Decoding of UGA selenocysteine codons in eubacteria is mediated by the specialized elongation factor SelB, which conveys the charged tRNASec to the A site of the ribosome, through binding to the SECIS mRNA hairpin. In an attempt to isolate the eukaryotic homolog of SelB, a database search in this work identified a mouse expressed sequence tag containing the complete cDNA encoding a novel protein of 583 amino acids, which we called mSelB. Several lines of evidence enabled us to establish that mSelB is the bona fide mammalian elongation factor for selenoprotein translation: it binds GTP, recognizes the Sec‐tRNASec in vitro and in vivo, and is required for efficient selenoprotein translation in vivo. In contrast to the eubacterial SelB, the recombinant mSelB alone is unable to bind specifically the eukaryotic SECIS RNA hairpin. However, complementation with HeLa cell extracts led to the formation of a SECIS‐dependent complex containing mSelB and at least another factor. Therefore, the role carried out by a single elongation factor in eubacterial selenoprotein translation is devoted to two or more specialized proteins in eukaryotes.

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Selenocysteine is incorporated into selenoproteins by an in-frame UGA codon whose readthrough requires the selenocysteine insertion sequence (SECIS), a conserved hairpin in the 3-untranslated region of eukaryotic selenoprotein mRNAs. To identify new selenoproteins, we developed a strategy that obviates the need for prior amino acid sequence information. A computational screen was used to scan nucleotide sequence data bases for sequences presenting a potential SECIS secondary structure. The computer-selected hairpins were then assayed in vivo for their functional capacities, and the cDNAs corresponding to the SECIS winners were identified. Four of them encoded novel selenoproteins as confirmed by in vivo experiments. Among these, SelZf1 and SelZf2 share a common domain with mitochondrial thioredoxin reductase-2. The three proteins, however, possess distinct N-terminal domains. We found that another protein, SelX, displays sequence similarity to a protein involved in bacterial pilus formation. For the first time, four novel selenoproteins were discovered based on a computational screen for the RNA hairpin directing selenocysteine incorporation.Selenium is an essential trace element whose deficiency can interfere with normal embryonic development and fertility or favor the appearance of certain cancers and viral diseases such as human immunodeficiency virus and coxsackievirus (1). The amino acid selenocysteine is the major biological form of selenium in bacteria and animals. It is found in the active site of selenoproteins and is directly involved in the catalytic reaction. In this regard, the capacity of the selenocysteine selenol group to become ionized at physiological pH, the cysteine thiol group requiring a higher pH, accounts for the higher rate of catalysis of selenoenzymes (2). Seven selenoprotein families have been characterized so far in mammals (3): the glutathione peroxidase and thioredoxin reductase families, involved in scavenging reactive oxygen species and maintaining the redox status of the cell; three iodothyronine deiodinases participating in the thyroid hormone metabolism; and last, SelW and SelP, which have not been attributed a function yet. More recently, a 15-kDa selenoprotein of unknown function has been purified (4). Selenophosphate synthetase-2, the seventh selenoprotein, is remarkable in that it contains selenocysteine, but is also a key actor in the biosynthesis of this amino acid (5).Selenocysteine is encoded by an in-frame UGA codon, implying the existence of a mechanism capable of distinguishing the UGA selenocysteine codon from a translational stop. This process requires, in eukaryotes, the presence of the selenocysteine insertion sequence (SECIS), 1 a hairpin residing in the 3Ј-untranslated region of selenoprotein mRNAs that is essential for readthrough of the UGA selenocysteine codon (6). Sequence comparisons and structure-function experiments generated a consensus secondary structure model for the SECIS element in which a functional motif could be identified (7,8).Compelling evid...

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The selenocysteine tRNA gene (tRNA(Sec)) is atypical. Though transcribed by RNA polymerase III like all other tRNA genes, its basal promoter elements are distinct and reside essentially upstream of the coding region. In addition, transcription from the basal promoter is activated by a 15 bp activator element. In this report we describe the cloning and functional characterization of Staf (selenocysteine tRNA gene transcription activating factor), a novel Xenopus laevis transcription factor which binds to the tRNA(Sec) activator element and mediates its activation properties. The 600 amino acid Staf protein contains seven zinc fingers and a separate acidic activation domain. Seven highly conserved regions were detected between Staf and human ZNF76, a protein of unknown function, thereby aiding in predicting the locations of the functional domains of Staf. With the use of a novel expression assay in X.laevis oocytes we succeeded in demonstrating that Staf can activate the RNA polymerase III promoter of the tRNA(Sec) gene. This constitutes the first demonstration of the capacity of a cloned factor to activate RNA polymerase III transcription in vivo.

