Mol. Cells 2017; 40(12): 945-953
Published online December 22, 2017
https://doi.org/10.14348/molcells.2017.0216
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: mrhee@cnu.ac.kr
We report the biological functions of a zebrafish homologue of RING-finger protein 152 (
Keywords delta-notch signaling, neurogenesis,
Ubiquitination is an important cellular process that affects protein homeostasis; it also controls complex processes during embryogenesis (Ro et al., 2015). Abnormal ubiquitination can result in the development of major human neurodegenerative diseases such as Parkinson’s and Alzheimer’s (Anuppalle et al., 2013). Ubiquitination involves three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3), which modify proteins and affect their localization and stability (Morreale and Walden, 2016). Among the three enzymes, E3 ligases are critical for determining substrate specificity. E3 ligases are classified into two major subtypes based on their structural characteristics: RING domain-containing and HECT domain-containing E3 ligases (Metzger et al., 2014). Furthermore, discrete ubiquitin chains on a substrate dictate whether the protein will alter its cellular localization or undergo proteasomal degradation (Swatek and Komander, 2016).
As a member of the RING domain-containing E3 ligase family, RNF152 is an important regulator of proteins functioning downstream of mechanistic target of rapamycin complex 1(mTORC1) signaling (Deng et al., 2015). In particular, mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates cellular growth and proliferation during the early stages of neurogenesis (Garza-Lombó et al., 2016). RNF152 targets RagA GTPase for K-63-mediated ubiquitination, which activates its inhibitor GATOR1, a GAP complex for Rag GTPase, thereby inactivating mTORC1 signaling (Deng et al., 2015). Although mTORC1 activity is critical in neonatal neuronal stem cells in the subventricular zone during neurogenesis (Hartman et al., 2013), the biological functions of RNF152 in embryogenesis have not yet been addressed.
Thus, we conducted molecular genetic studies on Rnf152 using zebrafish embryos to define the expression patterns, biological functions, and transcriptional regulation of
Phylogenetic analysis was performed to identify evolutionary relationship between zebrafish Rnf152 (NP_001014380) with human RNF152 (NP_775828), chimpanzee RNF152 (XP_001143948), mouse RNF152 (NP_848894), chicken RNF152 (NP_001291963), and
Wild type synchronized embryos were produced and maintained at 28.8°C (Westerfield, 2000). Embryos were collected with natural breeding in the cycle of 10-hour dark/14-hour light and morphological confirmation was done as described in Kimmel et al. (1995). Embryos were treated with 0.2 mM Phenylthiourea (PTU) after 9 hpf to stop melanogenesis.
Total RNA was isolated from various stages of zebrafish embryos using R&A-BLUE® Total RNA Extraction kit (iNtRON Biotechnology) and cDNA was synthesized with M-MLV Reverse Transcriptase (Enzynomics) with Oligo (dT)20 primer. The primers were used for PCR to analyze the zebrafish
After sequence confirmation, cloned
We selected a positive strand of
As we identified that zebrafish pre-mRNA has only a single exon, so a splice modifying Morpholino was not a possibility and we designed a translational blocking Morpholino; GCTC TGGGACAAGCTATCCATCGTC. For a specificity control, we designed 5′ mismatch oligo; GCTgTcGGAgAAcCTATCCATCcTC. We collected
The neighbor-joining phylogenetic relationships between the amino acid sequence of zebrafish Rnf152 and those of homologues from other species were analyzed using Phylogenetic and Molecular Evolutionary Analysis MEGA software package (version 7.0) (Kumar et al., 2016). Rnf152 (NP_001014380) comprised 198 amino acid residues. The chimpanzee, mouse, chicken, and
The temporal expression pattern of
To examine the biological functions of Rnf152, its expression was forced by microinjecting the
To identify the molecular elements in developing tissues affected by knockdown of
Notch-Delta signaling determines cell fate in the eyes, particularly the decision of neurons or Müller glia to differentiate or not differentiate (Taylor et al., 2015). In addition,
We examined
We showed that
Except for the telencephalon,
Among the
We report here that
NeuroD2 is a transcription factor harboring a Cdc20 recognition motif and destruction box (D-box) that regulates presynaptic development in the brain (Yang et al., 2009). The major mitotic E3 ubiquitin ligase, Cdc20-anaphase-promoting complex, activates presynaptic differentiation by promoting protein degradation, which negatively regulates presynaptic development (Yang et al., 2010). Given that NeuroD2 expression is inversely correlated with that of Cdc20, the cellular level of NeuroD2 is regulated by the ubiquitin-proteasome system (Yang et al., 2009).
The E3 ligase gene, Mib1, regulates neurogenesis in the developing spinal cord via Delta-Notch signaling (Kang et al., 2013). Furthermore, expression of Notch receptor family members such as
Mol. Cells 2017; 40(12): 945-953
Published online December 31, 2017 https://doi.org/10.14348/molcells.2017.0216
Copyright © The Korean Society for Molecular and Cellular Biology.
