In eukaryotes, the decoding of the UGA codon as selenocysteine (Sec) requires a Sec insertion sequence (SECIS) element in the 3Ј untranslated region of the mRNA. We purified a SECIS binding protein, SBP2, and obtained a cDNA clone that encodes this activity. SBP2 is a novel protein containing a putative RNA binding domain found in ribosomal proteins and a yeast suppressor of translation termination. By UV cross-linking and immunoprecipitation, we show that SBP2 specifically binds selenoprotein mRNAs both in vitro and in vivo. Using 75 Se-labeled Sec-tRNA Sec , we developed an in vitro system for analyzing Sec incorporation in which the translation of a selenoprotein mRNA was both SBP2 and SECIS element dependent. Immunodepletion of SBP2 from the lysates abolished Sec insertion, which was restored when recombinant SBP2 was added to the reaction. These results establish that SBP2 is essential for the cotranslational insertion of Sec into selenoproteins. We hypothesize that the binding activity of SBP2 may be involved in preventing termination at the UGA/ Sec codon.

Aminoacyl tRNA synthetases (ARS) catalyze the ligation of amino acids to cognate tRNAs. Chordate ARSs have evolved distinctive features absent from ancestral forms, including compartmentalization in a multisynthetase complex (MSC), noncatalytic peptide appendages, and ancillary functions unrelated to aminoacylation. Here, we show that glutamyl-prolyl-tRNA synthetase (GluProRS), a bifunctional ARS of the MSC, has a regulated, noncanonical activity that blocks synthesis of a specific protein. GluProRS was identified as a component of the interferon (IFN)-gamma-activated inhibitor of translation (GAIT) complex by RNA affinity chromatography using the ceruloplasmin (Cp) GAIT element as ligand. In response to IFN-gamma, GluProRS is phosphorylated and released from the MSC, binds the Cp 3'-untranslated region in an mRNP containing three additional proteins, and silences Cp mRNA translation. Thus, GluProRS has divergent functions in protein synthesis: in the MSC, its aminoacylation activity supports global translation, but translocation of GluProRS to an inflammation-responsive mRNP causes gene-specific translational silencing.

The C-to-U editing of apolipoprotein B (apo-B) mRNA is catalyzed by a multiprotein complex that recognizes an 11-nucleotide mooring sequence downstream of the editing site. The catalytic subunit of the editing enzyme, apobec-1, has cytidine deaminase activity but requires additional unidentified proteins to edit apo-B mRNA. We purified a 65-kDa protein that functionally complements apobec-1 and obtained peptide sequence information which was used in molecular cloning experiments. The apobec-1 complementation factor (ACF) cDNA encodes a novel 64.3-kDa protein that contains three nonidentical RNA recognition motifs. ACF and apobec-1 comprise the minimal protein requirements for apo-B mRNA editing in vitro. By UV cross-linking and immunoprecipitation, we show that ACF binds to apo-B mRNA in vitro and in vivo. Cross-linking of ACF is not competed by RNAs with mutations in the mooring sequence. Coimmunoprecipitation experiments identified an ACF-apobec-1 complex in transfected cells. Immunodepletion of ACF from rat liver extracts abolished editing activity. The immunoprecipitated complexes contained a functional holoenzyme. Our results support a model of the editing enzyme in which ACF binds to the mooring sequence in apo-B mRNA and docks apobec-1 to deaminate its target cytidine. The fact that ACF is widely expressed in human tissues that lack apobec-1 and apo-B mRNA suggests that ACF may be involved in other RNA editing or RNA processing events.Base modification editing of mRNAs involves the conversion of single nucleotides within the coding region of a transcript. A number of mRNAs undergo site-specific deamination reactions that convert A3I or C3U. These modifications result in the synthesis of alternative forms of the protein which have different biological functions (41). The A3I editing of the glutamate receptor, serotonin 5-HT 2C receptor, and hepatitis delta virus mRNAs is catalyzed by a family of adenosine deaminases known as ADAR. These enzymes act on doublestranded RNA and function as a single polypeptide which has both RNA-binding and catalytic activities (3,38,39). C-to-U conversions occur in mRNAs of Physarum polycephalum, plants, and mammals, but in most cases, the cis-acting sequences and trans-acting factors have not been identified (39).The best characterized example of C3U editing is the editing of mammalian apolipoprotein-B (apo-B) mRNA. Apo-B is a structural component of plasma lipoproteins and a significant risk factor for the development of atherosclerosis (7).

Selenium is an essential trace element that is incorporated into proteins as selenocysteine (Sec), the twenty-first amino acid. Sec is encoded by a UGA codon in the selenoprotein mRNA. The decoding of UGA as Sec requires the reprogramming of translation because UGA is normally read as a stop codon. The translation of selenoprotein mRNAs requires cis-acting sequences in the mRNA and novel trans-acting factors dedicated to Sec incorporation. Selenoprotein synthesis in vivo is highly selenium-dependent, and there is a hierarchy of selenoprotein expression in mammals when selenium is limiting. This review describes emerging themes from studies on the mechanism, kinetics, and efficiency of Sec insertion in prokaryotes. Recent developments that provide mechanistic insight into how the eukaryotic ribosome distinguishes between UGA/Sec and UGA/stop codons are discussed. The efficiency and regulation of mammalian selenoprotein synthesis are considered in the context of current models for Sec insertion.

