The α-arrestin SUP-13/ARRD-15 promotes isoform turnover of actin-interacting protein 1 in Caenorhabditis elegans striated muscle.

Precise arrangement of actin, myosin, and other regulatory components in a sarcomeric pattern is critical for producing contractile forces in striated muscles. Actin-interacting protein 1 (AIP1), also known as WD-repeat protein 1 (WDR1), is one of essential factors that regulate sarcomeric assembly of actin filaments. In the nematode Caenorhabditis elegans, mutation in unc-78, encoding one of the two AIP1 isoforms, causes severe disorganization of sarcomeric actin filaments and near paralysis, but mutation in sup-13 suppresses the unc-78-mutant phenotypes to restore nearly normal sarcomeric actin organization and worm motility. Here, we identified that sup-13 is a nonsense allele of arrd-15 encoding an α-arrestin. The sup-13/arrd-15 mutation suppressed the phenotypes of unc-78 null mutant but required aipl-1 that encodes a second AIP1 isoform. aipl-1 was normally expressed highly in embryos and downregulated in mature muscle. However, in the sup-13/arrd-15 mutant, the AIPL-1 protein was maintained at high levels in adult muscle to compensate for the absence of the UNC-78 protein. The sup-13/arrd-15 mutation caused accumulation of ubiquitinated AIPL-1 protein, suggesting that a normal function of sup-13/arrd-15 is to enhance degradation of ubiquitinated AIPL-1, thereby promoting transition of AIP1 isoforms from AIPL-1 to UNC-78 in developing muscle. These results suggest that α-arrestin is a novel factor to promote isoform turnover by enhancing protein degradation.

Kettin, the large actin-binding protein with multiple immunoglobulin domains, is essential for sarcomeric actin assembly and larval development in Caenorhabditis elegans.

Among many essential genes in the nematode Caenorhabditis elegans, let-330 is located on the left arm of chromosome V and was identified as the largest target of a mutagen in this region. However, let-330 gene has not been characterized at the molecular level. Here, we report that two sequenced let-330 alleles are nonsense mutations of ketn-1, a previously characterized gene encoding kettin. Kettin is a large actin-binding protein of 472 kDa with 31 immunoglobulin domains and is expressed in muscle cells in C. elegans. let-330/ketn-1 mutants are homozygous lethal at the first larval stage with mild defects in body elongation. These mutants have severe defects in sarcomeric actin and myosin assembly in striated muscle. However, α-actinin and vinculin, which are components of the dense bodies anchoring actin to the membranes, were not significantly disorganized by let-330/ketn-1 mutation. Kettin localizes to embryonic myofibrils before α-actinin is expressed, and α-actinin deficiency does not affect kettin localization in larval muscle. Depletion of vinculin minimally affects kettin localization but significantly reduces colocalization of actin with kettin in embryonic muscle cells. These results indicate that kettin is an essential protein for sarcomeric assembly of actin filaments in muscle cells.

let-65 is cytoplasmic methionyl tRNA synthetase in C. elegans.

Cytoplasmic methionyl tRNA synthetase (MetRS) is one of more than 20 cytoplasmic aminoacyl tRNA synthetase enzymes (ARS). This family of enzymes catalyzes a process fundamental for protein translation. Using a combination of genetic mapping, oligonucleotide array comparative genomic hybridization, and phenotypic correlation, we show that mutations in the essential gene, let-65, reside within the predicted Caenorhabditis elegans homologue of MetRS, which we have named mars-1. We demonstrate that the lethality associated with alleles of let-65 is fully rescued by a transgenic array that spans the mars-1 genomic region. Furthermore, sequence analysis reveals that six let-65 alleles lead to the alteration of highly conserved amino acids.

Allelic ratios and the mutational landscape reveal biologically significant heterozygous SNVs.

The issue of heterozygosity continues to be a challenge in the analysis of genome sequences. In this article, we describe the use of allele ratios to distinguish biologically significant single-nucleotide variants from background noise. An application of this approach is the identification of lethal mutations in Caenorhabditis elegans essential genes, which must be maintained by the presence of a wild-type allele on a balancer. The h448 allele of let-504 is rescued by the duplication balancer sDp2. We readily identified the extent of the duplication when the percentage of read support for the lesion was between 70 and 80%. Examination of the EMS-induced changes throughout the genome revealed that these mutations exist in contiguous blocks. During early embryonic division in self-fertilizing C. elegans, alkylated guanines pair with thymines. As a result, EMS-induced changes become fixed as either G→A or C→T changes along the length of the chromosome. Thus, examination of the distribution of EMS-induced changes revealed the mutational and recombinational history of the chromosome, even generations later. We identified the mutational change responsible for the h448 mutation and sequenced PCR products for an additional four alleles, correlating let-504 with the DNA-coding region for an ortholog of a NFκB-activating protein, NKAP. Our results confirm that whole-genome sequencing is an efficient and inexpensive way of identifying nucleotide alterations responsible for lethal phenotypes and can be applied on a large scale to identify the molecular basis of essential genes.

