Makorin-2 Is a Neurogenesis Inhibitor Downstream of Phosphatidylinositol 3-Kinase/Akt (PI3K/Akt) Signal

Abstract

Makorin-2 belongs to the makorin RING zinc finger gene family, which encodes putative ribonucleoproteins. Here we cloned the Xenopus makorin-2 (mkrn2) and characterized its function in Xenopus neurogenesis. Forced overexpression of mkrn2 produced tadpoles with dorso-posterior deficiencies and small-head/shorttail phenotype, whereas knockdown of mkrn2 by morpholino antisense oligonucleotides induced double axis in tadpoles. In Xenopus animal cap explant assay, mkrn2 inhibited activin, and retinoic acid induced animal cap neuralization, as evident from the suppression of a pan neural marker, neural cell adhesion molecule. Surprisingly, the anti-neurogenic activity of mkrn2 is independent of the two major neurogenesis signaling cascades, BMP-4 and Wnt8 pathways. Instead, mkrn2 works specifically through the phosphatidylinositol 3-kinase (PI3K) and Akt-mediated neurogenesis pathway. Overexpression of mkrn2 completely abrogated constitutively active PI3K-and Akt-induced, but not dominant negative glycogen synthase kinase-3␤ (GSK-3␤)-induced, neural cell adhesion molecule expression, indicating that mkrn2 acts downstream of PI3K and Akt and upstream of GSK-3␤. Moreover, mkrn2 upregulated the mRNA and protein levels of GSK-3␤. These results revealed for the first time the important role of mkrn2 as a new player in PI3K/Akt-mediated neurogenesis during Xenopus embryonic development. Makorin RING zinc finger (MKRN)3 gene family encodes putative ribonucleoproteins with a distinctive array of zinc finger domains. To date, nine MKRN family loci scattered throughout the human genome have been identified (1). The most studied member of this family is makorin-1 gene (MKRN1), which was recently documented to modulate telomere length homeostasis (2) and RNA polymerase II-dependent transcription (3). MKRN1 is widely transcribed in mammals, particularly in murine embryonic nervous system and adult testis (1). Such expression pattern suggests that members of this gene family may play an important role in embryonic development and neurogenesis.We had previously identified a new gene makorin-2 (MKRN2), formerly designated as HSPC070, from human CD34ϩ hematopoietic stem/progenitor cells (4). The nucleotide sequence of this gene shares 105 bp in the 3Ј-untranslated region with an oncogene c-RAF in reversed transcription orientation (4, 5). However, the function of MKRN2 remains elusive.Here we report the cloning of the Xenopus ortholog of human MKRN2 (mkrn2) and the characterization of its functions in Xenopus neurogenesis. In both gain-of-function and loss-of-function studies, we showed that dysregulation of mkrn2 produced morphological defects in neural development. To elucidate the underlying mechanism, we employed animal cap (AC) explant culture system to examine the effect of mkrn2 on the expression of a key neural marker, neural cell adhesion molecule (NCAM) (6). Xenopus AC is a unique model system for studying neural and mesodermal induction as it is capable of giving rise to either neural or mesodermal tissues in the presence o...

LEP-2/Makorin regulates LIN-28 stability to promote the juvenile-to-adult transition in Caenorhabditis elegans

“…During the reprogramming of mouse fibroblasts into induced pluripotent stem cells, Mkrn1 is co-regulated with a set of known differentiation markers (O'Malley et al, 2013). Also, ectopic Mkrn2 in Xenopus results in a smaller head and shorter tail than normal (Yang et al, 2008), a phenotype consistent with a precocious switch from proliferation to differentiation. These phenomena are consistent with a role for Mkrns in promoting differentiation by switching off LIN-28, which otherwise maintains an undifferentiated state.…”

“…Indeed, Mkrn1 in mammals functions as an E3 ligase, targeting several proteins and even itself, although has not yet been identified as one of its targets (Kim et al, 2005;Salvatico et al, 2010;Lee et al, 2009Lee et al, , 2012Ko et al, 2012;Shimada et al, 2009;Ko et al, 2010;Kim et al, 2014;Cassar et al, 2015). In other contexts, Mkrns are also RNA-binding proteins, are localized to ribonucleoprotein granules, and have been found to sequester some mRNAs or promote their translation (Cano et al, 2010;Cassar et al, 2015;Gajdos et al, 2010;Kwon et al, 2013;Miroci et al, 2012;Cheung et al, 2010;Yang et al, 2008). Thus, an alternative mechanism for LEP-2/Mkrn function could be to sequester, protect from degradation and/or promote the translation of an mRNA encoding another protein that promotes LIN-28 destruction.…”

Expression of a gene encoding a MKRN RING zinc finger protein in early germination stages of pea (Pisum sativum L.Var. Alaska) and its possible role in differentiation

“…Expression patterns of MKRN1 in murine embryonic nervous system and testis suggest that MKRN1 may have an important role in embryonic development and neurogenesis. Yang et al (2008) for the first time characterized the functional role of MKRN2 in Xenopus laevis and reported that MKRN2 plays a role in PI3 K/Akt-mediated neurogenesis during embryonic development of Xenopus. Expression studies of makorin genes (MKRN1 and MKRN2) in yellowtail fish (Chamnan et al 2003), mouse and humans (Gray et al 2000(Gray et al , 2001 and Xenopus (Yang et al 2008) have paved a way for an understanding of this gene family in animal systems.…”

“…Yang et al (2008) for the first time characterized the functional role of MKRN2 in Xenopus laevis and reported that MKRN2 plays a role in PI3 K/Akt-mediated neurogenesis during embryonic development of Xenopus. Expression studies of makorin genes (MKRN1 and MKRN2) in yellowtail fish (Chamnan et al 2003), mouse and humans (Gray et al 2000(Gray et al , 2001 and Xenopus (Yang et al 2008) have paved a way for an understanding of this gene family in animal systems. MKRN has been shown to act as an E3 ligase for hTERT (Kim et al 2005), as a transcriptional co-regulator of androgen and retinoic acid receptors (Omwancha et al 2006), and also for both p53 and p21 (Lee et al 2009).…”

“…The MKRN gene is characterized by a unique composition of C3H, RING and a novel Cys-His motif (Gray et al 2000) and it encodes putative ribonucleoproteins with a distinct array of zinc finger domains (Yang et al 2008). Gray et al (2000) have reported that nine MKRN family loci have been found scattered throughout the human genome.…”