Identification of a signature motif for the eIF4a3-SECIS interaction.

eIF4a3, a DEAD-box protein family member, is a component of the exon junction complex which assembles on spliced mRNAs. The protein also acts as a transcript-selective translational repressor of selenoprotein synthesis during selenium deficiency. Selenocysteine (Sec) incorporation into selenoproteins requires a Sec Insertion Sequence (SECIS) element in the 3' untranslated region. During selenium deficiency, eIF4a3 binds SECIS elements from non-essential selenoproteins, preventing Sec insertion. We identified a molecular signature for the eIF4a3-SECIS interaction using RNA gel shifts, surface plasmon resonance and enzymatic foot printing. Our results support a two-site interaction model, where eIF4a3 binds the internal and apical loops of the SECIS. Additionally, the stability of the complex requires uridine in the SECIS core. In terms of protein requirements, the two globular domains of eIF4a3, which are connected by a linker, are both critical for SECIS binding. Compared to full-length eIF4a3, the two domains in trans bind with a lower association rate but notably, the uridine is no longer important for complex stability. These results provide insight into how eIF4a3 discriminates among SECIS elements and represses translation.

Nucleolin binds to a subset of selenoprotein mRNAs and regulates their expression.

Selenium, an essential trace element, is incorporated into selenoproteins as selenocysteine (Sec), the 21st amino acid. In order to synthesize selenoproteins, a translational reprogramming event must occur since Sec is encoded by the UGA stop codon. In mammals, the recoding of UGA as Sec depends on the selenocysteine insertion sequence (SECIS) element, a stem-loop structure in the 3' untranslated region of the transcript. The SECIS acts as a platform for RNA-binding proteins, which mediate or regulate the recoding mechanism. Using UV crosslinking, we identified a 110 kDa protein, which binds with high affinity to SECIS elements from a subset of selenoprotein mRNAs. The crosslinking activity was purified by RNA affinity chromatography and identified as nucleolin by mass spectrometry analysis. In vitro binding assays showed that purified nucleolin discriminates among SECIS elements in the absence of other factors. Based on siRNA experiments, nucleolin is required for the optimal expression of certain selenoproteins. There was a good correlation between the affinity of nucleolin for a SECIS and its effect on selenoprotein expression. As selenoprotein transcript levels and localization did not change in siRNA-treated cells, our results suggest that nucleolin selectively enhances the expression of a subset of selenoproteins at the translational level.

Eukaryotic initiation factor 4a3 is a selenium-regulated RNA-binding protein that selectively inhibits selenocysteine incorporation.

The synthesis of selenoproteins requires the translational recoding of the UGA stop codon as selenocysteine. During selenium deficiency, there is a hierarchy of selenoprotein expression, with certain selenoproteins synthesized at the expense of others. The mechanism by which the limiting selenocysteine incorporation machinery is preferentially utilized to maintain the expression of essential selenoproteins has not been elucidated. Here we demonstrate that eukaryotic initiation factor 4a3 (eIF4a3) is involved in the translational control of a subset of selenoproteins. The interaction of eIF4a3 with the selenoprotein mRNA prevents the binding of SECIS binding protein 2, which is required for selenocysteine insertion, thereby inhibiting the synthesis of the selenoprotein. Furthermore, the expression of eIF4a3 is regulated in response to selenium. Based on knockdown and overexpression studies, eIF4a3 is necessary and sufficient to mediate selective translational repression in cells. Our results support a model in which eIF4a3 links selenium status with differential selenoprotein expression.

Known turnover and translation regulatory RNA-binding proteins interact with the 3' UTR of SECIS-binding protein 2.

The human selenoproteome is composed of approximately 25 selenoproteins, which cotranslationally incorporate selenocysteine, the 21st amino acid. Selenoprotein expression requires an unusual translation mechanism, as selenocysteine is encoded by the UGA stop codon. SECIS-binding protein 2 (SBP2) is an essential component of the selenocysteine insertion machinery. SBP2 is also the only factor known to differentiate among selenoprotein mRNAs, thereby modulating the relative expression of the individual selenoproteins. Here, we show that expression of SBP2 protein varies widely across tissues and cell types examined, despite previous observations of only modest variation in SBP2 mRNA levels. This discrepancy between SBP2 mRNA and protein levels implies translational regulation, which is often mediated via untranslated regions (UTRs) in regulated transcripts. We have identified multiple sequences in the SBP2 3' UTR that are highly conserved. The proximal short conserved region is GU rich and was subsequently shown to be a binding site for CUG-BP1. The distal half of the 3' UTR is largely conserved, and multiple proteins interact with this region. One of these proteins was identified as HuR. Both CUG-BP1 and HuR are members of the Turnover and Translation Regulatory RNA-Binding Protein family (TTR-RBP). Members of this protein family are linked by the common ability to rapidly effect gene expression through alterations in the stability and translatability of target mRNAs. The identification of CUG-BP1 and HuR as factors that bind to the SBP2 3' UTR suggests that TTR-RBPs play a role in the regulation of SBP2, which then dictates the expression of the selenoproteome.