H1 RNA, the RNA component of the human nuclear RNase P, is encoded by a unique gene transcribed by RNA polymerase III (Pol III). In this work, cis-acting elements and trans-acting factors involved in human H1 gene transcription were characterized by transcription assays of mutant templates and DNA binding assays of recombinant proteins. Four elements, lying within 100 bp of 5'-flanking sequences, were defined to be essential for maximal in vitro and in vivo expression, consisting of the octamer, Staf, proximal sequence element (PSE) and TATA motifs. These are also encountered in the promoter elements of vertebrate snRNA genes, where the first two constitute the distal sequence element (DSE). In all the genes examined so far, the DSE is distant from the PSE and TATA box that compose the basal promoter. However, we observed a fundamental difference in the organization of the H1 RNA and snRNA gene promoters with respect to the relative spacing of the DSE and PSE. Indeed, the H1 promoter is unusually compact, with the octamer motif and Staf binding site adjacent to the PSE and TATA motifs. It thus appears that the human RNase P RNA gene has adopted a unique promoter strategy placing the DSE immediately adjacent to the basal promoter.

The transcriptional activator Staf, originally identified in Xenopus laevis, is implicated in the enhanced transcription of small nuclear RNA (snRNA) and snRNAtype genes by RNA polymerases II (Pol II) and III (Pol III). This zinc finger protein also possesses the capacity to stimulate expression from a Pol II mRNA promoter. Here, we report a study on two human proteins, ZNF76 and ZNF143, that are 64 and 84% identical to their Xenopus counterpart, respectively. Northern blot analysis revealed that ZNF76 and ZNF143 mRNAs were expressed in all normal adult tissues examined. By using in vivo and in vitro assays, we have analyzed the DNA binding capacities and transcriptional properties of ZNF76 and ZNF143. The binding affinities of ZNF76 and ZNF143 for Staf divergent responsive elements were determined by gel shift assays, which revealed that the two proteins bound a same DNA motif with similar affinities. Also, polypeptide sequences containing the seven zinc fingers of ZNF76 and ZNF143 could efficiently repress in vivo the activated transcription from an snRNA-type promoter. Transfection experiments in Drosophila cells showed that ZNF76 and ZNF143 can activate transcription from an mRNA promoter through the Staf binding site. Finally, chimeric ZNF76 and ZNF143 proteins, carrying a heterologous DNA binding domain, are able to activate a Pol II mRNA promoter and snRNA Pol II and Pol III promoters in Xenopus oocytes, through the heterologous DNA binding site. Taken together, these findings demonstrate that ZNF76 and ZNF143 are two members of a same family of transactivator proteins. ZNF143 constitutes the human ortholog of the Xenopus Staf, and ZNF76 is a novel DNA binding protein related to Staf and ZNF143.Transcription is a major regulatory point in gene expression and depends largely on the interaction of regulatory proteins with their cognate DNA elements in gene promoters (1, 2). Analysis of promoters in a variety of snRNA 1 genes transcribed by either Pol II or Pol III has identified a number of distinct DNA elements required for gene expression. The Pol II and Pol III snRNA gene promoters both contain an essential PSE, which binds the basal transcription factor PTF also called SNAPc (3-5), and a DSE playing a major role in transcription efficiency. The DSE contains an octamer motif that binds the well characterized transcriptional activator Oct-1 (6, 7). In addition to Oct-1, Sp1 has been shown in some instances to be involved in mediating the activation properties of the DSE (8 -11). A number of other short transcription units, such as the 7SK, Y, MRP and tRNA Sec genes, have similar promoter organization and can be classified as snRNA-type genes (6). Recently, we have demonstrated that the zinc finger protein Staf, originally identified in Xenopus laevis as the transcriptional activator of the tRNA Sec gene (12, 13), is also involved in transcriptional activation of snRNA and snRNA-type genes transcribed by RNA Pol II and Pol III (14). In addition, Staf possesses the capacity to stimulate expression from an R...

Cotranslational insertion of selenocysteine into selenoenzymes is mediated by a specialized transfer RNA, the tRNA(Sec). We have carried out the determination of the solution structure of the eucaryotic tRNA(Sec). Based on the enzymatic and chemical probing approach, we show that the secondary structure bears a few unprecedented features like a 9 bp aminoacid-, a 4 bp thymine- and a 6 bp dihydrouridine-stems. Surprisingly, the eighth nucleotide, although being a uridine, is base-paired and cannot therefore correspond to the single-stranded invariant U8 found in all tRNAs. Rather, experimental evidence led us to propose that the role of the invariant U8 is actually played by the tenth nucleotide which is an A, numbered A8 to indicate this fact. The experimental data therefore demonstrate that the cloverleaf structure we derived experimentally resembles the hand-folded model proposed by Böck et al (ref. 3). Using the solution data and computer modelling, we derived a three-dimensional structure model which shows some unique aspects. Basically, A8, A14, U21 form a novel type of tertiary interaction in which A8 interacts with the Hoogsteen sites of A14 which itself forms a Watson-Crick pair with U21. No coherent model containing the canonical 15-48 interaction could be derived. Thus, the number of tertiary interactions appear to be limited, leading to an uncoupling of the variable stem from the rest of the molecule.