Alain Krol

Characterization of mSelB, a novel mammalian elongation factor for selenoprotein translation

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.

Novel selenoproteins identified in silico and in vivo by using a conserved RNA structural motif.

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 byin 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.

The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery

RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth.

U2 RNA shares a structural domain with U1, U4, and U5 RNAs.

We previously reported common structural features within the 3′‐terminal regions of U1, U4, and U5 RNAs. To check whether these features also exist in U2 RNA, the primary and secondary structures of the 3′‐terminal regions of chicken, pheasant, and rat U2 RNAs were examined. Whereas no difference was observed between pheasant and chicken, the chicken and rat sequences were only 82.5% homologous. Such divergence allowed us to propose a unique model of secondary structure based on maximum base‐pairing and secondary structure conservation. The same model was obtained from the results of limited digestion of U2 RNA with various nucleases. Comparison of this structure with those of U1, U4, and U5 RNAs shows that the four RNAs share a common structure designated as domain A, and consisting of a free single‐stranded region with the sequence Pu‐A‐(U)n‐G‐Pup flanked by two hairpins. The hairpin on the 3′ side is very stable and has the sequence Py‐N‐Py‐Gp in the loop. The presence of this common domain is discussed in connection with relationships among U RNAs and common protein binding sites.

The selenium to selenoprotein pathway in eukaryotes: More molecular partners than anticipated.

The amino acid selenocysteine (Sec) is the major biological form of the trace element selenium. Sec is co-translationally incorporated in selenoproteins. There are 25 selenoprotein genes in humans, and Sec was found in the active site of those that have been attributed a function. This review will discuss how selenocysteine is synthesized and incorporated into selenoproteins in eukaryotes. Sec biosynthesis from serine on the tRNA(Sec) requires four enzymes. Incorporation of Sec in response to an in-frame UGA codon, otherwise signaling termination of translation, is achieved by a complex recoding machinery to inform the ribosomes not to stop at this position on the mRNA. A number of the molecular partners acting in this machinery have been identified but their detailed mechanism of action has not been deciphered yet. Here we provide an overview of the literature in the field. Particularly striking is the higher than originally envisaged number of factors necessary to synthesize Sec and selenoproteins. Clearly, selenoprotein synthesis is an exciting and very active field of research.

Spatial and temporal expression patterns of selenoprotein genes during embryogenesis in zebrafish

Selenium is important for embryogenesis in vertebrates but little is known about the expression patterns and biological functions of most selenoprotein genes. Taking advantage of the zebrafish model, systematic analysis of selenoprotein gene expression was performed by in situ hybridization on whole-mount embryos at different developmental stages. Twenty-one selenoprotein mRNAs were analyzed and all of them exhibited expression patterns restricted to specific tissues. Moreover, we demonstrated that highly similar selenoprotein paralogs were expressed within distinct territories. Therefore, tissue- and development-specific expression patterns provided new information for selenoproteins of unknown function.

Changing the RNA polymerase specificity of U snRNA gene promoters.

The promoter of a Xenopus tropicalis U6 gene can be transcribed by both RNA polymerases II and III. Two distinct elements, a TATA-like sequence and the region of transcription initiation, are only required for transcription by RNA polymerase III, while further common elements are required for transcription by both polymerases. Based on the unusually stringent requirement for a purine at the normal position of polymerase III transcription initiation and on the properties of mutants in this region, we suggest that RNA polymerase III itself may recognize the site of transcription initiation and thus be directly involved in efficient promoter selection. We have used the information obtained on U6 promoter structure to manufacture a U6 promoter that is RNA polymerase II-specific and to change the Xenopus U2 gene promoter specificity from RNA polymerase II to RNA polymerase III.

Staf, a promiscuous activator for enhanced transcription by RNA polymerases II and III

Staf is a zinc finger protein that we recently identified as the transcriptional activator of the RNA polymerase III‐transcribed selenocysteine tRNA gene. In this work we demonstrate that enhanced transcription of the majority of vertebrate snRNA and snRNA‐type genes, transcribed by RNA polymerases II and III, also requires Staf. DNA binding assays and microinjection of mutant genes into Xenopus oocytes showed the presence of Staf‐responsive elements in the genes for human U4C, U6, Y4 and 7SK, Xenopus U1b1, U2, U5 and MRP and mouse U6 RNAs. Using recombinant Staf, we established that it mediates the activating properties of Staf‐responsive elements on RNA polymerase II and III snRNA promoters in vivo. Lastly a 19 bp consensus sequence for the Staf binding site, YY(A/T)CCC(A/G)N(A/C)AT(G/C)C(A/C)YYRCR, was derived by binding site selection. It enabled us to identify 23 other snRNA and snRNA‐type genes carrying potential Staf binding sites. Altogether, our results emphasize the prime importance of Staf as a novel activator for enhanced transcription of snRNA and snRNA‐type genes.

The N-terminal domain of the human TATA-binding protein plays a role in transcription from TATA-containing RNA polymerase II and III promoters

In eukaryotes, the TATA box binding protein (TBP) is an integral component of the transcription initiation complexes of all three classes of nuclear RNA polymerases. In this study we have investigated the role of the N‐terminal region of human TBP in transcription initiation from RNA polymerase (Pol) I, II and III promoters by using three monoclonal antibodies (mAbs). Each antibody recognizes a distinct epitope in the N‐terminal domain of human TBP. We demonstrate that these antibodies differentially affect transcription from distinct classes of promoters. One antibody, mAb1C2, and a synthetic peptide comprising its epitope selectively inhibited in vitro transcription from TATA‐containing, but not from TATA‐less promoters, irrespective of whether they were transcribed by Pol II or Pol III. Transcription by Pol I, on the other hand, was not affected. Two other antibodies and their respective epitope peptides did not affect transcription from any of the promoters tested. Order of addition experiments indicate that mAb1C2 did not prevent binding of TBP to the TATA box or the formation of the TBP‐TFIIA‐TFIIB complex but rather inhibited a subsequent step of preinitiation complex formation. These data suggest that a defined region within the N‐terminal domain of human TBP may be involved in specific protein‐protein interactions required for the assembly of functional preinitiation complexes on TATA‐containing, but not on TATA‐less promoters.

Reconsidering the evolution of eukaryotic selenoproteins: a novel nonmammalian family with scattered phylogenetic distribution

While the genome sequence and gene content are available for an increasing number of organisms, eukaryotic selenoproteins remain poorly characterized. The dual role of the UGA codon confounds the identification of novel selenoprotein genes. Here, we describe a comparative genomics approach that relies on the genome‐wide prediction of genes with in‐frame TGA codons, and the subsequent comparison of predictions from different genomes, wherein conservation in regions flanking the TGA codon suggests selenocysteine coding function. Application of this method to human and fugu genomes identified a novel selenoprotein family, named SelU, in the puffer fish. The selenocysteine‐containing form also occurred in other fish, chicken, sea urchin, green algae and diatoms. In contrast, mammals, worms and land plants contained cysteine homologues. We demonstrated selenium incorporation into chicken SelU and characterized the SelU expression pattern in zebrafish embryos. Our data indicate a scattered evolutionary distribution of selenoproteins in eukaryotes, and suggest that, contrary to the picture emerging from data available so far, other taxa‐specific selenoproteins probably exist.