1 assay (63) and the newly developed VL1 assay (for details, see Fig. ​Fig.3).3). Physical analysis of DSB formation and repair has been performed in meiotic time course experiments. It is proposed that S. pombe Hop1 and Mek1 are promoting interactions between homologous chromosomes rather than inhibiting interactions between sister chromatids.Open in a separate windowFIG. 3.PS1 and VL1 assay systems for intrachromosomal recombination. Strains with constructs carrying repeated DNA sequences have been assayed for prototroph formation either by intrachromatid recombination (ICR, yielding prototrophs only in PS1) or by unequal sister chromatid recombination (USCR, in PS1 and VL1). Crosses of the constructs were performed with strains carrying a deletion of the ade6 gene to exclude other homologous recombination events. (A) The PS1 assay involves copies of the ade6 gene inactivated by either the hot spot mutation M26 or the mutation 469. The repeated sequences are separated by the ura4⁺ marker (63). ICR (left) or USCR (right) between the repeated sequences can lead to formation of adenine prototrophs that have lost the ura4⁺ marker by crossover (CO) or single-strand annealing (SSA) events. Adenine prototrophs maintaining the ura4⁺ marker can derive from noncrossover (NCO) events. Both types of pairing may lead to CO or NCO products. (B) The newly constructed VL1 assay (see the supplemental material) involves different truncations of the ade6 gene separated by the hyg^R marker (also called hphMX6), conferring hygromycin resistance. The left truncation carries a 3′ portion of ade6; the right truncation carries a 5′ portion of ade6. While the gray parts of the truncations are not overlapping, the white sections of 500-bp length are of almost identical sequence, allowing for homologous pairing. CO and SSA products resulting from ICR retain only the central portion of ade6 and remain auxotrophic. Adenine prototrophic CO and NCO products resulting from USCR both retain hygromycin resistance. Note that NCO events may arise through loop formation of one sister chromatid and pairing with a single block (500 bp) of the repeated ade6 sequence (39).     

Identification of DSB-1, a Protein Required for Initiation of Meiotic Recombination in Caenorhabditis elegans,Illuminates a Crossover Assurance Checkpoint

Meiotic recombination, an essential aspect of sexual reproduction, is initiated by programmed DNA double-strand breaks (DSBs). DSBs are catalyzed by the widely-conserved Spo11 enzyme; however, the activity of Spo11 is regulated by additional factors that are poorly conserved through evolution. To expand our understanding of meiotic regulation, we have characterized a novel gene, dsb-1, that is specifically required for meiotic DSB formation in the nematode Caenorhabditis elegans. DSB-1 localizes to chromosomes during early meiotic prophase, coincident with the timing of DSB formation. DSB-1 also promotes normal protein levels and chromosome localization of DSB-2, a paralogous protein that plays a related role in initiating recombination. Mutations that disrupt crossover formation result in prolonged DSB-1 association with chromosomes, suggesting that nuclei may remain in a DSB-permissive state. Extended DSB-1 localization is seen even in mutants with defects in early recombination steps, including spo-11, suggesting that the absence of crossover precursors triggers the extension. Strikingly, failure to form a crossover precursor on a single chromosome pair is sufficient to extend the localization of DSB-1 on all chromosomes in the same nucleus. Based on these observations we propose a model for crossover assurance that acts through DSB-1 to maintain a DSB-permissive state until all chromosome pairs acquire crossover precursors. This work identifies a novel component of the DSB machinery in C. elegans, and sheds light on an important pathway that regulates DSB formation for crossover assurance.     

Systematic Analyses of rpm-1 Suppressors Reveal Roles for ESS-2 in mRNA Splicing in Caenorhabditis elegans

The PHR (Pam/Highwire/RPM-1) family of ubiquitin E3 ligases plays conserved roles in axon patterning and synaptic development. Genetic modifier analysis has greatly aided the discovery of the signal transduction cascades regulated by these proteins. In Caenorhabditis elegans, loss of function in rpm-1 causes axon overgrowth and aberrant presynaptic morphology, yet the mutant animals exhibit little behavioral deficits. Strikingly, rpm-1 mutations strongly synergize with loss of function in the presynaptic active zone assembly factors, syd-1 and syd-2, resulting in severe locomotor deficits. Here, we provide ultrastructural evidence that double mutants, between rpm-1 and syd-1 or syd-2, dramatically impair synapse formation. Taking advantage of the synthetic locomotor defects to select for genetic suppressors, previous studies have identified the DLK-1 MAP kinase cascade negatively regulated by RPM-1. We now report a comprehensive analysis of a large number of suppressor mutations of this screen. Our results highlight the functional specificity of the DLK-1 cascade in synaptogenesis. We also identified two previously uncharacterized genes. One encodes a novel protein, SUPR-1, that acts cell autonomously to antagonize RPM-1. The other affects a conserved protein ESS-2, the homolog of human ES2 or DGCR14. Loss of function in ess-2 suppresses rpm-1 only in the presence of a dlk-1 splice acceptor mutation. We show that ESS-2 acts to promote accurate mRNA splicing when the splice site is compromised. The human DGCR14/ES2 resides in a deleted chromosomal region implicated in DiGeorge syndrome, and its mutation has shown high probability as a risk factor for schizophrenia. Our findings provide the first functional evidence that this family of proteins regulate mRNA splicing in a context-specific manner.     

