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The 5'-3' exoribonuclease xrn-1 is essential for ventral epithelial enclosure during C. elegans embryogenesis.

ABSTRACT: Ribonucleases have been studied in yeast and bacteria, but their biological significance to multicellular organisms is virtually unknown. However, there is increasing evidence that specific, timed transcript degradation is critical for regulation of many cellular processes, including early development and RNA interference. In this report we have investigated the effects of the 5'-3' exoribonuclease xrn-1 on the development of the nematode worm Caenorhabditis elegans. Silencing of xrn-1 expression using RNA interference results in embryos that fail to complete ventral enclosure, where the outer layer of cells normally closes over the mesoderm in a purse-string movement. Our data suggest that xrn-1 is involved in a critical aspect of epithelial movement and reveal an unexpected link between RNA stability and morphogenesis. Because xrn-1 is highly conserved in all eukaryotes, it is possible that it plays a role in similar morphological processes such as dorsal or thorax closure in Drosophila and wound healing in humans. In contrast to work in human tissue culture cells, where the 3'-5' pathway has been shown to be the most important for degradation of mRNAs, our work shows that the 5'-3' degradation pathway is crucially important at a critical stage of development in C. elegans. We have also investigated whether xrn-1 can influence the response of C. elegans to RNA interference. Our data indicate that xrn-1 plays a facilitating, but not crucial role in this process.

SUBMITTER: Newbury S

PROVIDER: S-EPMC1370518 | biostudies-literature | 2004 Jan

REPOSITORIES: biostudies-literature

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The 5'-3' exoribonuclease xrn-1 is essential for ventral epithelial enclosure during C. elegans embryogenesis.

Newbury Sarah S Woollard Alison A

RNA (New York, N.Y.) 20040101 1

Ribonucleases have been studied in yeast and bacteria, but their biological significance to multicellular organisms is virtually unknown. However, there is increasing evidence that specific, timed transcript degradation is critical for regulation of many cellular processes, including early development and RNA interference. In this report we have investigated the effects of the 5'-3' exoribonuclease xrn-1 on the development of the nematode worm Caenorhabditis elegans. Silencing of xrn-1 expressio ...[more]

PMID: 14681585

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Project description:Glycosylphosphatidylinositol (GPI) is a post-translational modification resulting in the attachment of modified proteins to the outer leaflet of the plasma membrane. Tissue culture experiments have shown GPI-anchored proteins (GPI-APs) to be targeted to the apical membrane of epithelial cells. However, the in vivo importance of this targeting has not been investigated since null mutations in GPI biosynthesis enzymes in mice result in very early embryonic lethality. Missense mutations in the human GPI biosynthesis enzyme pigv are associated with a multiple congenital malformation syndrome with a high frequency of Hirschsprung disease and renal anomalies. However, it is currently unknown how these phenotypes are linked to PIGV function. Here, we identify a temperature-sensitive hypomorphic allele of PIGV in Caenorhabditis elegans, pigv-1(qm34), enabling us to study the role of GPI-APs in development. At the restrictive temperature we found a 75% reduction in GPI-APs at the surface of embryonic cells. Consequently, ~80% of pigv-1(qm34) embryos arrested development during the elongation phase of morphogenesis, exhibiting internal cysts and/or surface ruptures. Closer examination of the defects revealed them all to be the result of breaches in epithelial tissues: cysts formed in the intestine and excretory canal, and ruptures occurred through epidermal cells, suggesting weakening of the epithelial membrane or membrane-cortex connection. Knockdown of piga-1, another GPI biosynthesis enzymes resulted in similar phenotypes. Importantly, fortifying the link between the apical membrane and actin cortex by overexpression of the ezrin/radixin/moesin ortholog ERM-1, significantly rescued cyst formation and ruptures in the pigv-1(qm34) mutant. In conclusion, we discovered GPI-APs play a critical role in maintaining the integrity of the epithelial tissues, allowing them to withstand the pressure and stresses of morphogenesis. Our findings may help to explain some of the phenotypes observed in human syndromes associated with pigv mutations.

Project description:Understanding how oxidatively damaged RNA is handled intracellularly is of relevance due to the link between oxidized RNA and the progression/development of some diseases as well as aging. Among the ribonucleases responsible for the decay of modified (chemically or naturally) RNA is the exonuclease Xrn-1, a processive enzyme that catalyzes the hydrolysis of 5'-phosphorylated RNA in a 5'→3' direction. We set out to explore the reactivity of this exonuclease towards oligonucleotides (ONs, 20-nt to 30-nt long) of RNA containing 8-oxo-7,8-dihydroguanosine (8-oxoG), obtained via solid-phase synthesis. The results show that Xrn-1 stalled at sites containing 8-oxoG, evidenced by the presence of a slower moving band (via electrophoretic analyses) than that observed for the canonical analogue. The observed fragment(s) were characterized via PAGE and MALDI-TOF to confirm that the oligonucleotide fragment(s) contained a 5'-phosphorylated 8-oxoG. Furthermore, the yields for this stalling varied from app. 5-30% with 8-oxoG located at different positions and in different sequences. To gain a better understanding of the decreased nuclease efficiency, we probed: 1) H-bonding and spatial constraints; 2) anti-syn conformational changes; 3) concentration of divalent cation; and 4) secondary structure. This was carried out by introducing methylated or brominated purines (m1G, m6,6A, or 8-BrG), probing varying [Mg2+], and using circular dichroism (CD) to explore the formation of structured RNA. It was determined that spatial constraints imposed by conformational changes around the glycosidic bond may be partially responsible for stalling, however, the results do not fully explain some of the observed higher stalling yields. We hypothesize that altered π-π stacking along with induced H-bonding interactions between 8-oxoG and residues within the binding site may also play a role in the decreased Xrn-1 efficiency. Overall, these observations suggest that other factors, yet to be discovered/established, are likely to contribute to the decay of oxidized RNA. In addition, Xrn-1 degraded RNA containing m1G, and stalled mildly at sites where it encountered m6,6A, or 8-BrG, which is of particular interest given that the former two are naturally occurring modifications.

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Dataset Information

The 5'-3' exoribonuclease xrn-1 is essential for ventral epithelial enclosure during C. elegans embryogenesis.

Publications

The 5'-3' exoribonuclease xrn-1 is essential for ventral epithelial enclosure during C. elegans embryogenesis.

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