The multienzyme complex prolyl 4-hydroxylase catalyzes the hydroxylation of proline residues and acts as a chaperone during collagen synthesis in multicellular organisms. The ␤ subunit of this complex is identical to protein disulfide isomerase (PDI). The free-living nematode Caenorhabditis elegans is encased in a collagenous exoskeleton and represents an excellent model for the study of collagen biosynthesis and extracellular matrix formation. In this study, we examined prolyl 4-hydroxylase ␣-subunit (PHY; EC 1.14.11.2)-and ␤-subunit (PDI; EC 5.3.4.1)-encoding genes with respect to their role in collagen modification and formation of the C. elegans exoskeleton. We identified genes encoding two PHYs and a single associated PDI and showed that all three are expressed in collagen-synthesizing ectodermal cells at times of maximal collagen synthesis. Disruption of the pdi gene via RNA interference resulted in embryonic lethality. Similarly, the combined phy genes are required for embryonic development. Interference with phy-1 resulted in a morphologically dumpy phenotype, which we determined to be identical to the uncharacterized dpy-18 locus. Two dpy-18 mutant strains were shown to have null alleles for phy-1 and to have a reduced hydroxyproline content in their exoskeleton collagens. This study demonstrates in vivo that this enzyme complex plays a central role in extracellular matrix formation and is essential for normal metazoan development.The Caenorhabditis elegans exoskeleton (or cuticle) is a true extracellular matrix (ECM) (21) that is predominantly composed of highly cross-linked collagens (8). This exoskeleton, which is synthesized from the ectoderm (hypodermis) five times during nematode development, is responsible for the maintenance of postembryonic body shape, protection from the environment, and locomotion via opposed muscles (21). Approximately 1% of the C. elegans genome encodes cuticle collagens, representing over 150 small collagen genes (7), which encode short interrupted collagens most like vertebrate FACIT type IX cartilage collagens (30). This complex mixture of collagens constitutes Ͼ80% of the proteins in this resilient matrix (17). Mutations in individual cuticle collagen genes can result in dramatic morphological defects in this nematode, as seen for the dumpy, blister, squat, and roller mutations (21).Prolyl 4-hydroxylase (EC 1.14.11.2) is an endoplasmic reticulum (ER) enzyme responsible for the co-and posttranslational hydroxylation of proline in the Xaa-Pro-Gly repeats of procollagen. In vitro studies have demonstrated that 4-hydroxyproline is required for the thermal stability of the folded triple helix at physiological temperatures (19). An additional function of this enzyme is to act as a chaperone by retaining unfolded procollagen chains in the ER, releasing them for secretion only when they have folded correctly (37). In humans and other vertebrates, two isoforms of prolyl 4-hydroxylase exist (type I and type II) (19); these associate with a single ␤ subunit to form a catalytically...

BackgroundMicroRNAs (miRNAs) play key roles in regulating post-transcriptional gene expression and are essential for development in the free-living nematode Caenorhabditis elegans and in higher organisms. Whether microRNAs are involved in regulating developmental programs of parasitic nematodes is currently unknown. Here we describe the the miRNA repertoire of two important parasitic nematodes as an essential first step in addressing this question.ResultsThe small RNAs from larval and adult stages of two parasitic species, Brugia pahangi and Haemonchus contortus, were identified using deep-sequencing and bioinformatic approaches. Comparative analysis to known miRNA sequences reveals that the majority of these miRNAs are novel. Some novel miRNAs are abundantly expressed and display developmental regulation, suggesting important functional roles. Despite the lack of conservation in the miRNA repertoire, genomic positioning of certain miRNAs within or close to specific coding genes is remarkably conserved across diverse species, indicating selection for these associations. Endogenous small-interfering RNAs and Piwi-interacting (pi)RNAs, which regulate gene and transposon expression, were also identified. piRNAs are expressed in adult stage H. contortus, supporting a conserved role in germline maintenance in some parasitic nematodes.ConclusionsThis in-depth comparative analysis of nematode miRNAs reveals the high level of divergence across species and identifies novel sequences potentially involved in development. Expression of novel miRNAs may reflect adaptations to different environments and lifestyles. Our findings provide a detailed foundation for further study of the evolution and function of miRNAs within nematodes and for identifying potential targets for intervention.

The integral role that collagens play in the morphogenesis of the nematode exoskeleton or cuticle makes them a useful marker in the examination of the collagen synthesizing machinery. In this study, a green fluorescent protein-collagen fusion has been constructed by using the Caenorhabditis elegans adult-specific, hypodermally synthesized collagen COL-19. In wild-type nematodes, this collagen marker localized to the circumferential annular rings and the lateral trilaminar alae of the cuticle. Crosses carried out between a COL-19::GFP integrated strain and several morphologically mutant strains, including blister, dumpy, long, small, squat, and roller revealed significant COL-19 disruption that was predominantly strain-specific and provided a structural basis for the associated phenotypes. Disruption was most notable in the cuticle overlying the lateral seam cell syncytium, and confirmed the presence of two distinct forms of hypodermis, namely the circumferentially contracting lateral seam cells and the laterally contracting ventral-dorsal hypodermis. The effect of a single aberrant collagen being sufficient to mediate widespread collagen disruption was exemplified by the collagen mutant strain dpy-5 and its disrupted COL-19::GFP and DPY-7 collagen expression patterns. Through the disrupted pattern of COL-19 and DPY-7 in a thioredoxin mutant, dpy-11, and through RNA interference of a dual oxidase enzyme and a vesicular transport protein, we also show the efficacy of the COL-19::GFP strain as a marker for aberrant cuticle collagen synthesis and, thus, for the identification of factors involved in the construction of collagenous extracellular matrices. Developmental Dynamics 226:523-539, 2003.

