MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the posttranscriptional level 1 . Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex2 -4. These pre-miRNAs are cleaved by the RNase III Dicer5 -8 to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence9. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein10), which contains three double-stranded, RNAbinding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer-TRBP with Argonaute 2 (Ago2)11 , 12, the catalytic engine of RISC. The physical association of Dicer-TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer-TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer-TRBP complex not only in miRNA processing but also as a platform for RISC assembly.To gain an insight into the components of the miRNA/siRNA processing machinery, we isolated a Dicer-containing complex from human cells. This was accomplished by developing HEK293-derived stable cell lines expressing Dicer tagged with Flag (Flag-Dicer). Flag-Dicer was isolated using affinity chromatography, and the affinity eluate was subjected to SDSpolyacrylamide gel electrophoresis (PAGE) followed by silver staining and western blot analysis.Western blot and mass spectrometric analyses indicated that most polypeptides in the Dicer affinity eluate were products of the proteolytic break down of Dicer (Fig. 1a). However, mass spectroscopy identified a 50-kDa band (six peptide sequences that migrated slightly above the contaminating MEP50 band) corresponding to the human immunodeficiency virus (HIV)-1 transactivating response (TAR) RNA-binding protein (TRBP) 10 . The TRBP gene encodes a protein with three double-stranded RNA-binding domains (dsRBDs). Analysis of the nonredundant protein database by Blast identified proteins with close homology to TRBP in both vertebrates and Drosophila (CG6866) (Fig. 1b) (Fig. 1c). The presence of Dicer was also confirmed by mass spectrometric sequencing. Moreover, additional bands (indicated with an asterisk in Fig. 1c) correspond to SKB1 and MEP50, common contaminants of Flag purification. Although most of TRBP eluted in smaller fractions (32 and beyond; perhaps as a consequence of overexpression), a minor portion of TRBP eluted as a large complex (fractions 16 and 18) not easily visualized by silver sta...

We have isolated a holoenzyme complex termed BRCC containing BRCA1, BRCA2, and RAD51. BRCC not only displays increased association with p53 following DNA damage but also ubiquitinates p53 in vitro. BRCC36 and BRCC45 are novel components of the complex with sequence homology to a subunit of the signalosome and proteasome complexes. Reconstitution of a recombinant four-subunit complex containing BRCA1/BARD1/BRCC45/BRCC36 revealed an enhanced E3 ligase activity compared to that of BRCA1/BARD1 heterodimer. In vivo, depletion of BRCC36 and BRCC45 by the small interfering RNAs (siRNAs) resulted in increased sensitivity to ionizing radiation and defects in G2/M checkpoint. BRCC36 shows aberrant expression in sporadic breast tumors. These findings identify BRCC as a ubiquitin E3 ligase complex that enhances cellular survival following DNA damage.

Selenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA[Ser]Sec gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA[Ser]Sec and activities of major selenoproteins. Total liver selenium was dramatically decreased, whereas levels of low molecular weight selenocompounds were little affected. Plasma selenoprotein P levels were reduced by about 75%, suggesting that selenoprotein P is primarily exported from the liver. Glutathione S-transferase levels were elevated in the selenoprotein-deficient liver, suggesting a compensatory activation of this detoxification program. Mice appeared normal until about 24 h before death. Most animals died between 1 and 3 months of age. Death appeared to be due to severe hepatocellular degeneration and necrosis with concomitant necrosis of peritoneal and retroperitoneal fat. These studies revealed an essential role of selenoproteins in liver function.

