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The Tristetraprolin (TTP) protein family includes four mammalian members (TTP, TIS11b, TIS11d, and ZFP36L3), but only one in Drosophila melanogaster (DTIS11). These proteins bind target mRNAs with AU-rich elements (AREs) via two C3H zinc finger domains and destabilize the mRNAs. We found that overexpression of mouse TIS11b or DTIS11 in the Drosophila retina dramatically reduced eye size, similar to the phenotype of eyes absent (eya) mutants. The eya transcript is one of many ARE-containing mRNAs in Drosophila. We showed that TIS11b reduced levels of eya mRNA in vivo. In addition, overexpression of Eya rescued the TIS11b overexpression phenotype. RNA pull-down and luciferase reporter analyses demonstrated that the DTIS11 RNA-binding domain is required for DTIS11 to bind the eya 3' UTR and reduce levels of eya mRNA. Moreover, ectopic expression of DTIS11 in Drosophila S2 cells decreased levels of eya mRNA and reduced cell viability. Consistent with these results, TTP proteins overexpressed in MCF7 human breast cancer cells were associated with eya homologue 2 (EYA2) mRNA, and caused a decrease in EYA2 mRNA stability and cell viability. Our results suggest that eya mRNA is a target of TTP proteins, and that downregulation of EYA by TTP may lead to reduced cell viability in Drosophila and human cells.

Human p29 has been demonstrated in the yeast two-hybrid method and in vitro GST pull-down assay to associate with GCIP, a cyclin D interacting protein. In this study, we describe the cloning and genomic structure of the mouse homologue, mp29. The overall mouse mp29 amino acid sequence is highly identical (91%) to human p29. Polyclonal antibody against mp29 was raised and the subcellular localization of mp29 was identified to be in the nucleus. Genomic clones containing mp29 gene were isolated and this gene was divided into seven exons spanning 9 kb of genomic DNA. The transcription initiation site of mp29 gene was determined to be 94 bp upstream of the translation initiation codon and the first 140 bp proximal TATA-less promoter region is required to activate minimal transcription of mouse mp29 gene in mammalian cells.

We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-alpha (M(r) = 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of alpha-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrin-like motif in the central region of the protein; two potential Ca(2+)-binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-alpha appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathione S-transferase fusion proteins comprising amino acids 9-486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-alpha in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.

A 3.7-kb cDNA encodes the carp JAK1 kinase of 1,156 amino acid residues. The overall amino acid sequence identity between carp JAK1 and murine JAK1, JAK2, JAK3, and human TYK2 is 57%, 35.5%, 31.3%, and 42.4%, respectively. In addition, carp JAK1 shows higher sequence homology to mammalian JAK1 in both the kinase-like (JH2) and kinase (JH1) domains (approximately 70% identity). Therefore, carp JAK1 is a homolog of mammalian JAK1. To investigate the possible function of JH2 domain, full-length, and various truncated forms of carp JAK1 were produced in the baculovirus system. Our results demonstrate that c-JH1 and c-JH2 associate with each other and c-JH2 can be tyrosine-phosphorylated by c-JAK1 and by c-JH(1 + 2). The JAK1 gene was also isolated from a carp genomic library and characterized. This gene is divided into 24 exons spanning at least 31 kb of genomic DNA. Exon 1 contains the 5'-untranslated region and exon 2 contains the putative translation initiation site. The 2.5-kb DNA region upstream of the transcription initiation site contains numerous potential binding sites for transcription factors including NF-IL6, HNF-5, AP1, GHF-5, and E2A. When this DNA fragment was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme 16 times more efficiently than the promoterless pCAT-Basic. Deletion analysis defined a positive regulatory region between -1,023 and -528. A smaller region (-181 to +59) without any typical TATA-box sequences, G + C-rich sequences, or other binding sequences for known transcription factors still had promoter activity. Constructs without this region did not have detectable promoter activity. This suggests that this region of DNA may play an important role in the expression of carp JAK1 gene.

Human hPuf-A/KIAA0020 was first identified as a new minor histocompatibility antigen in 2001. Its zebrafish orthologue contains six Pumilio-homology RNA-binding domains and has been shown to participate in the development of eyes and primordial germ cells, but the cellular function of hPuf-A remains unclear. In this report, we showed that hPuf-A predominantly localized in the nucleoli with minor punctate signals in the nucleoplasm. The nucleolar localization of hPuf-A would redistribute to the nucleoplasm after the treatment of RNA polymerase inhibitors (actinomycin D and 5,6-dichlorobenzimidazole riboside) and topoisomerase inhibitors [camptothecin (CPT) and etoposide]. Interestingly, knockdown of hPuf-A sensitized cells to CPT and UV treatment and cells constitutively overexpressing hPuf-A became more resistant to genotoxic exposure. Affinity gel pull-down coupled with mass spectrometric analysis identified PARP-1 as one of the hPuf-A interacting proteins. hPuf-A specifically interacts with the catalytic domain of PARP-1 and inhibits poly(ADP-ribosyl)ation of PARP-1 in vitro. Depletion of hPuf-A increased the cleaved PARP-1 and overexpression of hPuf-A lessened PARP-1 cleavage when cells were exposed to CPT and UV light. Collectively, hPuf-A may regulate cellular response to genotoxic stress by inhibiting PARP-1 activity and thus preventing PARP-1 degradation by caspase-3. Cancer Res; 71(3); 1126–34. ©2011 AACR.