Meiotic Mutants That Cause a Polar Decrease in Recombination on the X Chromosome in Caenorhabditis Elegans

Recessive mutations in three autosomal genes, him-1, him-5 and him-8, cause high levels of X chromosome nondisjunction in hermaphrodites of Caenorhabditis elegans, with no comparable effect on autosomal disjunction. Each of the mutants has reduced levels of X chromosome recombination, correlating with the increase in nondisjunction. However, normal or elevated levels of recombination occur at the end of the X chromosome hypothesized to contain the pairing region (the left end), with recombination levels decreasing in regions approaching the right end. Thus, both the number and the distribution of X chromosome exchange events are altered in these mutants. As a result, the genetic map of the X chromosome in the him mutants exhibits a clustering of genes due to reduced recombination, a feature characteristic of the genetic map of the autosomes in non-mutant animals. We hypothesize that these him genes are needed for some processive event that initiates near the left end of the X chromosome.     

Caenorhabditis elegans RMI2 functional homolog-2 (RMIF-2) and RMI1 (RMH-1) have both overlapping and distinct meiotic functions within the BTR complex

Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to genetic exchange, originating from crossover (CO) generation. In mitosis, COs are suppressed to prevent sister chromatid exchange. Here, the BTR complex, consisting of the Bloom helicase (HIM-6 in worms), topoisomerase 3 (TOP-3), and the RMI1 (RMH-1 and RMH-2) and RMI2 scaffolding proteins, is essential for dismantling joint DNA molecules to form non-crossovers (NCOs) via decatenation. In contrast, in meiosis COs are essential for accurate chromosome segregation and the BTR complex plays distinct roles in CO and NCO generation at different steps in meiotic recombination. RMI2 stabilizes the RMI1 scaffolding protein, and lack of RMI2 in mitosis leads to elevated sister chromatid exchange, as observed upon RMI1 knockdown. However, much less is known about the involvement of RMI2 in meiotic recombination. So far, RMI2 homologs have been found in vertebrates and plants, but not in lower organisms such as Drosophila, yeast, or worms. We report the identification of the Caenorhabditis elegans functional homolog of RMI2, which we named RMIF-2. The protein shows a dynamic localization pattern to recombination foci during meiotic prophase I and concentration into recombination foci is mutually dependent on other BTR complex proteins. Comparative analysis of the rmif-2 and rmh-1 phenotypes revealed numerous commonalities, including in regulating CO formation and directing COs toward chromosome arms. Surprisingly, the prevalence of heterologous recombination was several fold lower in the rmif-2 mutant, suggesting that RMIF-2 may be dispensable or less strictly required for some BTR complex-mediated activities during meiosis.     

Roles for two partially redundant alpha-tubulins during mitosis in early Caenorhabditis elegans embryos

The Caenorhabditis elegans genome encodes multiple isotypes of alpha-tubulin and beta-tubulin. Roles for a number of these tubulins in neuronal development have been described, but less is known about the isoforms that function during early embryonic development. Microtubules are required for multiple events after fertilization produces a one-cell zygote in C. elegans, including pronuclear migration, mitotic spindle assembly and function, and proper spindle positioning. Here we describe a conditional and dominant mis-sense mutation in the C. elegans alpha-tubulin gene tba-1 that disrupts pronuclear migration and positioning of the first mitotic spindle, and results in a highly penetrant embryonic lethality, at the restrictive temperature of 26 degrees C. Our analysis of the dominant tba-1 (or346ts) allele suggests that TBA-1 assembles into microtubules in early embryonic cells. However, we also show that reduction of tba-1 function using RNA interference results in defects much less severe than those caused by the dominant or346ts mutation, due to partial redundancy of TBA-1 and another alpha-tubulin called TBA-2. Reducing the function of both TBA-1 and TBA-2 results in severe defects in microtubule-dependent processes. We conclude that microtubules in the early C. elegans embryo are composed of both TBA-1 and TBA-2, and that the dominant tba-1(or346ts) mutation disrupts MT assembly or stability. Cell Motil.     

