Cloning and Characterization of a Gene (RFN22) Encoding a Novel Brain Expressed Ring Finger Protein (BERP) That Maps to Human Chromosome 11p15.5

We have recently identified a novel RING finger protein expressed in the rat brain, which associates with myosin V and α-actinin-4. Here we have cloned and characterized the orthologous human BERP cDNA and gene (HGMW-approved symbol RNF22). The human BERP protein is encoded by 11 exons ranging in size from 71 to 733 bp, and fluorescence in situ hybridization shows that the BERP gene maps to chromosome 11p15.5, 3′ to the FE65 gene. The human BERP protein is 98% identical to the rat and mouse proteins, and we have identified a highly conserved potential orthologue in Caenorhabditis elegans. BERP belongs to the RING finger–B-box–coiled coil (RBCC) subgroup of RING finger proteins, and a cluster of these RBCC protein genes is present in chromosome 11p15. Chromosome region 11p15 is thought to harbor tumor suppressor genes, and deletions of this region occur frequently in several types of human cancers. These observations indicate that BERP may be a novel tumor suppressor gene.     

A novel RING finger-B box-coiled-coil protein, GERP

The RING finger domain occurs in a wide variety of proteins involved in cellular regulation. The polymerase chain reaction was used to search for novel RING finger proteins, using primers derived from expressed sequence tags (ests). A cDNA encoding a novel RING finger protein expressed in brain, lung, breast, placenta, kidney, muscle, and germinal center B cells is described. The human gene is expressed in a variety of tumors, including anaplastic oligodendroglioma and maps to chromosome 10q24.3, a region showing frequent deletion or loss of heterozygosity in glioblastomas. It was therefore designated glioblastoma expressed RING finger protein (GERP). GERP contains an N-terminal RING finger, followed by two B-boxes and a coiled-coil, and thus belongs to the RBCC subfamily of RING finger proteins. The structure of this protein and its mapping to a locus thought to harbor tumor suppressor genes indicates that it may be a new tumor suppressor gene important in gliomas and other malignancies.     

BERP, a novel ring finger protein, binds to alpha-actinin-4

We recently identified BERP as a novel RING finger protein belonging to the RBCC protein family. It contains an N-terminal RING finger, followed by a B-box zinc finger and a coiled-coil domain. BERP interacts with the tail domain of the class V myosins through a beta-propeller structure in the BERP C-terminal. To identify other proteins interacting with BERP, the yeast two-hybrid strategy was employed, using the RBCC domain as bait. Screening of a rat brain cDNA library identified alpha-actinin-4 as a specific binding partner for the N-terminus of BERP. This actinin isoform could be immunoprecipitated together with BERP from HEK 293 cells transfected with expression constructs for BERP and alpha-actinin-4. These proteins could also be colocalized immunohistochemically in the cytoplasm of differentiated PC12 cells. We suggest that BERP may anchor class V myosins to particular cell domains via its interaction with alpha-actinin-4.     

Cloning and characterization of a novel RING finger protein that interacts with class V myosins.

We have identified a novel protein (BERP) that is a specific partner for the tail domain of myosin V. Class V myosins are a family of molecular motors thought to interact via their unique C-terminal tails with specific proteins for the targeted transport of organelles. BERP is highly expressed in brain and contains an N-terminal RING finger, followed by a B-box zinc finger, a coiled-coil (RBCC domain), and a unique C-terminal beta-propeller domain. A yeast two-hybrid screening indicated that the C-terminal beta-propeller domain mediates binding to the tail of the class V myosin myr6 (myosin Vb). This interaction was confirmed by immunoprecipitation, which also demonstrated that BERP could associate with myosin Va, the product of the dilute gene. Like myosin Va, BERP is expressed in a punctate pattern in the cytoplasm as well as in the neurites and growth cones of PC12 cells. We also found that the RBCC domain of BERP is involved in protein dimerization. Stable expression of a mutant form of BERP lacking the myosin-binding domain but containing the dimerization domain resulted in defective PC12 cell spreading and prevented neurite outgrowth in response to nerve growth factor. Our studies present a novel interaction for the beta-propeller domain and provide evidence for a role for BERP in myosin V-mediated cargo transport.     

Cloning and identification of a novel RNF6 transcriptional splice variant Spg2 in human development

RING finger proteins are zinc finger proteins containing the RING motifs. They act mainly as E3 ubiq-uitin ligases, bind the ubiquitin E2 conjugating enzyme and promote degradation of targeted proteins, Many novel genes have been isolated and differentially expressed in human adult and embryo testis by a testis cDNA-array differential display technique. A novel RING finger cDNA is highly expressed in adult testis and at low level in fetal testis. It was named Spg2. It contains a 2055 nucleotide ORF, en-codes a 685-amino-acid RNF6 protein, and has a RING finger in its C terminal. NCBI Blast shows that the gene is located on chromosome 13 and contains five exons. A multiple tissue expression profile also indicates that it is highly expressed in human testis, so we speculate that it may be associated with human spermatogenesis by virtue of the action of its RING domain.     