Selenium has been implicated in cancer prevention, but the mechanism and possible involvement of selenoproteins in this process are not understood. To elucidate whether the 15-kDa selenoprotein may play a role in cancer etiology, the complete sequence of the human 15-kDa protein gene was determined, and various characteristics associated with expression of the protein were examined in normal and malignant cells and tissues. The 51-kilobase pair gene for the 15-kDa selenoprotein consisted of five exons and four introns and was localized on chromosome 1p31, a genetic locus commonly mutated or deleted in human cancers. Two stemloop structures resembling selenocysteine insertion sequence elements were identified in the 3-untranslated region of the gene, and only one of these was functional. Two alleles in the human 15-kDa protein gene were identified that differed by two single nucleotide polymorphic sites that occurred within the selenocysteine insertion sequence-like structures. These 3-untranslated region polymorphisms resulted in changes in selenocysteine incorporation into protein and responded differently to selenium supplementation. Human and mouse 15-kDa selenoprotein genes manifested the highest level of expression in prostate, liver, kidney, testis, and brain, and the level of the selenoprotein was reduced substantially in a malignant prostate cell line and in hepatocarcinoma. The expression pattern of the 15-kDa protein in normal and malignant tissues, the occurrence of polymorphisms associated with protein expression, the role of selenium in differential regulation of polymorphisms, and the chromosomal location of the gene may be relevant to a role of this protein in cancer.The essential trace element selenium occurs in several proteins found in bacteria (1), archaea (2), and eukaryotes (1, 3). All of the known selenoproteins described to date incorporate selenium into protein co-translationally as the amino acid selenocysteine (Sec) 1 in response to the UGA codon (1, 3, 4), with the exception of several bacterial molybdenum-containing enzymes such as Clostridium barkeri nicotinic acid hydroxylase that contains an active center dissociable selenium species (5-7). Among the selenoproteins thus far identified in mammals are four glutathione peroxidases, three thioredoxin reductases, three thyroid hormone deiodinases, selenophosphate synthetase, selenoprotein P, selenoprotein W, selenoprotein T, selenoprotein R (also called selenoprotein X), selenoprotein N, and a 15-kDa selenoprotein (1, 3, 8 -10).The 15-kDa selenoprotein was initially identified as a strongly labeled protein that was detected when human T cells were grown in the presence of [75 Se]selenite (11). The open reading frame within the human 15-kDa protein cDNA coded for 162 residues and contained an in-frame TGA codon that would result in the incorporation of Sec at codon position 93. A putative stem-loop structure, designated the Sec insertion sequence (SECIS) element, was predicted in the 3Ј-untranslated region (3Ј-UTR). SECIS elements are ...

Mice homozygous for an allele encoding the selenocysteine (Sec) tRNA [Ser]Sec gene (Trsp) flanked by loxP sites were generated. Cre recombinase-dependent removal of Trsp in these mice was lethal to embryos. To investigate the role of Trsp in mouse mammary epithelium, we deleted this gene by using transgenic mice carrying the Cre recombinase gene under control of the mouse mammary tumor virus (MMTV) long terminal repeat or the whey acidic protein promoter. While both promoters target Cre gene expression to mammary epithelium, MMTV-Cre is also expressed in spleen and skin. Sec tRNA[Ser]Sec amounts were reduced by more than 70% in mammary tissue with either transgene, while in skin and spleen, levels were reduced only with MMTV-Cre. The selenoprotein population was selectively affected with MMTV-Cre in breast and skin but not in the control tissue, kidney. Moreover, within affected tissues, expression of specific selenoproteins was regulated differently and often in a contrasting manner, with levels of Sep15 and the glutathione peroxidases GPx1 and GPx4 being substantially reduced. Expression of the tumor suppressor genes BRCA1 and p53 was also altered in a contrasting manner in MMTV-Cre mice, suggesting greater susceptibility to cancer and/or increased cell apoptosis. Thus, the conditional Trsp knockout mouse allows tissue-specific manipulation of Sec tRNA and selenoprotein expression, suggesting that this approach will provide a useful tool for studying the role of selenoproteins in health.Selenium is an essential micronutrient in the diet of mammals and numerous other life forms (see reference 26 for a review). Many health benefits have been attributed to this element, including a role in the prevention of cancer (10) and heart disease and other cardiovascular and muscle disorders (11), in delaying the aging process (33) and the onset of AIDS in human immunodeficiency virus-positive patients (1), in male reproduction (17), in mammalian development (5), in immune function (33), and as an antiviral agent (2). Selenium is incorporated into protein in the form of selenocysteine (Sec), and Sec has its own tRNA (designated Sec tRNA[Ser]Sec ) and its own code word, UGA (26). Sec is indeed the 21st naturally occurring amino acid in the genetic code. Most certainly, the health benefits of selenium are due in large part to its presence in protein (26).Sec tRNA [Ser]Sec is the only known tRNA that governs the expression of an entire class of proteins, the selenoproteins (26). This provides a unique opportunity to study the expression of selenoproteins by manipulating the levels and characteristics of Sec tRNA [Ser]Sec . For example, the levels of numerous selenoproteins were reduced in a protein-and tissue-specific manner in transgenic mice carrying mutant Sec tRNA [Ser]Sec transgenes lacking the highly modified base isopentenyladenosine in its anticodon (37). Glutathione peroxidase 1 (GPx1) and thioredoxin reductases 1 (TR1) and 3 (TR3) were the most and least affected selenoproteins, respectively, and selenoprotein expre...