The collagen prolyl 4-hydroxylases (P4Hs, EC 1.14.11.2) play a critical role in the synthesis of the extracellular matrix. The enzymes characterized from vertebrates and Drosophila are ␣ 2 ␤ 2 tetramers, in which protein disulfide isomerase (PDI) serves as the ␤ subunit. Two conserved ␣ subunit isoforms, PHY-1 and PHY-2, have been identified in Caenorhabditis elegans. We report here that three unique P4H forms are assembled from these polypeptides and the single ␤ subunit PDI-2, both in a recombinant expression system and in vivo, namely a PHY-1/PHY-2/(PDI-2) 2 mixed tetramer and PHY-1/PDI-2 and PHY-2/PDI-2 dimers. The mixed tetramer is the main P4H form in wild-type C. elegans but phy-2 ؊/؊ and phy-1 ؊/؊ (dpy-18) mutant nematodes can compensate for its absence by increasing the assembly of the PHY-1/PDI-2 and PHY-2/PDI-2 dimers, respectively. All three of the mixed tetramer-forming polypeptides PHY-1, PHY-2, and PDI-2 are coexpressed in the cuticle collagen-synthesizing hypodermal cells. The catalytic properties of the mixed tetramer are similar to those of other P4Hs, and analogues of 2-oxoglutarate were found to produce severe temperature-dependent effects on P4H mutant strains. Formation of the novel mixed tetramer was species-specific, and studies with hybrid recombinant PHY polypeptides showed that residues Gln 121 -Ala 271 and Asp 1 -Leu 122 in PHY-1 and PHY-2, respectively, are critical for its assembly.The free-living nematode Caenorhabditis elegans represents an excellent model system for studying the extracellular matrix (ECM) 1 and the enzymes involved in its biosynthesis and modification (1-3). The major ECM formed in C. elegans is the collagenous cuticle or exoskeleton, a protective structure that is synthesized repeatedly during development. Over 150 small collagen genes are involved in the formation of this structure (2), producing stage-specific cuticles that are both structurally and chemically distinct (3).Collagen prolyl 4-hydroxylases (P4Hs) are enzymes resident in the endoplasmic reticulum (ER) (4, 5) that play a critical role in the synthesis and processing of all collagens (6). Recently, an additional family of cytoplasmic P4Hs has been shown to play an essential role in O 2 sensing and the hypoxia response (7,8).The collagen P4Hs present in vertebrates (4, 5) and in Drosophila (9) are ␣ 2 ␤ 2 tetramers in which the multifunctional chaperone protein disulfide isomerase (PDI) serves as the ␤ subunit. The ␣ subunit binds Fe 2ϩ , 2-oxoglutarate, and ascorbate (4, 5) and possesses the peptide substrate-binding site (10). The main role of PDI is to retain the ␣ subunits in a catalytically active, nonaggregated conformation within the ER (11-13). Two conserved P4H ␣ subunit isoforms have been described in human and mouse tissues, which form [␣(I)] 2 ␤ 2 or [␣(II)] 2 ␤ 2 tetramers (14 -16). Initial characterization of a recombinant C. elegans ␣ subunit isoform (PHY-1) revealed a unique association in that it formed an active ␣␤ dimer with both human PDI and its C. elegans orthologue PDI-2 but no...

The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as molting. A targeted RNA interference screen using a cuticle collagen marker has been employed to identify components of the cuticle biosynthetic pathway. We have characterized an essential peroxidase, MoLT-7 (MLT-7), that is responsible for proper cuticle molting and re-synthesis. MLT-7 is an active, inhibitable peroxidase that is expressed in the cuticle-synthesizing hypodermis coincident with each larval molt. mlt-7 mutants show a range of body morphology defects, most notably molt, dumpy, and early larval stage arrest phenotypes that can all be complemented with a wild type copy of mlt-7. The cuticles of these mutants lacks di-tyrosine cross-links, becomes permeable to dye and accessible to tyrosine iodination, and have aberrant collagen protein expression patterns. Overexpression of MLT-7 causes mutant phenotypes further supporting its proposed enzymatic role. In combination with BLI-3, an H 2 O 2 -generating NADPH dual oxidase, MLT-7 is essential for post-embryonic development. Disruption of mlt-7, and particularly bli-3, via RNA interference also causes dramatic changes to the in vivo cross-linking patterns of the cuticle collagens DPY-13 and COL-12. This points toward a functionally cooperative relationship for these two hypodermally expressed proteins that is essential for collagen cross-linking and proper extracellular matrix formation. Collagenous extracellular matrices (ECMs)3 serve numerous critical roles and are found throughout the animal kingdom. The cuticle is an ECM that makes up the most external surface of nematodes and its roles are diverse. This tough but flexible exoskeleton maintains body shape, provides a protective barrier to the environment, and permits motility via its attachments to muscles (1). There are two distinct sets of discernible cuticular structures on the surface of the nematode Caenorhabditis elegans, circumferential indentations termed annulae and longitudinal ridges termed alae. The latter are present only in the first and alternative third stage (dauer) larvae and in adult C. elegans (2). At the transition between consecutive larval stages, a new cuticle is synthesized from the underlying hypodermis. The term molting is used for the removal and re-synthesis of the cuticle. The cuticle is a complex and versatile tissue made up of multiple layers, each of which has distinct components and levels of structural integrity (3, 4). The principal components of the cuticle are the collagens. Over 180 cuticle collagens are encoded in the C. elegans genome, and their temporal expression is cyclical and corresponds to the larval molts (5). Each of these synthetic periods is further subdivided into distinct peaks as follows: 4 h prior to, 2 h prior to, and coincident with the molt. Collagens ...

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