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Reprogramming the Ribosome for Selenoprotein Expression: RNA Elements and Protein Factors

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Reprogramming the Ribosome for Selenoprotein Expression: RNA Elements and Protein Factors Chapter 2 Reprogramming the Ribosome for Selenoprotein Expression: RNA Elements and Protein Factors Marla J. Berry and Michael T. Howard Abstract Many of the benefits of the antioxidant selenium can be attributed to its incorporation into selenoenzymes as the 21st amino acid, selenocysteine. Selenocysteine incorporation occurs cotranslationally at UGA codons in a subset of messages in prokaryotes, eukaryotes, and archaea. UGA codons are recoded to specify selenocysteine, rather than termination, by the presence of specialized cis- and trans-acting factors. Here we discuss the mechanism of selenocysteine inser- tion, the factors which affect efficiency of incorporation, and regulation of mRNA levels. Although much remains to be learned about the multiple factors affecting gene and tissue-specific regulation of the selenoenzymes, significant advances in this regard have been made in understanding the role of selenium status, the expres- sion and selective modification of specific trans-acting factors, and the cis-acting sequences associated with each selenoenzyme message. Contents 2.1 Selenium, Selenocysteine, and Selenoproteins .................. 30 2.2 The Mechanism of Selenocysteine Incorporation in Eukaryotes .......... 30 2.2.1 Identification of Cis-Acting Factors in Eukaryotes ............. 30 2.2.2 Identification of Trans-Acting Factors in Eukaryotes ............ 35 2.3 Efficiency of Selenocysteine Incorporation in Eukaryotes ............. 39 2.4 Hierarchy of Selenoprotein Synthesis ...................... 41 2.5 Other Factors Effecting Differential Selenoprotein Expression .......... 43 2.6 Where do Selenoprotein mRNA Decoding Complexes Assemble? ......... 44 2.7 Elucidating the Functions of Selenoproteins ................... 45 2.8 Summary .................................. 46 References ................................... 47 M.J. Berry (B) Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA e-mail: [email protected] J.F. Atkins, R.F. Gesteland (eds.), Recoding: Expansion of Decoding Rules Enriches 29 Gene Expression, Nucleic Acids and Molecular Biology 24, DOI 10.1007/978-0-387-89382-2_2, C Springer Science+Business Media, LLC 2010 30 M.J. Berry and M.T. Howard 2.1 Selenium, Selenocysteine, and Selenoproteins Selenium has long been known for its antioxidant properties, but it has only in recent years come to light that the beneficial effects of this trace element in our diet are attributable to selenoenzymes. In animals approximately 80% of selenium is cova- lently associated with proteins in the form of the 21st amino acid selenocysteine (Hawkes et al., 1985). This amino acid has a lower pKa than cysteine, producing a highly reactive group at physiological pH which is often responsible for catalyz- ing reduction/oxidation reactions. The known functions of selenoenzymes include protecting cell membranes, proteins, and nucleic acids from cumulative oxidative damage. These functions are carried out by the glutathione peroxidases, enzymes that break down hydroperoxides and lipid peroxides, the thioredoxin reductases, which catalyze regeneration of the essential thiol cofactor, thioredoxin, and other recently identified selenoproteins. Selenoenzymes function in preserving mam- malian sperm integrity and in thyroid hormone homeostasis, highlighting essential roles for the trace element in development and metabolism. Selenium deficiency has been linked to cardiovascular disease in deficient regions of rural China, and cumu- lative oxidative damage has been implicated in the pathogenesis of cancers, diabetes, Alzheimer’s and Parkinson’s diseases. Further, the oxidative stress caused by sele- nium deficiency has been shown in experimental animals to increase susceptibility to infection by influenza and other viruses. 2.2 The Mechanism of Selenocysteine Incorporation in Eukaryotes The mechanism of selenocysteine incorporation in eukaryotes has, for the last ∼15 years, been assumed to be inherently different from that in prokaryotes due to differences in the architecture of selenoprotein mRNAs and in the factors catalyzing selenocysteine biosynthesis and incorporation. After extensive efforts spanning the same time frame, many of the essential differences in these mechanisms are being revealed through identification of the cis- and trans-acting factors catalyzing seleno- cysteine biosynthesis and its cotranslational insertion in eukaryotes. Additional insights into the efficiency of selenoprotein synthesis are being unveiled through studies of the interactions among these factors. 