Kinesin-1 and Cytoplasmic Dynein Act Sequentially to Move the Meiotic Spindle to the Oocyte Cortex in Caenorhabditis elegans

During female meiosis in animals, the meiotic spindle is attached to the egg cortex by one pole during anaphase to allow selective disposal of half the chromosomes in a polar body. In Caenorhabditis elegans, this anaphase spindle position is achieved sequentially through kinesin-1–dependent early translocation followed by anaphase-promoting complex (APC)-dependent spindle rotation. Partial depletion of cytoplasmic dynein heavy chain by RNA interference blocked spindle rotation without affecting early translocation. Dynein depletion also blocked the APC-dependent late translocation that occurs in kinesin-1–depleted embryos. Time-lapse imaging of green fluorescent protein-tagged dynein heavy chain as well as immunofluorescence with dynein-specific antibodies revealed that dynein starts to accumulate at spindle poles just before the initiation of rotation or late translocation. Accumulation of dynein at poles was kinesin-1 independent and APC dependent, just like dynein driven spindle movements. This represents a case of kinesin-1/dynein coordination in which these two motors of opposite polarity act sequentially and independently on a cargo to move it in the same direction.     

SPDL-1 functions as a kinetochore receptor for MDF-1 in Caenorhabditis elegans

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation by delaying anaphase onset until all sister kinetochores are attached to bipolar spindles. An RNA interference screen for synthetic genetic interactors with a conserved SAC gene, san-1/MAD3, identified spdl-1, a Caenorhabditis elegans homologue of Spindly. SPDL-1 protein localizes to the kinetochore from prometaphase to metaphase, and this depends on KNL-1, a highly conserved kinetochore protein, and CZW-1/ZW10, a component of the ROD–ZW10–ZWILCH complex. In two-cell–stage embryos harboring abnormal monopolar spindles, SPDL-1 is required to induce the SAC-dependent mitotic delay and localizes the SAC protein MDF-1/MAD1 to the kinetochore facing away from the spindle pole. In addition, SPDL-1 coimmunoprecipitates with MDF-1/MAD1 in vivo. These results suggest that SPDL-1 functions in a kinetochore receptor of MDF-1/MAD1 to induce SAC function.     

Small RNAs, RNAi and the Inheritance of Gene Silencing in Caenorhabditis elegans

Invasive nucleic acids such as transposons and viruses usually exhibit aberrant characteristics, e.g., unpaired DNA or abnormal double-stranded RNA. Organisms employ a variety of strategies to defend themselves by distinguishing self and nonself substances and disabling these invasive nucleic acids. Furthermore, they have developed ways to remember this exposure to invaders and transmit the experience to their descendants. The mechanism underlying this inheritance has remained elusive. Recent research has shed light on the initiation and maintenance of RNA-mediated inherited gene silencing. Small regulatory RNAs play a variety of crucial roles in organisms, including gene regulation, developmental timing, antiviral defense, and genome integrity, via a process termed as RNA interference (RNAi). Recent research has revealed that small RNAs and the RNAi machinery are engaged in establishing and promoting transgenerational gene silencing. Small RNAs direct the RNAi and chromatin modification machinery to the cognate nucleic acids to regulate gene expression and epigenetic alterations. Notably, these acquired small RNAs and epigenetic changes persist and are transmitted from parents to offspring for multiple generations. Thus, RNAi is a vital determinant of the inheritance of gene silencing and acts as a driving force of evolution.     

Roles for Caenorhabditis elegans rad-51 in meiosis and in resistance to ionizing radiation during development

We have investigated the role of Caenorhabditis elegans RAD-51 during meiotic prophase and embryogenesis, making use of the silencing effect of RNA interference (RNAi). rad-51 RNAi leads to severe defects in chromosome morphology in diakinesis oocytes. We have explored the effect of rad-51 RNAi in mutants lacking fundamental components of the recombination machinery. If double-strand breaks are prevented by spo-11 mutation, rad-51 RNAi does not affect chromosome appearance. This is consistent with a role for RAD-51 downstream of the initiation of recombination. In the absence of MRE-11, as in the absence of SPO-11, RAD-51 depletion has no effect on the chromosomes, which appear intact, thus indicating a role for MRE-11 in DSB induction. Intriguingly, rad-51 silencing in oocytes that lack MSH-5 leads to chromosome fragmentation, a novel trait that is distinct from that seen in msh-5 mutants and in rad-51 RNAi oocytes, suggesting new potential roles for the msh-5 gene. Silencing of the rad-51 gene also causes a reduction in fecundity, which is suppressed by mutation in the DNA damage checkpoint gene rad-5, but not in the cell death effector gene ced-3. Finally, RAD-51 depletion is also seen to affect the soma, resulting in hypersensitivity to ionizing radiation in late embryogenesis.