Characterization of the octamer, a cis-regulatory element that modulates excretory cell gene-expression in Caenorhabditis elegans.

BACKGROUND: We have previously demonstrated that the POU transcription factor CEH-6 is required for driving aqp-8 expression in the C. elegans excretory (canal) cell, an osmotic regulatory organ that is functionally analogous to the kidney. This transcriptional regulation occurs through a CEH-6 binding to a cis-regulatory element called the octamer (ATTTGCAT), which is located in the aqp-8 promoter. RESULTS: Here, we further characterize octamer driven transcription in C. elegans. First, we analyzed the positional requirements of the octamer. To do so, we assayed the effects on excretory cell expression by placing the octamer within the well-characterized promoter of vit-2. Second, using phylogenetic footprinting between three Caenorhabditis species, we identified a set of 165 genes that contain conserved upstream octamers in their promoters. Third, we used promoter::GFP fusions to examine the expression patterns of 107 of the 165 genes. This analysis demonstrated that conservation of octamers in promoters increases the likelihood that the gene is expressed in the excretory cell. Furthermore, we found that the sequences flanking the octamers may have functional importance. Finally, we altered the octamer using site-directed mutagenesis. Thus, we demonstrated that some nucleotide substitutions within the octamer do not affect the expression pattern of nearby genes, but change their overall expression was changed. Therefore, we have expanded the core octamer to include flanking regions and variants of the motif. CONCLUSIONS: Taken together, we have demonstrated that octamer-containing regions are associated with excretory cell expression of several genes that have putative roles in osmoregulation. Moreover, our analysis of the octamer sequence and its sequence variants could aid in the identification of additional genes that are expressed in the excretory cell and that may also be regulated by CEH-6.

Polymorphic segmental duplication in the nematode Caenorhabditis elegans.

BACKGROUND: The nematode Caenorhabditis elegans was the first multicellular organism to have its genome fully sequenced. Over the last 10 years since the original publication in 1998, the C. elegans genome has been scrutinized and the last gaps were filled in November 2002, which present a unique opportunity for examining genome-wide segmental duplications. RESULTS: Here, we performed analysis of the C. elegans genome in search for segmental duplications using a new tool -- OrthoCluster -- we have recently developed. We detected 3,484 duplicated segments -- duplicons -- ranging in size from 234 bp to 108 Kb. The largest pair of duplicons, 108 kb in length located on the left arm of Chromosome V, was further characterized. They are nearly identical at the DNA level (99.7% identity) and each duplicon contains 26 putative protein coding genes. Genotyping of 76 wild-type strains obtained from different labs in the C. elegans community revealed that not all strains contain this duplication. In fact, only 29 strains carry this large segmental duplication, suggesting a very recent duplication event in the C. elegans genome. CONCLUSION: This report represents the first demonstration that the C. elegans laboratory wild-type N2 strains has acquired large-scale differences.

High-resolution array comparative genomic hybridization analysis reveals unanticipated complexity of genetic deficiencies on chromosome V in Caenorhabditis elegans.

Genomic rearrangements are widely used in Caenorhabditis elegans research but many remain incompletely characterized at the physical level. We have used oligo-array comparative genomic analysis to assess the physical structure of 20 deficiencies and a single duplication of chromosome V. We find that while deletions internal to the chromosome appear simple in structure, terminal deletions are complex, containing duplications in addition to the deletion. Additionally, we confirm that transposon-induced deficiencies contain breakpoints that initiate at Tc1 elements. Finally, 13 of these deficiencies are known to suppress recombination far beyond the extent of the deletion. These deficiencies fall into two classes: strong and weak suppressors of adjacent recombination. Analysis of the deleted regions in these deficiencies reveals no common physical sites to explain the observed differences in recombination suppression. However, we find a strong correlation between the size of the rearranged chromosome and the severity of recombination suppression. Rearranged chromosomes that have a minor effect on recombination fall within 2% of normal chromosome size. Our observations highlight the use of array-based approaches for the analysis of rearranged genomes, revealing previously unidentified deficiency characteristics and addressing biologically relevant questions.

Transcriptional regulation of AQP-8, a Caenorhabditis elegans aquaporin exclusively expressed in the excretory system, by the POU homeobox transcription factor CEH-6.

