Donna M. Driscoll

A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs

In eukaryotes, the decoding of the UGA codon as selenocysteine (Sec) requires a Sec insertion sequence (SECIS) element in the 3′ untranslated region of the mRNA. We purified a SECIS binding protein, SBP2, and obtained a cDNA clone that encodes this activity. SBP2 is a novel protein containing a putative RNA binding domain found in ribosomal proteins and a yeast suppressor of translation termination. By UV cross‐linking and immunoprecipitation, we show that SBP2 specifically binds selenoprotein mRNAs both in vitro and in vivo. Using 75Se‐labeled Sec‐tRNASec, we developed an in vitro system for analyzing Sec incorporation in which the translation of a selenoprotein mRNA was both SBP2 and SECIS element dependent. Immunodepletion of SBP2 from the lysates abolished Sec insertion, which was restored when recombinant SBP2 was added to the reaction. These results establish that SBP2 is essential for the co‐translational insertion of Sec into selenoproteins. We hypothesize that the binding activity of SBP2 may be involved in preventing termination at the UGA/Sec codon.

Decoding apparatus for eukaryotic selenocysteine insertion

Decoding UGA as selenocysteine requires a unique tRNA, a specialized elongation factor, and specific secondary structures in the mRNA, termed SECIS elements. Eukaryotic SECIS elements are found in the 3′ untranslated region of selenoprotein mRNAs while those in prokaryotes occur immediately downstream of UGA. Consequently, a single eukaryotic SECIS element can serve multiple UGA codons, whereas prokaryotic SECIS elements only function for the adjacent UGA, suggesting distinct mechanisms for recoding in the two kingdoms. We have identified and characterized the first eukaryotic selenocysteyl‐tRNA‐specific elongation factor. This factor forms a complex with mammalian SECIS binding protein 2, and these two components function together in selenocysteine incorporation in mammalian cells. Expression of the two functional domains of the bacterial elongation factor–SECIS binding protein as two separate proteins in eukaryotes suggests a mechanism for rapid exchange of charged for uncharged selenocysteyl‐tRNA–elongation factor complex, allowing a single SECIS element to serve multiple UGA codons.

Molecular Cloning of Apobec-1 Complementation Factor, a Novel RNA-Binding Protein Involved in the Editing of Apolipoprotein B mRNA

ABSTRACT The C-to-U editing of apolipoprotein B (apo-B) mRNA is catalyzed by a multiprotein complex that recognizes an 11-nucleotide mooring sequence downstream of the editing site. The catalytic subunit of the editing enzyme, apobec-1, has cytidine deaminase activity but requires additional unidentified proteins to edit apo-B mRNA. We purified a 65-kDa protein that functionally complements apobec-1 and obtained peptide sequence information which was used in molecular cloning experiments. The apobec-1 complementation factor (ACF) cDNA encodes a novel 64.3-kDa protein that contains three nonidentical RNA recognition motifs. ACF and apobec-1 comprise the minimal protein requirements for apo-B mRNA editing in vitro. By UV cross-linking and immunoprecipitation, we show that ACF binds to apo-B mRNA in vitro and in vivo. Cross-linking of ACF is not competed by RNAs with mutations in the mooring sequence. Coimmunoprecipitation experiments identified an ACF-apobec-1 complex in transfected cells. Immunodepletion of ACF from rat liver extracts abolished editing activity. The immunoprecipitated complexes contained a functional holoenzyme. Our results support a model of the editing enzyme in which ACF binds to the mooring sequence in apo-B mRNA and docks apobec-1 to deaminate its target cytidine. The fact that ACF is widely expressed in human tissues that lack apobec-1 and apo-B mRNA suggests that ACF may be involved in other RNA editing or RNA processing events.

Rat phospholipid-hydroperoxide glutathione peroxidase. cDNA cloning and identification of multiple transcription and translation start sites.

