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XB-ART-57216
PLoS One January 1, 2020; 15 (7): e0235433.

Defective heart chamber growth and myofibrillogenesis after knockout of adprhl1 gene function by targeted disruption of the ancestral catalytic active site.

Smith SJ , Towers N , Demetriou K , Mohun TJ .


Abstract
ADP-ribosylhydrolase-like 1 (Adprhl1) is a pseudoenzyme expressed in the developing heart myocardium of all vertebrates. In the amphibian Xenopus laevis, knockdown of the two cardiac Adprhl1 protein species (40 and 23 kDa) causes failure of chamber outgrowth but this has only been demonstrated using antisense morpholinos that interfere with RNA-splicing. Transgenic production of 40 kDa Adprhl1 provides only part rescue of these defects. CRISPR/Cas9 technology now enables targeted mutation of the adprhl1 gene in G0-generation embryos with routine cleavage of all alleles. Testing multiple gRNAs distributed across the locus reveals exonic locations that encode critical amino acids for Adprhl1 function. The gRNA recording the highest frequency of a specific ventricle outgrowth phenotype directs Cas9 cleavage of an exon 6 sequence, where microhomology mediated end-joining biases subsequent DNA repairs towards three small in-frame deletions. Mutant alleles encode discrete loss of 1, 3 or 4 amino acids from a di-arginine (Arg271-Arg272) containing peptide loop at the centre of the ancestral ADP-ribosylhydrolase site. Thus despite lacking catalytic activity, it is the modified (adenosine-ribose) substrate binding cleft of Adprhl1 that fulfils an essential role during heart formation. Mutation results in striking loss of myofibril assembly in ventricle cardiomyocytes. The defects suggest Adprhl1 participation from the earliest stage of cardiac myofibrillogenesis and are consistent with previous MO results and Adprhl1 protein localization to actin filament Z-disc boundaries. A single nucleotide change to the gRNA sequence renders it inactive. Mice lacking Adprhl1 exons 3-4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion.

PubMed ID: 32726316
PMC ID: PMC7390403
Article link: PLoS One
Grant support: [+]
Genes referenced: actc1 adprh adprhl1 inpp5k myl7 npm1 tcp1 tyr
GO keywords: heart morphogenesis [+]
Antibodies: Adprhl1 Ab1
Morpholinos: adprhl1 MO1 adprhl1 MO10 adprhl1 MO11 adprhl1 MO12 adprhl1 MO13 adprhl1 MO2 adprhl1 MO3 adprhl1 MO4 adprhl1 MO5 adprhl1 MO6 adprhl1 MO7 adprhl1 MO8 adprhl1 MO9

Disease Ontology terms: congenital heart disease
Phenotypes: Xla Wt + adprhl1 MO (Fig. 2.C,D) [+]

Article Images: [+] show captions
References [+] :
Afgan, The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. 2019, Pubmed


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