SECISaln, a web-based tool for the creation of structure-based alignments of eukaryotic SECIS elements

Summary: Selenoproteins contain the 21st amino acid selenocysteine which is encoded by an inframe UGA codon, usually read as a stop. In eukaryotes, its co-translational recoding requires the presence of an RNA stem–loop structure, the SECIS element in the 3 untranslated region of (UTR) selenoprotein mRNAs. Despite little sequence conservation, SECIS elements share the same overall secondary structure. Until recently, the lack of a significantly high number of selenoprotein mRNA sequences hampered the identification of other potential sequence conservation. In this work, the web-based tool SECISaln provides for the first time an extensive structure-based sequence alignment of SECIS elements resulting from the well-defined secondary structure of the SECIS RNA and the increased size of the eukaryotic selenoproteome. We have used SECISaln to improve our knowledge of SECIS secondary structure and to discover novel, conserved nucleotide positions and we believe it will be a useful tool for the selenoprotein and RNA scientific communities

Selenoprotein N is dynamically expressed during mouse development and detected early in muscle precursors

<p>Abstract</p> <p>Background</p> <p>In humans, mutations in the <it>SEPN1 </it>gene, encoding selenoprotein N (SelN), are involved in early onset recessive neuromuscular disorders, referred to as <it>SEPN1</it>-related-myopathies. The mechanisms behind these pathologies are poorly understood since the function of SelN remains elusive. However, previous results obtained in humans and more recently in zebrafish pointed to a potential role for SelN during embryogenesis. Using qRT-PCR, Western blot and whole mount <it>in situ </it>hybridization, we characterized in detail the spatio-temporal expression pattern of the murine <it>Sepn1 </it>gene during development, focusing particularly on skeletal muscles.</p> <p>Results</p> <p>In whole embryos, <it>Sepn1 </it>transcripts were detected as early as E5.5, with expression levels peaking at E12.5, and then strongly decreasing until birth. In isolated tissues, only mild transcriptional variations were observed during development, whereas a striking reduction of the protein expression was detected during the perinatal period. Furthermore, we demonstrated that <it>Sepn1 </it>is expressed early in somites and restricted to the myotome, the sub-ectodermal mesenchyme and the dorsal root ganglia at mid-gestation stages. Interestingly, <it>Sepn1 </it>deficiency did not alter somitogenesis in embryos, suggesting that SelN is dispensable for these processes in mouse.</p> <p>Conclusion</p> <p>We characterized for the first time the expression pattern of <it>Sepn1 </it>during mammalian embryogenesis and we demonstrated that its differential expression is most likely dependent on major post-transcriptional regulations. Overall, our data strongly suggest a potential role for selenoprotein N from mid-gestation stages to the perinatal period. Interestingly, its specific expression pattern could be related to the current hypothesis that selenoprotein N may regulate the activity of the ryanodine receptors.</p

A short motif in Drosophila SECIS Binding Protein 2 provides differential binding affinity to SECIS RNA hairpins

Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA Sec codon. Recoding UGA Sec requires a complex mechanism, comprising the cis-acting SECIS RNA hairpin in the 3′UTR of selenoprotein mRNAs, and trans-acting factors. Among these, the SECIS Binding Protein 2 (SBP2) is central to the mechanism. SBP2 has been so far functionally characterized only in rats and humans. In this work, we report the characterization of the Drosophila melanogaster SBP2 (dSBP2). Despite its shorter length, it retained the same selenoprotein synthesis-promoting capabilities as the mammalian counterpart. However, a major difference resides in the SECIS recognition pattern: while human SBP2 (hSBP2) binds the distinct form 1 and 2 SECIS RNAs with similar affinities, dSBP2 exhibits high affinity toward form 2 only. In addition, we report the identification of a K (lysine)-rich domain in all SBP2s, essential for SECIS and 60S ribosomal subunit binding, differing from the well-characterized L7Ae RNA-binding domain. Swapping only five amino acids between dSBP2 and hSBP2 in the K-rich domain conferred reversed SECIS-binding properties to the proteins, thus unveiling an important sequence for form 1 binding