Decoding UGA as selenocysteine requires a unique tRNA, a specialized elongation factor, and specific secondary structures in the mRNA, termed SECIS elements. Eukaryotic SECIS elements are found in the 3′ untranslated region of selenoprotein mRNAs while those in prokaryotes occur immediately downstream of UGA. Consequently, a single eukaryotic SECIS element can serve multiple UGA codons, whereas prokaryotic SECIS elements only function for the adjacent UGA, suggesting distinct mechanisms for recoding in the two kingdoms. We have identified and characterized the first eukaryotic selenocysteyl-tRNA-specific elongation factor. This factor forms a complex with mammalian SECIS binding protein 2, and these two components function together in selenocysteine incorporation in mammalian cells. Expression of the two functional domains of the bacterial elongation factor-SECIS binding protein as two separate proteins in eukaryotes suggests a mechanism for rapid exchange of charged for uncharged selenocysteyl-tRNA-elongation factor complex, allowing a single SECIS element to serve multiple UGA codons.

Data taken during the final shallow-site run of the first tower of the Cryogenic Dark Matter Search (CDMS II) detectors have been reanalyzed with improved sensitivity to small energy depositions. Four ∼224 g germanium and two ∼105 g silicon detectors were operated at the Stanford Underground Facility (SUF) between December 2001 and June 2002, yielding 118 live days of raw exposure. Three of the germanium and both silicon detectors were analyzed with a new low-threshold technique, making it possible to lower the germanium and silicon analysis thresholds down to the actual trigger thresholds of ∼1 keV and ∼2 keV, respectively. Limits on the spin-independent cross section for weakly interacting massive particles (WIMPs) to elastically scatter from nuclei based on these data exclude interesting parameter space for WIMPs with masses below 9 GeV/c 2 . Under standard halo assumptions, these data partially exclude parameter space favored by interpretations of the DAMA/LIBRA and CoGeNT experiments' data as WIMP signals, and exclude new parameter space for WIMP masses between 3 GeV/c 2 and 4 GeV/c 2 .PACS numbers: 14.80. Ly, 95.35.+d, 95.30.Cq, 85.25.Oj, 29.40.Wk

The Cryogenic Dark Matter Search (CDMS) employs Ge and Si detectors to search for weakly interacting massive particles (WIMPs) via their elastic-scattering interactions with nuclei while discriminating against interactions of background particles. CDMS data, accounting for the neutron background, give limits on the spin-independent WIMP-nucleon elastic-scattering cross section that exclude unexplored parameter space above 10 GeV͞c 2 WIMP mass and, at .75% C.L., the entire 3s allowed region for the WIMP signal reported by the DAMA experiment. Extensive evidence indicates that a large fraction of the matter in the universe is nonluminous, nonbaryonic, and "cold"-nonrelativistic at the time matter began to dominate the energy density of the universe [1][2][3]. Weakly interacting massive particles (WIMPs) are an excellent candidate for nonbaryonic, cold dark matter [2,4]. Minimal supersymmetry provides a natural WIMP candidate in the form of the lightest superpartner, with a typical mass M ϳ 100 GeV͞c 2 [5][6][7][8]. WIMPs are expected to have collapsed into a roughly isothermal, spherical halo within which the visible portion of our galaxy resides. WIMPs scatter off nuclei via the weak interaction, potentially allowing their direct detection [9,10]. The expected spectrum of recoil energies (energy given to the recoiling nucleus during the interaction) is exponential with a characteristic energy of a few to tens of keV [11]. The expected event rate is model dependent, but is generically 1 kg 21 d 21 or lower [10].This Letter reports new exclusion limits on the spinindependent WIMP-nucleon elastic-scattering cross section by the Cryogenic Dark Matter Search (CDMS). The rate of rare WIMP-nucleon interactions is constrained by extended exposure of detectors that discriminate WIMPinduced nuclear recoils from electron recoils caused by interactions of background particles [12,13].The ionization yield Y (the ratio of ionization production to recoil energy in a semiconductor) of a particle interaction differs greatly for nuclear and electron recoils. CDMS detectors measure phonon and electron-hole pair production to determine recoil energy and ionization yield for each event. The data discussed here were obtained with two types of detectors, Berkeley Large Ionization-and Phonon-mediated (BLIP) and Z-sensitive Ionization-and Phonon-mediated (ZIP) detectors [12][13][14][15][16][17][18]. For both types, the drift field for the ionization measurement is supplied by radially segmented electrodes on the faces of the disk-shaped crystals [19]. In BLIP detectors, phonon production is determined from the detector's calorimetric temperature change. In ZIP detectors, athermal phonons are collected to determine phonon production and xy position. Detector performance is discussed in detail elsewhere [14,[16][17][18][19][20].Photons cause most bulk electron recoils, while lowenergy electrons incident on the detector surfaces cause low-Y electron recoils in a thin surface layer ("surface events"). Neutron, photon, and electron sources ar...

The translational recoding of UGA as selenocysteine (Sec) is directed by a SECIS element in the 3' untranslated region (UTR) of eukaryotic selenoprotein mRNAs. The selenocysteine insertion sequence (SECIS) contains two essential tandem sheared G.A pairs that bind SECIS-binding protein 2 (SBP2), which recruits a selenocysteine-specific elongation factor and Sec-tRNA(Sec) to the ribosome. Here we show that ribosomal protein L30 is a component of the eukaryotic selenocysteine recoding machinery. L30 binds SECIS elements in vitro and in vivo, stimulates UGA recoding in transfected cells and competes with SBP2 for SECIS binding. Magnesium, known to induce a kink-turn in RNAs that contain two tandem G.A pairs, decreases the SBP2-SECIS complex in favor of the L30-SECIS interaction. We propose a model in which SBP2 and L30 carry out different functions in the UGA recoding mechanism, with the SECIS acting as a molecular switch upon protein binding.