BACH1 (also known as FANCJ and BRIP1) is a DNA helicase that directly interacts with the C-terminal BRCT repeat of the breast cancer susceptibility protein BRCA1. Previous biochemical and functional analyses have suggested a role for the BACH1 homolog in Caenorhabditis elegans during DNA replication. Here, we report the association of BACH1 with a distinct BRCA1/BRCA2-containing complex during the S phase of the cell cycle. Depletion of BACH1 or BRCA1 using small interfering RNAs results in delayed entry into the S phase of the cell cycle. Such timely progression through S phase requires the helicase activity of BACH1. Importantly, cells expressing a dominant negative mutation in BACH1 that results in a defective helicase displayed increased activation of DNA damage checkpoints and genomic instability. BACH1 helicase is silenced during the G 1 phase of the cell cycle and is activated through a dephosphorylation event as cells enter S phase. These results point to a critical role for BACH1 helicase activity not only in the timely progression through the S phase but also in maintaining genomic stability.Inherited defects in the breast cancer susceptibility genes BRCA1 and BRCA2 are associated with increased risk for hereditary breast, ovarian, and other cancers (1,9,23,25). BRCA1 and BRCA2 encode very large proteins with little resemblance to one another or to proteins of known function (23, 37). BRCA1 encodes a 1,863-amino-acid nuclear phosphoprotein with an N-terminal RING domain and C-terminal BRCT motifs (6,14,23,30,32).The BRCA1 carboxyl-terminal domain, BRCT, is approximately 90 amino acids in length and plays an important role in the tumor suppressor functions of BRCA1 (2). Apart from BRCA1, more than 30 other BRCT-containing proteins have been documented in the human genome and appear to interact with proteins involved in DNA repair and checkpoint control (2, 4). Recent evidence suggests that the BRCT domain represents a new class of modules that mediate phosphorylationdependent protein-protein interactions (29). The BRCT motif of BRCA1 plays a critical role in its ability to mediate doublestrand break repair and homologous recombination (24,33,44). Mutations which disrupt or delete the C-terminal BRCT domain, but not other regions of BRCA1, have been shown to cause significant relocalization of BRCA1 from nucleus to cytoplasm (28). Loss of the BRCA1 BRCT domain has been attributed to tumor formation in mice (19). Cancer-causing missense mutations have been identified at the interface between the two BRCT repeats of BRCA1, which destabilize the structure (39, 40).BACH1 (BRCA1-associated C-terminal helicase, also known as FANCJ and BRIP1), a member of the DEAH family of DNA helicases, directly interacts with the BRCT domain of the breast cancer gene product BRCA1 (5). More recently, it was shown that the BRCA1 interaction with BACH1 depends on the phosphorylation status of BACH1, and this phosphorylation-specific interaction is likely involved in the double-strand break repair function of BACH1 (41). BACH1 ...