Due to the ever changing environmental conditions in soil, regulation of osmotic homeostasis in the soil-dwelling nematode Caenorhabditis elegans is critical. AQP-8 is a C. elegans aquaporin that is expressed in the excretory cell, a renal equivalent tissue, where the protein participates in maintaining water balance. To better understand the regulation of AQP-8, we undertook a promoter analysis to identify the aqp-8 cis-regulatory elements. Using progressive 5' deletions of upstream sequence, we have mapped an essential regulatory region to roughly 300 bp upstream of the translational start site of aqp-8. Analysis of this region revealed a sequence corresponding to a known DNA functional element (octamer motif), which interacts with POU homeobox transcription factors. Phylogenetic footprinting showed that this site is perfectly conserved in four nematode species. The octamer site's function was further confirmed by deletion analyses, mutagenesis, functional studies, and electrophoretic mobility shift assays. Of the three POU homeobox proteins encoded in the C. elegans genome, CEH-6 is the only member that is expressed in the excretory cell. We show that expression of AQP-8 is regulated by CEH-6 by performing RNA interference experiments. CEH-6's mammalian ortholog, Brn1, is expressed both in the kidney and the central nervous system and binds to the same octamer consensus binding site to drive gene expression. These parallels in transcriptional control between Brn1 and CEH-6 suggest that C. elegans may well be an appropriate model for determining gene-regulatory networks in the developing vertebrate kidney.

Caenorhabditis elegans dpy-14: an essential collagen gene with unique expression profile and physiological roles in early development.

We describe the molecular characterisation of Caenorhabditis elegans dpy-14, a gene encoding an essential cuticular collagen annotated as col-59. Expression of dpy-14 starts at the 16 E cell stage, making it the earliest-expressing collagen reported to date. SAGE data and dpy-14 promoter::GFP reporter constructs indicate that the gene is transcribed mainly during embryogenesis, specifically in ciliated neurons and hypoderm. Water permeability assays and lectin staining showed that a mutation in the DPY-14 collagen results in defects in the channels of the amphids, which are a class of ciliated neuron, while the amphids appear morphologically normal by dye filling methods. Behavioural assays showed that the ciliated neurons expressing the gene are functional in dpy-14 mutants. All together, our data suggest that ciliated neurons and their hypodermal support cells collaborate in the transcription and synthesis of DPY-14, which then becomes a component of the amphid channels but not of the amphids proper. Interestingly, seam cells of dpy-14 mutants do not properly fuse to form a syncytium. This novel phenotype due to collagen mutations further stresses that dpy-14 plays a fundamental role in C. elegans physiology, since it is required for the proper development of the hypoderm.

Distinct regulatory elements mediate similar expression patterns in the excretory cell of Caenorhabditis elegans.

Identification of cis-regulatory elements and their binding proteins constitutes an important part of understanding gene function and regulation. It is well accepted that co-expressed genes tend to share transcriptional elements. However, recent findings indicate that co-expression data show poor correlation with co-regulation data even in unicellular yeast. This motivates us to experimentally explore whether it is possible that co-expressed genes are subject to differential regulatory control using the excretory cell of Caenorhabditis elegans as an example. Excretory cell is a functional equivalent of human kidney. Transcriptional regulation of gene expression in the cell is largely unknown. We isolated a 10-bp excretory cell-specific cis-element, Ex-1, from a pgp-12 promoter. The significance of the element has been demonstrated by its capacity of converting an intestine-specific promoter into an excretory cell-specific one. We also isolated a cDNA encoding an Ex-1 binding transcription factor, DCP-66, using a yeast one-hybrid screen. Role of the factor in regulation of pgp-12 expression has been demonstrated both in vitro and in vivo. Search for occurrence of Ex-1 reveals that only a small portion of excretory cell-specific promoters contain Ex-1. Two other distinct cis-elements isolated from two different promoters can also dictate the excretory cell-specific expression but are independent of regulation by DCP-66. The results indicate that distinct regulatory elements are able to mediate the similar expression patterns.

Autosomal genes of autosomal/X-linked duplicated gene pairs and germ-line proliferation in Caenorhabditis elegans.

We report molecular genetic studies of three genes involved in early germ-line proliferation in Caenorhabditis elegans that lend unexpected insight into a germ-line/soma functional separation of autosomal/X-linked duplicated gene pairs. In a genetic screen for germ-line proliferation-defective mutants, we identified mutations in rpl-11.1 (L11 protein of the large ribosomal subunit), pab-1 [a poly(A)-binding protein], and glp-3/eft-3 (an elongation factor 1-alpha homolog). All three are members of autosome/X gene pairs. Consistent with a germ-line-restricted function of rpl-11.1 and pab-1, mutations in these genes extend life span and cause gigantism. We further examined the RNAi phenotypes of the three sets of rpl genes (rpl-11, rpl-24, and rpl-25) and found that for the two rpl genes with autosomal/X-linked pairs (rpl-11 and rpl-25), zygotic germ-line function is carried by the autosomal copy. Available RNAi results for highly conserved autosomal/X-linked gene pairs suggest that other duplicated genes may follow a similar trend. The three rpl and the pab-1/2 duplications predate the divergence between C. elegans and C. briggsae, while the eft-3/4 duplication appears to have occurred in the lineage to C. elegans after it diverged from C. briggsae. The duplicated C. briggsae orthologs of the three C. elegans autosomal/X-linked gene pairs also display functional differences between paralogs. We present hypotheses for evolutionary mechanisms that may underlie germ-line/soma subfunctionalization of duplicated genes, taking into account the role of X chromosome silencing in the germ line and analogous mammalian phenomena.

