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Cloning and characterization of mr-s, a novel SAM domain protein, predominantly expressed in retinal photoreceptor cells.

ABSTRACT: BACKGROUND: Sterile alpha motif (SAM) domains are approximately 70 residues long and have been reported as common protein-protein interaction modules. This domain is found in a large number of proteins, including Polycomb group (PcG) proteins and ETS family transcription factors. In this work, we report the cloning and functional characterization of a novel SAM domain-containing protein, which is predominantly expressed in retinal photoreceptors and the pineal gland and is designated mouse mr-s (major retinal SAM domain protein). RESULTS: mr-s is evolutionarily conserved from zebrafish through human, organisms through which the mechanism of photoreceptor development is also highly conserved. Phylogenetic analysis suggests that the SAM domain of mr-s is most closely related to a mouse polyhomeotic (ph) ortholog, Mph1/Rae28, which is known as an epigenetic molecule involved in chromatin modifications. These findings provide the possibility that mr-s may play a critical role by regulating gene expression in photoreceptor development. mr-s is preferentially expressed in the photoreceptors at postnatal day 3-6 (P3-6), when photoreceptors undergo terminal differentiation, and in the adult pineal gland. Transcription of mr-s is directly regulated by the cone-rod homeodomain protein Crx. Immunoprecipitation assay showed that the mr-s protein self-associates mainly through the SAM domain-containing region as well as ph. The mr-s protein localizes mainly in the nucleus, when mr-s is overexpressed in HEK293T cells. Moreover, in the luciferase assays, we found that mr-s protein fused to GAL4 DNA-binding domain functions as a transcriptional repressor. We revealed that the repression activity of mr-s is not due to a homophilic interaction through its SAM domain but to the C-terminal region. CONCLUSION: We identified a novel gene, mr-s, which is predominantly expressed in retinal photoreceptors and pineal gland. Based on its expression pattern and biochemical analysis, we predict that mr-s may function as a transcriptional repressor in photoreceptor cells and in pinealocytes of the pineal gland.

SUBMITTER: Inoue T

PROVIDER: S-EPMC1435744 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Cloning and characterization of mr-s, a novel SAM domain protein, predominantly expressed in retinal photoreceptor cells.

Inoue Tatsuya T Terada Koji K Furukawa Akiko A Koike Chieko C Tamaki Yasuhiro Y Araie Makoto M Furukawa Takahisa T

BMC developmental biology 20060316

<h4>Background</h4>Sterile alpha motif (SAM) domains are approximately 70 residues long and have been reported as common protein-protein interaction modules. This domain is found in a large number of proteins, including Polycomb group (PcG) proteins and ETS family transcription factors. In this work, we report the cloning and functional characterization of a novel SAM domain-containing protein, which is predominantly expressed in retinal photoreceptors and the pineal gland and is designated mous ...[more]

PMID: 16539743

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Project description:BackgroundIn fish, molecular mechanisms that control follicle-enclosed oocyte progression throughout oogenesis and oocyte developmental competence acquisition remain poorly understood. Existing data in mammals have indicated that the so called "oocyte-specific" genes play an important role in oogenesis, fertilization, and early embryo development. In teleost species, very little is known about "oocyte-specific" genes. The present study therefore aimed at identifying and characterizing oocyte-specific genes in fish.ResultsUsing digital differential display PCR, mouse ESTs exhibiting an oocyte-predominant expression were identified. Those murine ESTs were subsequently used to identify cognate rainbow trout (Oncorhynchus mykiss) ESTs using a reciprocal Blast search strategy. In the present study we report the identification of five previously uncharacterized rainbow trout cDNAs exhibiting a oocyte-specific, oocyte-predominant, or gonad-specific expression: zygote arrest 1 (zar1), v-mos Moloney murine sarcoma viral oncogene-like protein (mos), B-cell translocation gene (btg3), growth differentiation factor 9 (gdf9), and mutS homolog 4 (msh4). The orthology relationship of each of these genes with vertebrate counterparts was verified by phylogenetic analysis. Among those five genes, three had never been characterized in any fish species. In addition, we report the oocyte-predominant expression of btg3 for the first time in any vertebrate species. Finally, those five genes are present in unfertilized eggs as maternally-inherited mRNAs thus suggesting that they could participate in ovarian folliculogenesis as well as early embryonic development.ConclusionThe expression patterns of zar1, mos, btg3, gdf9 and msh4 in rainbow trout and the functions of their orthologs in higher vertebrates strongly suggest that they might play an important role in follicle-enclosed oocyte development, meiosis control and early embryonic development in fish. Future investigations are however required to unravel the participation of those strong candidates in the molecular processes that control folliculogenesis and/or oocyte developmental competence in fish.

Project description:A novel microtubule-associated protein (MAP) of M(r) 115,000 has been identified by screening of a HeLa cell cDNA expression library with an anti-serum raised against microtubule-binding proteins from HeLa cells. Monoclonal and affinity-purified polyclonal antibodies were generated for the further characterization of this MAP. It is different from the microtubule-binding proteins of similar molecular weights, characterized so far, by its nucleotide-insensitive binding to microtubules and different sedimentation behavior. Since it is predominantly expressed in cells of epithelial origin (Caco-2, HeLa, MDCK), and rare (human skin, A72) or not detectable (Vero) in fibroblastic cells, we name it E-MAP-115 (epithelial MAP of 115 kD). In HeLa cells, E-MAP-115 is preferentially associated with subdomains or subsets of perinuclear microtubules. In Caco-2 cells, labeling for E-MAP-115 increases when they polarize and form blisters. The molecular characterization of E-MAP-115 reveals that it is a novel protein with no significant homologies to other known proteins. The secondary structure predicted from its sequence indicates two domains connected by a putative hinge region rich in proline and alanine (PAPA region). E-MAP-115 has two highly charged regions with predicted alpha-helical structure, one basic with a pI of 10.9 in the NH2-terminal domain and one neutral with a pI of 7.6 immediately following the PAPA region in the acidic COOH-terminal half of the molecule. A novel microtubule-binding site has been localized to the basic alpha-helical region in the NH2-terminal domain using in vitro microtubule-binding assays and expression of mutant polypeptides in vivo. Overexpression of this domain of E-MAP-115 by transfection of fibroblasts lacking significant levels of this protein with its cDNA renders microtubules stable to nocodazole. We conclude that E-MAP-115 is a microtubule-stabilizing protein that may play an important role during reorganization of microtubules during polarization and differentiation of epithelial cells.

Dataset Information

Cloning and characterization of mr-s, a novel SAM domain protein, predominantly expressed in retinal photoreceptor cells.

Publications

Cloning and characterization of mr-s, a novel SAM domain protein, predominantly expressed in retinal photoreceptor cells.

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