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Biochem Biophys Res Commun
2010 Mar 26;3941:18-23. doi: 10.1016/j.bbrc.2010.02.041.
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Identification of protein domains required for makorin-2-mediated neurogenesis inhibition in Xenopus embryos.
Cheung WK
,
Yang PH
,
Huang QH
,
Chen Z
,
Chen SJ
,
Lin MC
,
Kung HF
.
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Makorin-2, consisting of four highly conserved C(3)H zinc fingers, a Cys-His motif and a C(3)HC(4) RING zinc finger domain, is a putative ribonucleoprotein. We have previously reported that Xenopus makorin-2 (mkrn2) is a neurogenesis inhibitor acting upstream of glycogen synthase kinase-3beta (GSK-3beta) in the phosphatidylinositol 3-kinase/Akt pathway. In an effort to identify the functional domains required for its anti-neurogenic activity, we designed and constructed a series of N- and C-terminal truncation mutants of mkrn2. Concurred with the full-length mkrn2, we showed that overexpression of one of the truncation mutants mkrn2(s)-7, which consists of only the third C(3)H zinc finger, Cys-His motif and C(3)HC(4) RING zinc finger, is essential and sufficient to produce the phenotypical dorso-posterior deficiencies and small-head/short-tail phenotype in tadpoles. In animal cap explant assay, we further demonstrated that mkrn2(s)-7 not only inhibits activin and retinoic acid-induced animal cap neuralization and the expression of a pan-neural marker neural cell adhesion molecule, but also induces GSK-3beta expression. These results collectively suggest that the third C(3)H zinc finger, Cys-His motif and C(3)HC(4) RING zinc finger are indispensable for the anti-neurogenic activity of mkrn2.
Fig. 1.
Abnormal phenotypes induced by microinjection of mkrn2 truncation mutants and MOa in Xenopus embryos. (A) Schematic illustration of the protein domains harboured by mkrn2 truncation mutants. (B) Summary of normal, defective, and dead phenotypes in different injection groups. Values were the combined data of three independent experiments with each experiment consisting of 20 embryos. Bracketed figures indicated the number of embryos with specified phenotype.
Fig. 2.
mkrn2(s)-3 affects neural development in Xenopus embryos and suppresses NCAM expression in animal cap. Embryos at two-cell stage were injected into the animal pole areas with mRNAs encoding β-gal (control), mkrn2(s), C-terminal truncation mutants and MOa. (A) Embryos injected with mkrn2(s) and mkrn2(s)-3 showed dorso-posterior deficiencies with small head and short-tail phenotype at stage 33. (B) Dissected ACs were cultured in the absence of activin and retinoic acid to stage 22 equivalent for RT-PCR analysis of NCAM expression. NCAM expression in whole embryos (WE) was used as positive control. Reaction without the addition of reverse transcriptase (No RT) served as a negative control to confirm the absence of contaminating genomic DNA. Elongation factor 1α (EF-1α) expression was used as a loading control. (C) and (D) Dissected ACs were cultured in the presence of activin and retinoic acid until the equivalent of stage 22 for photography or RT-PCR analysis.
Fig. 3.
mkrn2(s)-7 inhibits neurogenesis, animal cap neuralization and NCAM expression. Embryos were injected with mRNA encoding β-gal (control), mkrn2(s), N-terminal truncation mutants and MOa. (A) Morphological observations of tadpoles at stage 33. (B) NCAM expression in ACs cultured in the absence of activin and retinoic acid. (C) and (D) Neural tissue induction and NCAM expression in ACs cultured in the presence of activin and retinoic acid. WE, whole embryo; No RT, reaction without the addition of reverse transcriptase.
Fig. 4.
mkrn2(s)-7 induces GSK-3β mRNA expression. WE, whole embryo; No RT, reaction without the addition of reverse transcriptase.
Fig. s1. Amino acid sequence alignment of ten makorin-2 orthologs. Makorin-2 from frogs (Xenopus laevis, Xenopus tropicalis), mammals (Homo sapiens, Bos taurus, Mus musculus, Rattus norvegicus), chicken (Gallus gallus), and fishes (Danio rerio, Seriola quinqueradiata, Takifugu rubripes) were aligned by T-coffee [17]. Residues of ⩾70% identity or similarity were reverse shaded. Critical amino acids in the conserved zinc finger motifs and percentage sequence identity with respect to Home sapiens were indicated.
Fig. s2. Phylogenetic relationship of makorin-2 orthologs. The phylogenetic tree was constructed using Neighbor-Joining algorithm implemented in MEGA version 3.1 [18]. The confidence of topology was assessed by bootstrap resampling analysis with 10,000 replicates. Bootstrap value of each branch was indicated.