2.2.1 Identification of Cis-Acting Factors in Eukaryotes Selenocysteine incorporation occurs cotranslationally at UGA codons in a subset of messages in prokaryotes, eukaryotes, and archaea. UGA codons are recoded to spec- ify selenocysteine, rather than termination, by the presence of specific secondary structures in selenoprotein mRNAs termed selenocysteine insertion sequences, or SECIS, elements. In prokaryotes, SECIS elements are located in the cod- ing region, immediately downstream of the UGA codons they serve (Fig. 2.1A). 2 Reprogramming the Ribosome for Selenoprotein Expression 31 Fig. 2.1 Models for selenocysteine insertion in bacteria, archaea, and eukaryotes. (A) The bac- terial selenocysteine elongation factor (green) binds the Sec-tRNA and also binds directly to the bacterial SECIS element (red) located adjacent to and downstream of the UGA codon to deliver the Sec-tRNA to the ribosome. (B) Similarly, the archaeal elongation factor binds to the Sec-tRNA and interacts with the 3 UTR SECIS element analogous to the situation in eukaryotes. (C) In eukary- otes the SECIS element binds to SBP2 (orange) which binds to Sec-tRNA-bound EFsec. SBP2 also binds to the ribosome. Consequently it is unclear if the ribosome is loaded with SBP2 and possibly other selenocysteine insertion factors prior to decoding the UGA codon (1) or if the fac- tors assemble during decoding of the UGA codon (2). L30 (magenta) exists bound to the ribosome and in a free form. A structure downstream of the UGA codon (yellow) stimulates selenocysteine insertion by a yet to be determined mechanism. L30 can compete with SBP2 for binding to the SECIS element under conditions which favor the kink-turn conformation at the GA:AG quartet (D). It has been suggested that this may trigger conformational changes which allow delivery of the Sec-tRNA to the A-site by EFsec. Decoding of the UGA codon is required to remove the exon junction complex (EJC) downstream to protect selenoprotein messages from nonsense-mediated decay (see Fig. 2.2) 32 M.J. Berry and M.T. Howard Selenocysteine incorporation occurs via a bifunctional protein, SELB, consisting of a Sec-tRNA[Ser]Sec-specific elongation factor (EF) domain and a SECIS RNA- binding domain (Kromayer et al., 1996). In archaea and eukaryotes (Fig. 2.1B and C, respectively), SECIS elements are typically located in the 3 untranslated region (UTR), but at least one SECIS element has been identified in the 5 UTR in an archaea selenoprotein gene (Wilting et al., 1997). In eukaryotes, SECIS elements have been shown to recode the entire message, functioning for any upstream in- frame UGA (Berry et al., 1993; Hill et al., 1993; Shen et al., 1993), provided a minimal spacing requirement is met (Martin et al., 1996). In addition, information is encoded locally near the UGA codon which influences the efficiency of seleno- cysteine insertion (Grundner-Culemann et al., 2001; Gupta and Copeland, 2007; McCaughan et al., 1995). At least a subset of eukaryotic selenoprotein messages contain a highly conserved RNA secondary structure, referred to as the seleno- cysteine codon redefinition element or SRE, which resides just downstream of the UGA codon and modulates selenocysteine insertion efficiency (Howard et al., 2005, 2007). Eukaryotic SECIS elements: Eukaryotic SECIS elements consist of a stem-loop structure that contains several conserved sequence and structural features. The sequence features initially identified include AUGA and GA at the 5 and 3 bases of the stem, respectively, and a conserved AAR motif in a loop at the top of the stem (Berry et al., 1991, 1993). The sequences at the base of the stem were shown to form a quartet of non-Watson–Crick base pairs, with a central tandem of sheared G.A pairs (Walczak et al., 1996). The stem separating the SECIS core from the con- served adenosines is typically fixed at 9–11 base pairs (Grundner-Culemann et al., 1999). An open loop below the quartet and an additional helix below this were subsequently delineated. As additional selenoprotein sequences were elucidated, compilation revealed variation in the conserved features, including substitution of G for the first A at the 5 base of the upper stem (Buettner et al., 1999), the presence of the AAR motif in an internal bulge rather than an apical loop (Grundner-Culemann et al., 1999), and substitution of C’s for A’s in the AAR motif (Kryukov et al., 2003). Nonetheless, with the variations and subsequent refinements, these features allowed the generation of search programs for SECIS elements such that the entire seleno- proteome of an organism could be predicted from the genome sequence (Kryukov et al., 2003). Delineation of the conserved or semiconserved features also proved essential in identifying cognate binding proteins, as discussed below. The SRE and UGA codon context: Although the distal 3 UTR SECIS element is sufficient for UGA to encode selenocysteine, the efficiency of selenocysteine insertion varies
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