Phospholipid-hydroperoxide glutathione peroxidase (PhGPx) is a selenoenzyme that reduces hydroperoxides of phospholipid, cholesterol, and cholesteryl ester. Previous studies suggested that both the mitochondrial and nonmitochondrial forms of PhGPx are 170 amino acids long. In this study, we isolated a full-length cDNA clone encoding rat testis PhGPx. Based on sequence analysis, the cDNA encodes a protein of 197 amino acids, with translation initiating at AUG. The additional 27 amino acids at the N terminus contain the features of a mitochondrial targeting sequence. In vitro translation of the full-length PhGPx mRNA initiated predominantly at AUG. However, translation initiated at AUG when AUG was deleted. An RNase protection assay was used to map the 5′-ends of PhGPx mRNAs in rat tissues. We identified two major windows of transcription initiation that are tissue-specific. Rat testis predominantly expresses larger transcripts that encode the 197-amino acid protein containing the potential mitochondrial targeting signal. The predominant smaller transcripts in somatic tissues lack AUG and encode a 170-amino acid protein, which may represent the nonmitochondrial forms of PhGPx. Our results suggest that the use of alternative transcription and translation start sites determines the subcellular localization of PhGPx in different tissues.

Ribosomal protein L30 is a component of the UGA- selenocysteine recoding machinery in eukaryotes

The translational recoding of UGA as selenocysteine (Sec) is directed by a SECIS element in the 3′ untranslated region (UTR) of eukaryotic selenoprotein mRNAs. The selenocysteine insertion sequence (SECIS) contains two essential tandem sheared G·A pairs that bind SECIS-binding protein 2 (SBP2), which recruits a selenocysteine-specific elongation factor and Sec-tRNASec to the ribosome. Here we show that ribosomal protein L30 is a component of the eukaryotic selenocysteine recoding machinery. L30 binds SECIS elements in vitro and in vivo, stimulates UGA recoding in transfected cells and competes with SBP2 for SECIS binding. Magnesium, known to induce a kink-turn in RNAs that contain two tandem G·A pairs, decreases the SBP2–SECIS complex in favor of the L30-SECIS interaction. We propose a model in which SBP2 and L30 carry out different functions in the UGA recoding mechanism, with the SECIS acting as a molecular switch upon protein binding.

Purification, redox sensitivity, and RNA binding properties of SECIS-binding protein 2, a protein involved in selenoprotein biosynthesis.

In mammalian selenoprotein mRNAs, the highly structured 3′ UTR contains selenocysteine insertion sequence (SECIS) elements that are required for the recognition of UGA as the selenocysteine codon. Our previous work demonstrated a tight correlation between codon-specific translational read-through and the activity of a 120-kDa RNA-binding protein that interacted specifically with the SECIS element in the phospholipid hydroperoxide glutathione peroxidase mRNA. This study reports the RNA binding and biochemical properties of this protein, SECIS-binding protein 2 (SBP2). We detected SBP2 binding activity in liver, hepatoma cell, and testis extracts from which SBP2 has been purified by anion exchange and RNA affinity chromatography. This scheme has allowed us to identify a 120-kDa polypeptide that co-elutes with SBP2 binding activity from wild-type but not mutant RNA affinity columns. A characterization of SBP2 biochemical properties reveals that SBP2 binding is sensitive to oxidation and the presence of heparin, rRNA, and poly(G). SBP2 activity elutes with a molecular mass of ∼500 kDa during gel filtration chromatography, suggesting the existence of a large functional complex. Direct cross-linking and competition experiments demonstrate that the minimal phospholipid hydroperoxide glutathione peroxidase 3′ UTR binding site is between 82 and 102 nucleotides, which correlates with the minimal sequence necessary for translational read-through. SBP2 also interacts specifically with the minimally functional 3′ UTR of another selenoprotein mRNA, deiodinase 1.