Mammalian selenocysteine-containing proteins characterized with respect to function are involved in redox processes and exhibit distinct expression patterns and cellular locations. A recently identified 15-kDa selenoprotein (Sep15) has no homology to previously characterized proteins, and its function is not known. Here we report the intracellular localization and identification of a binding partner for this selenoprotein which implicate Sep15 in the regulation of protein folding. The native Sep15 isolated from rat prostate and mouse liver occurred in a complex with a 150-kDa protein. The latter protein was identified as UDP-glucose:glycoprotein glucosyltransferase (UGTR), the endoplasmic reticulum (ER)-resident protein, which was previously shown to be involved in the quality control of protein folding. UGTR functions by glucosylating misfolded proteins, retaining them in the ER until they are correctly folded or transferring them to degradation pathways. To determine the intracellular localization of Sep15, we expressed a green fluorescent protein-Sep15 fusion protein in CV-1 cells, and this protein was localized to the ER and possibly other perinuclear compartments. We determined that Sep15 contained the N-terminal signal peptide that was essential for translocation and that it was cleaved in the mature protein. However, C-terminal sequences of Sep15 were not involved in trafficking and retention of Sep15. The data suggest that the association between Sep15 and UGTR is responsible for maintaining the selenoprotein in the ER. This report provides the first example of the ER-resident selenoprotein and suggests a possible role of the trace element selenium in the quality control of protein folding.

Expression of functional breast cancer susceptibility gene 1 (BRCA1) in human breast and ovarian cancers is associated with resistance to platinum-based chemotherapeutics and poly(ADP ribose) polymerase (PARP) inhibitors. BRCA1 is a nuclear tumor suppressor that is critical for resolving double-strand DNA breaks (DSBs) and interstrand crosslinks (ICLs) by homologous recombination (HR). In vitro, animal and human clinical data have demonstrated that BRCA1-deficient cancers are highly sensitive to ICL-inducing chemotherapeutic agents, are amenable to synthetic lethal approaches that exploit defects in DSB/ ICL repair, and may be associated with improved survival. Conversely, high or restored expression of BRCA1 in breast and ovarian cancer is associated with therapeutic resistance and poor prognosis. There has been much interest in identifying agents that interfere with BRCA1-dependent DSB/ICL repair to restore or enhance sensitivity to cancer therapeutics. We demonstrate that the heat-shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin [17-AAG (Tanespimycin)], currently in Phase II/III clinical evaluation for several cancers, induces BRCA1 ubiquitination and proteasomal degradation, resulting in compromised repair of ionizing radiation-and platinum-induced DNA damage. We show that loss of HSP90 function abolishes BRCA1-dependent DSB repair and that BRCA1-deficient cells are hypersensitive to 17-AAG due to impaired Gap 2/Mitosis (G2/M) checkpoint activation and resultant mitotic catastrophe. In summary, we document an upstream HSP90-dependent regulatory point in the Fanconi anemia/BRCA DSB/ICL repair pathway, illuminate the role of BRCA1 in regulating damage-associated checkpoint and repair responses to HSP90 inhibitors, and identify BRCA1 as a clinically relevant target for enhancing sensitivity in refractory and/or resistant malignancies. chemosensitivity | DNA repair I nherited mutations in the breast cancer susceptibility gene 1 (BRCA1) predispose the development of breast, ovarian, and other malignancies (1-5). BRCA1 is a nuclear tumor suppressor critical for repair of double-strand DNA breaks (DSBs) and interstrand crosslinks (ICLs) by homologous recombination (HR) (6). BRCA1 is phosphorylated by ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 related (ATR), and checkpoint kinase 2 (CHK2) kinases in response to DNA damage and recruits and organizes multiple distinct protein complexes that recognize and repair damaged DNA and activate cell cycle checkpoints (7,8). In vitro and in vivo studies have shown that tumor cells expressing high levels of BRCA1 are resistant to both ionizing radiation (IR) and several classes of chemotherapeutic agents, and that ablation of BRCA1 expression can restore sensitivity to these agents (9, 10). Indeed, cancers arising in BRCA1 mutation carriers are relatively hypersensitive to platinum-based therapies, and high BRCA1 mRNA expression in sporadic cancers is a biomarker for poor response to these same agents (11-13).DSB-repair-deficient...