Genome-wide evidence for an essential role of the human Staf/ZNF143 transcription factor in bidirectional transcription

In the human genome, ∼10% of the genes are arranged head to head so that their transcription start sites reside within <1 kbp on opposite strands. In this configuration, a bidirectional promoter generally drives expression of the two genes. How bidirectional expression is performed from these particular promoters constitutes a puzzling question. Here, by a combination of in silico and biochemical approaches, we demonstrate that hStaf/ZNF143 is involved in controlling expression from a subset of divergent gene pairs. The binding sites for hStaf/ZNF143 (SBS) are overrepresented in bidirectional versus unidirectional promoters. Chromatin immunoprecipitation assays with a significant set of bidirectional promoters containing putative SBS revealed that 93% of them are associated with hStaf/ZNF143. Expression of dual reporter genes directed by bidirectional promoters are dependent on the SBS integrity and requires hStaf/ZNF143. Furthermore, in some cases, functional SBS are located in bidirectional promoters of gene pairs encoding a noncoding RNA and a protein gene. Remarkably, hStaf/ZNF143 per se exhibits an inherently bidirectional transcription activity, and together our data provide the demonstration that hStaf/ZNF143 is indeed a transcription factor controlling the expression of divergent protein–protein and protein–non-coding RNA gene pairs

The SBP2 protein central to selenoprotein synthesis contacts the human ribosome at expansion segment 7L of the 28S rRNA.

SBP2 is a pivotal protein component in selenoprotein synthesis. It binds the SECIS stem-loop in the 3' UTR of selenoprotein mRNA and interacts with both the specialized translation elongation factor and the ribosome at the 60S subunit. In this work, our goal was to identify the binding partners of SBP2 on the ribosome. Cross-linking experiments with bifunctional reagents demonstrated that the SBP2-binding site on the human ribosome is mainly formed by the 28S rRNA. Direct hydroxyl radical probing of the entire 28S rRNA revealed that SBP2 bound to 80S ribosomes or 60S subunits protects helix ES7L-E in expansion segment 7 of the 28S rRNA. Diepoxybutane cross-linking confirmed the interaction of SBP2 with helix ES7L-E. Additionally, binding of SBP2 to the ribosome led to increased reactivity toward chemical probes of a few bases in ES7L-E and in the universally conserved helix H89, indicative of conformational changes in the 28S rRNA in response to SBP2 binding. This study revealed for the first time that SBP2 makes direct contacts with a discrete region of the human 28S rRNA

A novel insight into the mechanism of mammalian selenoprotein synthesis

IThe amino acid selenocysteine is encoded by UGA, usually a stop codon, thus requiring a specialized machinery to enable its incorporation into selenoproteins. The machinery comprises the tRNASec, a 3′-UTR mRNA stemloop termed SElenoCysteine Insertion Sequence (SECIS), which is mandatory for recoding UGA as a Sec codon, the SECIS Binding Protein 2 (SBP2), and other proteins. Little is known about the molecular mechanism and, in particular, when, where, and how the SECIS and SBP2 contact the ribosome. Previous work by others used the isolated SECIS RNA to address this question. Here, we developed a novel approach using instead engineered minimal selenoprotein mRNAs containing SECIS elements derivatized with photoreactive groups. By cross-linking experiments in rabbit reticulocyte lysate, new information could be gained about the SBP2 and SECIS contacts with components of the translation machinery at various translation steps. In particular, we found that SBP2 was bound only to the SECIS in 48S pre-initiation and 80S pretranslocation complexes. In the complex where the Sec-tRNASec was accommodated to the A site but transpeptidation was blocked, SBP2 bound the ribosome and possibly the SECIS element as well, and the SECIS had flexible contacts with the 60S ribosomal subunit involving several ribosomal proteins. Altogether, our findings led to broadening our understanding about the unique mechanism of selenocysteine incorporation in mammals

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

Selenoprotein gene nomenclature

The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4 and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine-R-sulfoxide reductase 1) and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15 kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV) and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates