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Exon 2 skipping eliminates ?-glutamyl carboxylase activity, indicating a partial splicing defect in a patient with vitamin K clotting factor deficiency.

ABSTRACT: Essentials A carboxylase mutation that impairs splicing to delete exon 2 sequences was previously reported. We found that the mutant was inactive for vitamin K-dependent (VKD) protein carboxylation. An incomplete splicing defect likely accounts for VKD clotting activity observed in the patient. The results indicate the importance of proper carboxylase embedment in the membrane for function. BACKGROUND:Mutations in the ?-glutamyl carboxylase (GGCX), which is required for vitamin K-dependent (VKD) protein activation, can result in vitamin K clotting factor deficiency (VKCFD1). A recent report described a VKCFD1 patient with a homozygous carboxylase mutation that altered splicing and deleted exon 2 (?2GGCX). Only ?2GGCX RNA was observed in the patient. OBJECTIVES:Loss of exon 2 results in the deletion of carboxylase sequences thought to be important for membrane topology and consequent function. Carboxylase activity is required for life, and we therefore tested whether the ?2GGCX mutant is active. METHODS:HEK 293 cells were edited by the use of CRISPR-Cas9 to eliminate endogenous carboxylase. Recombinant wild-type GGCX and recombinant ?2GGCX were then expressed and tested for carboxylation of the VKD protein factor IX. A second approach was used to monitor carboxylation biochemically, using recombinant carboxylases expressed in insect cells that lack endogenous carboxylase. RESULTS AND CONCLUSIONS:?2GGCX activity was undetectable in both assays, which is strikingly different from the low levels of carboxylase activity observed with other VKCFD1 mutants. The similarity in clotting function between patients with ?2GGCX and these mutations must therefore arise from a novel mechanism. Low levels of properly spliced carboxylase RNA that produce full-length protein would not have been observed in the previous study. The results suggest that the splicing defect is incomplete. ?2GGCX RNA has been detected in normal human liver, and has been designated carboxylase isoform 2; however, ?2GGCX protein was not observed in normal human liver. The lack of activity and protein expression suggest that isoform 2 is not physiologically relevant to normal VKD protein carboxylation.

SUBMITTER: Rishavy MA

PROVIDER: S-EPMC7181818 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Exon 2 skipping eliminates γ-glutamyl carboxylase activity, indicating a partial splicing defect in a patient with vitamin K clotting factor deficiency.

Rishavy Mark A MA Hallgren Kevin W KW Zhang Haitao H Runge Kurt W KW Berkner Kathleen L KL

Journal of thrombosis and haemostasis : JTH 20190607 7

Essentials A carboxylase mutation that impairs splicing to delete exon 2 sequences was previously reported. We found that the mutant was inactive for vitamin K-dependent (VKD) protein carboxylation. An incomplete splicing defect likely accounts for VKD clotting activity observed in the patient. The results indicate the importance of proper carboxylase embedment in the membrane for function.<h4>Background</h4>Mutations in the γ-glutamyl carboxylase (GGCX), which is required for vitamin K-dependen ...[more]

PMID: 31009158

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Project description:BackgroundCircular RNAs (circRNAs) play diverse roles in different biological and physiological environments and are always expressed in a tissue-specific manner. Especially, circRNAs are enriched in the brain tissues of almost all investigated species, including humans, mice, Drosophila, etc. Although circRNAs were found in C. elegans, the neuron-specific circRNA data is not available yet. Exon-skipping is found to be correlated to circRNA formation, but the mechanisms that link them together are not clear.ResultsHere, through large-scale neuron isolation from the first larval (L1) stage of C. elegans followed by RNA sequencing with ribosomal RNA depletion, the neuronal circRNA data in C. elegans were obtained. Hundreds of novel circRNAs were annotated with high accuracy. circRNAs were highly expressed in the neurons of C. elegans and were positively correlated to the levels of their cognate linear mRNAs. Disruption of reverse complementary match (RCM) sequences in circRNA flanking introns effectively abolished circRNA formation. In the zip-2 gene, deletion of either upstream or downstream RCMs almost eliminated the production of both the circular and the skipped transcript. Interestingly, the 13-nt RCM in zip-2 is highly conserved across five nematode ortholog genes, which show conserved exon-skipping patterns. Finally, through in vivo one-by-one mutagenesis of all the splicing sites and branch points required for exon-skipping and back-splicing in the zip-2 gene, I showed that back-splicing still happened without exon-skipping, and vice versa.ConclusionsThrough protocol optimization, total RNA obtained from sorted neurons is increased to hundreds of nanograms. circRNAs highly expressed in the neurons of C. elegans are more likely to be derived from genes also highly expressed in the neurons. RCMs are abundant in circRNA flanking introns, and RCM-deletion is an efficient way to knockout circRNAs. More importantly, these RCMs are not only required for back-splicing but also promote the skipping of exon(s) to be circularized. Finally, RCMs in circRNA flanking introns can directly promote both exon-skipping and back-splicing, providing a new explanation for the correlation between them.

Dataset Information

Exon 2 skipping eliminates ?-glutamyl carboxylase activity, indicating a partial splicing defect in a patient with vitamin K clotting factor deficiency.

Publications

Exon 2 skipping eliminates γ-glutamyl carboxylase activity, indicating a partial splicing defect in a patient with vitamin K clotting factor deficiency.

Similar Datasets

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