Ajeet Kumar1, Tae-Lin Huh2, Joonho Choe3, and Myungchull Rhee1,*
1Department of Life Science, BK21 Plus Program, Graduate School, Chungnam National University, Daejeon 34134, Korea, 2School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 41566, Korea, 3Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Korea
Correspondence to:*Correspondence: mrhee@cnu.ac.kr
We report the biological functions of a zebrafish homologue of RING-finger protein 152 (
Keywords: delta-notch signaling, neurogenesis,
Ubiquitination is an important cellular process that affects protein homeostasis; it also controls complex processes during embryogenesis (Ro et al., 2015). Abnormal ubiquitination can result in the development of major human neurodegenerative diseases such as Parkinson’s and Alzheimer’s (Anuppalle et al., 2013). Ubiquitination involves three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3), which modify proteins and affect their localization and stability (Morreale and Walden, 2016). Among the three enzymes, E3 ligases are critical for determining substrate specificity. E3 ligases are classified into two major subtypes based on their structural characteristics: RING domain-containing and HECT domain-containing E3 ligases (Metzger et al., 2014). Furthermore, discrete ubiquitin chains on a substrate dictate whether the protein will alter its cellular localization or undergo proteasomal degradation (Swatek and Komander, 2016).
As a member of the RING domain-containing E3 ligase family, RNF152 is an important regulator of proteins functioning downstream of mechanistic target of rapamycin complex 1(mTORC1) signaling (Deng et al., 2015). In particular, mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates cellular growth and proliferation during the early stages of neurogenesis (Garza-Lombó et al., 2016). RNF152 targets RagA GTPase for K-63-mediated ubiquitination, which activates its inhibitor GATOR1, a GAP complex for Rag GTPase, thereby inactivating mTORC1 signaling (Deng et al., 2015). Although mTORC1 activity is critical in neonatal neuronal stem cells in the subventricular zone during neurogenesis (Hartman et al., 2013), the biological functions of RNF152 in embryogenesis have not yet been addressed.
Thus, we conducted molecular genetic studies on Rnf152 using zebrafish embryos to define the expression patterns, biological functions, and transcriptional regulation of
Phylogenetic analysis was performed to identify evolutionary relationship between zebrafish Rnf152 (NP_001014380) with human RNF152 (NP_775828), chimpanzee RNF152 (XP_001143948), mouse RNF152 (NP_848894), chicken RNF152 (NP_001291963), and
Wild type synchronized embryos were produced and maintained at 28.8°C (Westerfield, 2000). Embryos were collected with natural breeding in the cycle of 10-hour dark/14-hour light and morphological confirmation was done as described in Kimmel et al. (1995). Embryos were treated with 0.2 mM Phenylthiourea (PTU) after 9 hpf to stop melanogenesis.
Total RNA was isolated from various stages of zebrafish embryos using R&A-BLUE® Total RNA Extraction kit (iNtRON Biotechnology) and cDNA was synthesized with M-MLV Reverse Transcriptase (Enzynomics) with Oligo (dT)20 primer. The primers were used for PCR to analyze the zebrafish
After sequence confirmation, cloned
We selected a positive strand of
As we identified that zebrafish pre-mRNA has only a single exon, so a splice modifying Morpholino was not a possibility and we designed a translational blocking Morpholino; GCTC TGGGACAAGCTATCCATCGTC. For a specificity control, we designed 5′ mismatch oligo; GCTgTcGGAgAAcCTATCCATCcTC. We collected
The neighbor-joining phylogenetic relationships between the amino acid sequence of zebrafish Rnf152 and those of homologues from other species were analyzed using Phylogenetic and Molecular Evolutionary Analysis MEGA software package (version 7.0) (Kumar et al., 2016). Rnf152 (NP_001014380) comprised 198 amino acid residues. The chimpanzee, mouse, chicken, and
The temporal expression pattern of
To examine the biological functions of Rnf152, its expression was forced by microinjecting the
To identify the molecular elements in developing tissues affected by knockdown of
Notch-Delta signaling determines cell fate in the eyes, particularly the decision of neurons or Müller glia to differentiate or not differentiate (Taylor et al., 2015). In addition,
We examined
We showed that
Except for the telencephalon,
Among the
We report here that
NeuroD2 is a transcription factor harboring a Cdc20 recognition motif and destruction box (D-box) that regulates presynaptic development in the brain (Yang et al., 2009). The major mitotic E3 ubiquitin ligase, Cdc20-anaphase-promoting complex, activates presynaptic differentiation by promoting protein degradation, which negatively regulates presynaptic development (Yang et al., 2010). Given that NeuroD2 expression is inversely correlated with that of Cdc20, the cellular level of NeuroD2 is regulated by the ubiquitin-proteasome system (Yang et al., 2009).
The E3 ligase gene, Mib1, regulates neurogenesis in the developing spinal cord via Delta-Notch signaling (Kang et al., 2013). Furthermore, expression of Notch receptor family members such as
Khadija Habib, Kausik Bishayee, Jieun Kang, Ali Sadra*, and Sung-Oh Huh*
Mol. Cells 2022; 45(8): 588-602 https://doi.org/10.14348/molcells.2022.0044Zobia Umair, Shiv Kumar, Daniel H. Kim, Khezina Rafiq, Vijay Kumar, SungChan Kim, Jae-Bong Park, Jae-Yong Lee, Unjoo Lee, and Jaebong Kim
Mol. Cells 2018; 41(12): 1061-1071 https://doi.org/10.14348/molcells.2018.0341Jeong-Oh Shin, Jong-Joo Lee, Mikyoung Kim, Youn Wook Chung, Hyehyun Min, Jae-Yoon Kim, Hyoung-Pyo Kim, and Jinwoong Bok
Mol. Cells 2018; 41(7): 695-702 https://doi.org/10.14348/molcells.2018.0230