Haprin,a novel haploid germ cell-specific RING finger protein involved in the acrosome reaction

The acrosome reaction (i.e. the exocytosis of the sperm vesicle) is a prerequisite for fertilization, but its molecular mechanism is largely unknown. We have identified a cDNA clone for a gene named haprin, which encodes a haploid germ cell-specific RING finger protein. This protein is a novel member of the RBCC (RING finger, B-box type zinc finger, and coiled-coil domain) motif family that has roles in several cellular processes, such as exocytosis. It is transcribed exclusively in testicular germ cells after meiotic division. Western blot and immunohistochemical analyses showed the molecular weight of Haprin protein to be Mr approximately 82,000. It was localized in the acrosomal region of elongated spermatids and mature sperm and was not present in acrosome-reacted sperm. The specific antibody against the RING finger domain of Haprin inhibited the acrosome reaction in permeabilized sperm. These results indicated that the novel RBCC protein Haprin plays a key role in the acrosome reaction and fertilization.     

HLS5, a novel RBCC (ring finger, B box, coiled-coil) family member isolated from a hemopoietic lineage switch, is a candidate tumor suppressor

Hemopoietic cells, apparently committed to one lineage, can be reprogrammed to display the phenotype of another lineage. The J2E erythroleukemic cell line has on rare occasions developed the features of monocytic cells. Subtractive hybridization was used in an attempt to identify genes that were up-regulated during this erythroid to myeloid transition. We report here on the isolation of hemopoietic lineage switch 5 (Hls5), a gene expressed by the monocytoid variant cells, but not the parental J2E cells. Hls5 is a novel member of the RBCC (Ring finger, B box, coiled-coil) family of genes, which includes Pml, Herf1, Tif-1alpha, and Rfp. Hls5 was expressed in a wide range of adult tissues; however, at different stages during embryogenesis, Hls5 was detected in the branchial arches, spinal cord, dorsal root ganglia, limb buds, and brain. The protein was present in cytoplasmic granules and punctate nuclear bodies. Isolation of the human cDNA and genomic DNA revealed that the gene was located on chromosome 8p21, a region implicated in numerous leukemias and solid tumors. Enforced expression of Hls5 in HeLa cells inhibited cell growth, clonogenicity, and tumorigenicity. It is conceivable that HLS5 is one of the tumor suppressor genes thought to reside at the 8p21 locus.     

Molecular cloning and characterization of RNF26 on human chromosome 11q23 region, encoding a novel RING finger protein with leucine zipper

Genetic alterations of RING finger genes, encoding an ubiquitin-protein ligase, are implicated in several types of human cancer through dysregulation of growth regulators. Here, a novel RING finger gene, RNF26, was cloned and characterized. The RNF26 gene on human chromosome 11q23 region was found to encode a polypeptide of 433 amino acids with the N-terminal leucine zipper domain and the C-terminal RING finger domain. Among the RING finger protein family, RING finger domains of RNF26, CGR19, NEURL, KIAA0554, and AK022937 were found to constitute a novel C3HC5 subfamily, which is distinct from C3H2C3 or C3HC4 subfamilies. RING finger domain of RNF26 was most homologous to that of CGR19 (49% amino-acid identity). The 3.2-kb RNF26 mRNA was expressed ubiquitously in normal human tissues, but was upregulated in several human cancer cell lines, including HL-60 (promyelocytic leukemia), HeLa S3 (cervical uterus cancer), SW480 (colorectal cancer), and MKN7 (gastric cancer). In addition, RNF26 was upregulated in 50% of primary gastric cancer examined in this study. Although substrates of ubiquitination mediated by RNF26 remain to be elucidated, RNF26 upregulation in several types of human cancer might be implicated in carcinogenesis through dysregulation of its substrates.     

Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3

We have previously isolated and characterized murine MYB binding protein (p160) 1a, a protein that specifically interacts with the leucine zipper motif within the negative regulatory domain of the c-Myb proto-oncoprotein. We now describe the molecular cloning of the human MYBBP1A cDNA and chromosomal localization to 17p13.3 by fluorescence in situ hybridization analysis. Given the likely presence of a tumor suppressor gene (or genes) within this region of chromosome 17, the position of MYBBP1A was further mapped by radiation hybrid analysis and was found to lie between markers D17S1828 and D17S938. A P1 artificial chromosome clone containing the 5' region of MYBBP1A was isolated and indicates a physical linkage between MYBBP1A and the 15-lipoxygenase gene (ALOX15). A novel, polymorphic (CA)(25) dinucleotide repeat was also isolated from this PAC and may serve as a useful marker for MYBBP1A and this region of chromosome 17.     