Methylated histone readers are critical for chromatin dynamics, transcription, and DNA repair. Human PHRF1 contains a plant homeodomain (PHD) that recognizes methylated histones and a RING domain, which ubiquitinates substrates. A recent study reveals that PHRF1 is a tumor suppressor that promotes TGF-β cytostatic signaling through TGIF ubiquitination. Also, PHRF1 is a putative phosphorylation substrate of ataxia telangiectasia-mutated/ataxia telangiectasia and Rad3-related kinases; however, the role of PHRF1 in DNA damage response is unclear. Here we report a novel function of PHRF1 in modulating non-homologous end-joining (NHEJ). PHRF1 quickly localizes to DNA damage lesions upon genotoxic insults. Ablation of PHRF1 decreases the efficiency of plasmid-based end-joining, whereas PHRF1 overexpression leads to an elevated NHEJ in H1299 reporter cells. Immunoprecipitation and peptide pull-down assays verify that PHRF1 constitutively binds to di- and trimethylated histone H3 lysine 36 (H3K36) (H3K36me2 and H3K36me3) via its PHD domain. Substitution of S915DT917E to ADAE in PHRF1 decreases its affinity for NBS1. Both PHD domain and SDTE motif are required for its NHEJ-promoting activity. Furthermore, PHRF1 mediates PARP1 polyubiquitination for proteasomal degradation. These results suggest that PHRF1 may combine with H3K36 methylation and NBS1 to promote NHEJ and stabilize genomic integrity upon DNA damage insults.

Early detection and identification of SARS-CoV-infected patients and actions to prevent transmission are absolutely critical to prevent another SARS outbreak. Antibodies that specifically recognize the SARS-CoV spike and nucleocapsid proteins may provide a rapid screening method to allow accurate identification and isolation of patients with the virus early in their infection. For this reason, we raised peptide-induced polyclonal antibodies against SARS-CoV spike protein and polyclonal antibodies against SARS-CoV nucleocapsid protein using 6x His nucleocapsid recombinant protein. Western blot analysis and immunofluorescent staining showed that these antibodies specifically recognized SARS-CoV.

Human astrin is a newly identified microtubule-associated protein, which is highly expressed in the testis. Silencing of astrin has resulted in growth arrest and apoptotic cell death. In this study, we describe the cloning and genomic structure of mastrin, the mouse counterpart to astrin. The overall mouse mastrin amino-acid sequence is 66% identical to human astrin. Mastrin protein was demonstrated to localize to mitotic spindles during mitosis. Genomic clones containing mastrin gene were isolated; the gene was found to have 24 exons spanning 24kb of genomic DNA. Deletion analysis of 5(')-flanking sequences demonstrated that the first 120bp proximal to the TATA-less promoter region is necessary for minimal transcription of the mouse mastrin gene.

The BH3-only proapoptotic protein, BAD, was cloned from zebrafish embryos and its properties were characterized. Zebrafish BAD (zBAD) is a protein with 147 amino acids that contains a BH3 domain and a putative 14-3-3 binding site with the sequence of RPRSRS84AP, corresponding to S136 in mouse BAD (mBAD). zBAD shares 34%, 28%, and 29% amino acid sequence identity to the human, mouse, and rat BAD, respectively. RT-PCR analysis revealed that the expression of zBAD gene is found in various parts of zebrafish tissues. The treatment with the z-VAD fmk, a broad-range caspase inhibitor, in COS-1 cells significantly increased the expression of zebrafish BAD fusion proteins (GFP-zBAD and HA-zBAD), indicating that zebrafish BAD fusion proteins may be cleaved by caspase(s). zBAD was shown to induce apoptosis when it was overexpressed in COS-1 cells. In addition, zBAD was also expressed in muscle cells under the muscle-specific promoter from zebrafish α-actin gene. Abnormality in the skeletal muscles and the loss of green fluorescence signal in the same region were observed. Taken together, our results indicate that zBAD could induce apoptosis in vitro and in vivo and may have biological implications in apoptosis during zebrafish development.

One novel gene product, hMAP126, was demonstrated to interact with p29 in the yeast two-hybrid assay. The full-length cDNA of hMAP126 has been obtained and encodes a protein of 1120 amino acids. Multiple tissue Northern blot analysis showed that hMAP126 was abundantly expressed in the testis. Polyclonal antiserum against hMAP126 was raised and affinity-purification of anti-hMAP126 antibodies was performed. The subcellular distribution of hMAP126 was localized to the mitotic spindle. Furthermore, hMAP126 was identified to be post-translationally modified and phosphorylated by p34 cdc2 kinase in vitro. Taken together, we have isolated a novel protein, hMAP126, which may be involved in the functional and dynamic regulation of mitotic spindles.

The rad17 gene of Schizosaccharomyces pombe plays an important role as a checkpoint protein following DNA damage and during DNA replication. The human homologue of S. pombe rad17, Hrad17, was recently identified, but its function has not yet been established. Using the yeast two-hybrid system, we determined that HRad17 can interact with a nucleolar protein, NHP2L1. This interaction was also demonstrated biochemically, in human cells. Immunofluorescence studies revealed that HRad17 and NHP2L1 colocalize to the nucleolus, and immunogold labeling further resolved the location of NHP2L1 to the dense fibrillar component of the nucleolus. Interestingly, the localization of HRad17 in the nucleolus was altered in response to UV irradiation. These results provide some insight into the DNA damage and replication checkpoint mechanisms of HRad17.