Identification of a nematode chemosensory gene family.

Taking advantage of the recent availability of the whole genome sequence of Caenorhabditis briggsae, a closely related nematode to Caenorhabditis elegans, we have examined the chemosensory gene superfamily by using comparative genomic methods. We have identified a chemosensory gene family, serpentine receptor class ab (srab), which exists in both species with 25 members in C. elegans and 14 members in C. briggsae. More than 20% of these gene models are reannotated. The srab family is similar to, but distinct from, the previously described serpentine receptor class a (sra) family and shows a differential expansion in C. elegans similar to that previously described for sra. The cellular expression patterns for multiple members of the srab family in both phasmid neurons in the tail and amphid neurons in the head supports the conclusion that they are chemosensory genes and suggests that they may play a role in integrating chemosensory inputs from both ends of the organism. The expansion of both the srab and sra gene families in C. elegans relative to C. briggsae is due to multiple rounds of tandem duplication and translocation of individual genes.

The RNA-binding protein SUP-12 controls muscle-specific splicing of the ADF/cofilin pre-mRNA in C. elegans.

Tissue-specific alternative pre-mRNA splicing is essential for increasing diversity of functionally different gene products. In Caenorhabditis elegans, UNC-60A and UNC-60B, nonmuscle and muscle isoforms of actin depolymerizing factor (ADF)/cofilin, are expressed by alternative splicing of unc-60 and regulate distinct actin-dependent developmental processes. We report that SUP-12, a member of a new family of RNA recognition motif (RRM) proteins, including SEB-4, regulates muscle-specific splicing of unc-60. In sup-12 mutants, expression of UNC-60B is decreased, whereas UNC-60A is up-regulated in muscle. sup-12 mutations strongly suppress muscle defects in unc-60B mutants by allowing expression of UNC-60A in muscle that can substitute for UNC-60B, thus unmasking their functional redundancy. SUP-12 is expressed in muscle and localized to the nuclei in a speckled pattern. The RRM domain of SUP-12 binds to several sites of the unc-60 pre-mRNA including the UG repeats near the 3'-splice site in the first intron. Our results suggest that SUP-12 is a novel tissue-specific splicing factor and regulates functional redundancy among ADF/cofilin isoforms.

Loss of C. elegans BBS-7 and BBS-8 protein function results in cilia defects and compromised intraflagellar transport.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous developmental disorder whose molecular basis is largely unknown. Here, we show that mutations in the Caenorhabditis elegans bbs-7 and bbs-8 genes cause structural and functional defects in cilia. C. elegans BBS proteins localize predominantly at the base of cilia, and like proteins involved in intraflagellar transport (IFT), a process necessary for cilia biogenesis and maintenance, move bidirectionally along the ciliary axoneme. Importantly, we demonstrate that BBS-7 and BBS-8 are required for the normal localization/motility of the IFT proteins OSM-5/Polaris and CHE-11, and to a notably lesser extent, CHE-2. We propose that BBS proteins play important, selective roles in the assembly and/or function of IFT particle components. Our findings also suggest that some of the cardinal and secondary symptoms of BBS, such as obesity, diabetes, cardiomyopathy, and learning defects may result from cilia dysfunction.

Basal body dysfunction is a likely cause of pleiotropic Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder characterized primarily by retinal dystrophy, obesity, polydactyly, renal malformations and learning disabilities. Although five BBS genes have been cloned, the molecular basis of this syndrome remains elusive. Here we show that BBS is probably caused by a defect at the basal body of ciliated cells. We have cloned a new BBS gene, BBS8, which encodes a protein with a prokaryotic domain, pilF, involved in pilus formation and twitching mobility. In one family, a homozygous null BBS8 mutation leads to BBS with randomization of left-right body axis symmetry, a known defect of the nodal cilium. We have also found that BBS8 localizes specifically to ciliated structures, such as the connecting cilium of the retina and columnar epithelial cells in the lung. In cells, BBS8 localizes to centrosomes and basal bodies and interacts with PCM1, a protein probably involved in ciliogenesis. Finally, we demonstrate that all available Caenorhabditis elegans BBS homologues are expressed exclusively in ciliated neurons, and contain regulatory elements for RFX, a transcription factor that modulates the expression of genes associated with ciliogenesis and intraflagellar transport.