Using molecular dynamics to map interaction networks in an aminoacyl-tRNA synthetase.

Long-range functional communication is a hallmark of many enzymes that display allostery, or action-at-a-distance. Many aminoacyl-tRNA synthetases can be considered allosteric, in that their trinucleotide anticodons bind the enzyme at a site removed from their catalytic domains. Such is the case with E. coli methionyl-tRNA synthase (MetRS), which recognizes its cognate anticodon using a conserved tryptophan residue 50 A away from the site of tRNA aminoacylation. The lack of details regarding how MetRS and tRNA(Met) interact has limited efforts to deconvolute the long-range communication that occurs in this system. We have used molecular dynamics simulations to evaluate the mobility of wild-type MetRS and a Trp-461 variant shown previously by experiment to be deficient in tRNA aminoacylation. The simulations reveal that MetRS has significant mobility, particularly at structural motifs known to be involved in catalysis. Correlated motions are observed between residues in distant structural motifs, including the active site, zinc binding motif, and anticodon binding domain. Both mobility and correlated motions decrease significantly but not uniformly upon substitution at Trp-461. Mobility of some residues is essentially abolished upon removal of Trp-461, despite being tens of Angstroms away from the site of mutation and solvent exposed. This conserved residue does not simply participate in anticodon binding, as demonstrated experimentally, but appears to mediate the protein's distribution of structural ensembles. Finally, simulations of MetRS indicate that the ligand-free protein samples conformations similar to those observed in crystal structures with substrates and substrate analogs bound. Thus, there are low energetic barriers for MetRS to achieve the substrate-bound conformations previously determined by structural methods.

DNA minor groove adducts formed by a platinum-acridine conjugate inhibit association of tata-binding protein with its cognate sequence.

PT-ACRAMTU ([PtCl(en)(ACRAMTU-S)](NO(3))(2), en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) is a cytotoxic platinum-acridine conjugate previously shown to form adducts with the N3 endocyclic nitrogen of adenine in the DNA minor groove. This unusual observation and our prior determination of the pronounced 5'-TA/TA base-step affinity of the drug have prompted us to investigate effects of these adducts on DNA minor groove binding proteins. Here, we used electrophoretic mobility shift assays to study the recognition of a PT-ACRAMTU-modified TATA box sequence by TATA-binding protein (TBP). The frequency of PT-ACRAMTU adducts in the minor groove of the TATA box was varied by selective elimination of potential major groove and minor groove binding sites in a 24-bp probe sequence through incorporation of deaza nucleobases. The most dramatic effect on TBP binding was observed in a duplex substituted with 7-deaza-G and 7-deaza-A, which reduced binding by as much as 73% compared to an unplatinated duplex. In contrast, elimination of A-N3 binding sites had no significant effect on TBP binding, suggesting that minor groove adducts of PT-ACRAMTU are the cause of inhibition. This notion was further corroborated by efficient platinum-mediated photo-cross-linking of the drug-modified DNA to TBP. PT-ACRAMTU appears to be the first platinum-based drug capable of targeting DNA sequences critical for transcription initiation. The biological consequences of PT-ACRAMTU's minor groove adducts are discussed.

Unique base-step recognition by a platinum-acridinylthiourea conjugate leads to a DNA damage profile complementary to that of the anticancer drug cisplatin.

The sequence specificity and time course of covalent DNA adduct formation of the novel platinum-acridine conjugate [PtCl(en)(ACRAMTU)](NO(3))(2) [PT-ACRAMTU, 2; en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea] have been investigated using restriction enzyme cleavage and transcription footprinting assays and compared to the damage produced by the clinical agent cis-diamminedichloroplatinum(II) (cisplatin, 1). The rate of DNA binding of 1 and 2 was also monitored by atomic emission spectrometry. Restriction enzymes were chosen that cleave the phosphodiester linkage at, or adjacent to, the predicted damage sites. While conjugate 2 selectively protected supercoiled plasmid from cleavage by EcoRI and DraI enzymes at their respective restriction sites, G downward arrow AATTC and TTT downward arrow AAA, 1 inhibited DNA hydrolysis by HindIII and PspOMI at A downward arrow AGCTT and G downward arrow GGCCC (arrows mark cleavage sites) more efficiently. Transcription footprinting using T7 RNA polymerase revealed major single-base damage sites for 2 at adenine in 5'-TA and 5'-GA sequences. In addition, the enzyme is efficiently stalled at guanine bases, primarily in the sequence 5'-CGA where the damaged nucleobase is flanked by two high-affinity intercalation sites of ACRAMTU. While 1 targets poly(G) sequences, the binding of 2 appears to be dominated by the groove and sequence recognition of the intercalator. The biochemical assays used confirm previous structural information extracted from mass spectra of DNA fragments modified by 2 isolated from enzymatic digests [Barry, C. G., et al. (2003) J. Am. Chem. Soc. 125, 9629-9637]. Possible DNA-binding mechanisms and biological consequences of the unprecedented modification of alternating TA sequences by 2, which occurred at a faster rate than binding to G, are discussed.