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.