FXY2/MID2, a gene related to the X-linked Opitz syndrome gene FXY/MID1, maps to Xq22 and encodes a FNIII domain-containing protein that associates with microtubules

Opitz G/BBB syndrome (OS) is a genetically heterogeneous disorder with an X-linked locus and an autosomal locus linked to 22q11.2. OS affects multiple organ systems with often variable severity even between siblings. The clinical features, which include hypertelorism, cleft lip and palate, defects of cardiac septation, hypospadias, and anorectal anomalies, indicate an underlying disturbance of the developing ventral midline of the embryo. The gene responsible for X-linked OS, FXY/MID1, is located on the short arm of the human X chromosome within Xp22.3 and encodes a protein with both an RBCC (RING finger, B-box, coiled coil) and a B30.2 domain. The Fxy gene in mice is also located on the X chromosome but spans the pseudoautosomal boundary in this species. Here we describe a gene closely related to FXY/MID1, called FXY2, which also maps to the X chromosome within Xq22. The mouse Fxy2 gene is located on the distal part of the mouse X chromosome within a region syntenic to Xq22. Analysis of genes flanking both FXY/MID1 and FXY2 (as well as their counterparts in mouse) suggests that these regions may have arisen as a result of an intrachromosomal duplication on an ancestral X chromosome. We have also identified in both FXY2 and FXY/MID1 proteins a conserved fibronectin type III domain located between the RBCC and B30.2 domains that has implications for understanding protein function. The FXY/MID1 protein has previously been shown to colocalize with microtubules, and here we show that the FXY2 protein similarly associates with microtubules in a manner that is dependent on the carboxy-terminal B30.2 domain.     

Chromosomal localization of two human zinc finger protein genes, ZNF24 (KOX17) and ZNF29 (KOX26), to 18q12 and 17p13-p12, respectively

Two members of the zinc finger Krüppel family, ZNF24 (KOX17) and ZNF29 (KOX26), have been localized by somatic cell hybrid analysis and in situ chromosomal hybridization to human chromosomes 18q12 and 17p13-p12, respectively. The mapping of ZNF29 together with the previously reported localization of ZFP3 suggests that a zinc finger gene complex is located on human chromosome 17p. ZNF29 maps centromeric to the human p53 tumor antigen gene (TP53). In the analogous murine position, the two mouse zinc finger genes Zfp2 and Zfp3 have recently been assigned to the distal region of mouse chromosome 11, the murine homolog of human chromosome 17. Both human zinc finger genes ZNF24 and ZNF29 are in chromosomal regions that have been noted to be deleted in neoplasms of the lung and of the central nervous system at chromosome 17p and in colorectal neoplasia at chromosomes 17p and 18q.     

Isolation and characterization of a novel human gene (HFB30) which encodes a protein with a RING finger motif.

A human cDNA, HFB30, encoding a novel protein that contains a RING finger (C3HC4-type zinc finger) motif was isolated. This cDNA clone consists of 3056 nucleotides and encodes an open reading frame of a 474 amino acid protein. From RT-PCR analysis, the messenger RNA was ubiquitously expressed in various human tissues. The gene was located to the chromosome 5q23.3-q31.1 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. Furthermore, the gene consists of nine exons that span about 20 kb of genome DNA.     

FXY2/MID2, a Gene Related to the X-Linked Opitz Syndrome Gene FXY/MID1, Maps to Xq22 and Encodes a FNIII Domain-Containing Protein That Associates with Microtubules

Opitz G/BBB syndrome (OS) is a genetically heterogeneous disorder with an X-linked locus and an autosomal locus linked to 22q11.2. OS affects multiple organ systems with often variable severity even between siblings. The clinical features, which include hypertelorism, cleft lip and palate, defects of cardiac septation, hypospadias, and anorectal anomalies, indicate an underlying disturbance of the developing ventral midline of the embryo. The gene responsible for X-linked OS, FXY/MID1, is located on the short arm of the human X chromosome within Xp22.3 and encodes a protein with both an RBCC (RING finger, B-box, coiled coil) and a B30.2 domain. The Fxy gene in mice is also located on the X chromosome but spans the pseudoautosomal boundary in this species. Here we describe a gene closely related to FXY/MID1, called FXY2, which also maps to the X chromosome within Xq22. The mouse Fxy2 gene is located on the distal part of the mouse X chromosome within a region syntenic to Xq22. Analysis of genes flanking both FXY/MID1 and FXY2 (as well as their counterparts in mouse) suggests that these regions may have arisen as a result of an intrachromosomal duplication on an ancestral X chromosome. We have also identified in both FXY2 and FXY/MID1 proteins a conserved fibronectin type III domain located between the RBCC and B30.2 domains that has implications for understanding protein function. The FXY/MID1 protein has previously been shown to colocalize with microtubules, and here we show that the FXY2 protein similarly associates with microtubules in a manner that is dependent on the carboxy-terminal B30.2 domain.