An in vitro system for the editing of apolipoprotein B mRNA

A novel form of RNA editing generates two forms of apolipoprotein B (apo-B) mRNA by converting C at nucleotide 6666 to U or a U-like base. We have established an in vitro system for the editing of apo-B mRNA using synthetic RNAs and S100 extracts from rat hepatoma cells. Editing was detected by a sensitive primer extension assay and confirmed by DNA sequencing. The in vitro editing activity is specific and sensitive to proteinase K. Apo-B100 RNAs were synthesized in vitro from deletion mutants spanning nucleotide 6666. Synthetic RNAs containing 2383, 483, and 55 nucleotides of apo-B mRNA sequence were edited in vitro with similar efficiency, but an RNA containing 26 nucleotides was not edited.

Insight into mammalian selenocysteine insertion: Domain structure and ribosome binding properties of Sec insertion sequence binding protein 2

ABSTRACT The cotranslational incorporation of the unusual amino acid selenocysteine (Sec) into both prokaryotic and eukaryotic proteins requires the recoding of a UGA stop codon as one specific for Sec. The recognition of UGA as Sec in mammalian selenoproteins requires a Sec insertion sequence (SECIS) element in the 3′ untranslated region as well as the SECIS binding protein SBP2. Here we report a detailed analysis of SBP2 structure and function using truncation and site-directed mutagenesis. We have localized the RNA binding domain to a conserved region shared with several ribosomal proteins and eukaryotic translation termination release factor 1. We also identified a separate and novel functional domain N-terminal to the RNA binding domain which was required for Sec insertion but not for SECIS binding. Conversely, we showed that the RNA binding domain was necessary but not sufficient for Sec insertion and that the conserved glycine residue within this domain was required for SECIS binding. Using glycerol gradient sedimentation, we found that SBP2 was stably associated with the ribosomal fraction of cell lysates and that this interaction was not dependent on its SECIS binding activity. This interaction also occurred with purified components in vitro, and we present data which suggest that the SBP2-ribosome interaction occurs via 28S rRNA. SBP2 may, therefore, have a distinct function in selecting the ribosomes to be used for Sec insertion.

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.

Molecular cloning and expression of lipid transfer inhibitor protein reveals its identity with apolipoprotein F

Published studies demonstrate that lipid transfer inhibitor protein (LTIP) is an important regulator of cholesteryl ester transfer protein (CETP) activity. Although LTIP inhibits CETP activity among different lipoprotein classes, it preferentially suppresses transfer events involving low density lipoprotein (LDL), whereas transfers involving high density lipoprotein as donor are less affected. In this study, we report the purification of LTIP and the expression of its cDNA in cultured cells. Purification of LTIP, in contrast to other published protocols, took advantage of the tight association of this protein with LDL. Ultracentrifugally isolated LDL was further purified on anti-apoE and apoA-I affinity columns. Affinity purified LDL was delipidated by tetramethylurea, and the tetramethylurea-soluble proteins were separated by SDS-polyacrylamide gel electrophoresis. The protein migrating at a molecular mass of ∼33 kDa was excised from the gel and its N-terminal amino acid sequence determined. The 14-amino acid sequence obtained showed complete homology with the sequence deduced for apolipoprotein F (apoF) cDNA isolated from Hep G2 cells. On Western blots, peptide-specific antibodies raised against synthetic fragments of apoF reacted with the same 33-kDa protein in LTIP-containing fractions purified from LDL and from lipoprotein-deficient plasma. In contrast to that previously reported, apoF was shown to be associated almost exclusively with LDL, identical to the distribution of LTIP activity. The cDNA for apoF was cloned from a human liver cDNA library, ligated into a mammalian expression vector, and transiently transfected into COS-7 cells. Conditioned media containing secreted apoF demonstrated CETP inhibitor activity, whereas cells transfected with vector alone did not. This CETP inhibitor activity was efficiently removed from the media by nickel-Sepharose, consistent with the 6-His tag incorporated into recombinant apoF. By Western blot, the 6-His-tagged protein had a molecular weight slightly larger than native apoF. The CETP inhibitor activity of recombinant apoF possessed the same LDL specificity, oleate sensitivity, and dependence on lipoprotein concentration as previously noted for LTIP. We conclude that LTIP and apoF are identical.