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Biomedical subjects

D M Driscoll

Publications and source records attributed to D M Driscoll.

At least 19 recordsLinked to original sources
PubMed2001

RNA binding proteins and selenocysteine.

Selenocysteine is incorporated into protein by a complex co-translational mechanism that involves both cis and trans acting factors. Among the trans-acting factors are RNA binding proteins that interact with the selenoprotein 3' UTRs at a sequence known as the selenocysteine insertion sequence (SECIS). These factors are generally referred to as SBPs, and in this review we will discuss the history of the SBPs, and give a detailed description of the recently identified SBP2 which is the only SBP known to be required for Sec insertion. The mechanism by which SBP2 may be involved in this process will be discussed.

3' Untranslated Regions↗
PubMed2001

Selenocysteine incorporation directed from the 3'UTR: characterization of eukaryotic EFsec and mechanistic implications.

The mechanism of selenocysteine incorporation in eukaryotes has been assumed for almost a decade to be inherently different from that in prokaryotes, due to differences in the architecture of selenoprotein mRNAs in the two kingdoms. After extensive efforts in a number of laboratories spanning the same time frame, some of the essential differences between these mechanisms are finally being revealed, through identification of the factors catalyzing cotranslational selenocysteine insertion in eukaryotes. A single factor in prokaryotes recognizes both the selenoprotein mRNA, via sequences in the coding region, and the unique selenocysteyl-tRNA, via both its secondary structure and amino acid. The corresponding functions in eukaryotes are conferred by two distinct but interacting factors, one recognizing the mRNA, via structures in the 3' untranslated region, and the second recognizing the tRNA. Now, with these factors in hand, crucial questions about the mechanistic details and efficiency of this intriguing process can begin to be addressed.

3' Untranslated Regions↗
PubMed2001

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

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.

3' Untranslated Regions↗
PubMed2001

The selenocysteine incorporation machinery: interactions between the SECIS RNA and the SECIS-binding protein SBP2.

The decoding of UGA as a selenocysteine (Sec) codon in mammalian selenoprotein mRNAs requires a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region. The SECIS is a hairpin structure that contains a non-Watson-Crick base-pair quartet with a conserved G.A/A.G tandem in the core of the upper helix. Another essential component of the Sec insertion machinery is SECIS-binding protein 2 (SBP2). In this study, we define the binding site of SBP2 on six different SECIS RNAs using enzymatic and hydroxyl radical footprinting, gel mobility shift analysis, and phosphate-ethylation binding interference. We show that SBP2 binds to a variety of mammalian SECIS elements with similar affinity and that the SBP2 binding site is conserved across species. Based on footprinting studies, SBP2 protects the proximal part of the hairpin and both strands of the lower half of the upper helix that contains the non-Watson-Crick base pair quartet. Gel mobility shift assays showed that the G.A/A.G tandem and internal loop are critical for the binding of SBP2. Modification of phosphates by ethylnitrosourea along both strands of the non-Watson-Crick base pair quartet, on the 5' strand of the lower helix and part of the 5' strand of the internal loop, prevented binding of SBP2. We propose a model in which SBP2 covers the central part of the SECIS RNA, binding to the non-Watson-Crick base pair quartet and to the 5' strands of the lower helix and internal loop. Our results suggest that the affinity of SBP2 for different SECIS elements is not responsible for the hierarchy of selenoprotein expression that is observed in vivo.

Animals↗
PubMed2000

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 (75)Se-labeled Sec-tRNA(Sec), 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.

Amino Acid Sequence↗
PubMed2000

Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.

The translation of mammalian selenoprotein mRNAs requires the 3' untranslated region that contains a selenocysteine insertion sequence (SECIS) element necessary for decoding an in-frame UGA codon as selenocysteine (Sec). Selenoprotein biosynthesis is inefficient, which may be due to competition between Sec insertion and termination at the UGA/Sec codon. We analyzed the polysome distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, a member of the glutathione peroxidase family of selenoproteins, in rat hepatoma cell and mouse liver extracts. In linear sucrose gradients, the sedimentation velocity of PHGPx mRNA was impeded compared to CuZn superoxide dismutase (SOD) mRNA, which has a coding region of similar size. Selenium supplementation increased the loading of ribosomes onto PHGPx mRNA, but not CuZn SOD mRNA. To determine whether the slow sedimentation velocity of PHGPx mRNA is due to a block in elongation, we analyzed the polysome distribution of wild-type and mutant mRNAs translated in vitro. Mutation of the UGA/Sec codon to UGU/cysteine increased ribosome loading and protein synthesis. When UGA/Sec was replaced with UAA or when the SECIS element core was deleted, the distribution of the mutant mRNAs was similar to the wild-type mRNA. Addition of SECIS-binding protein SBP2, which is essential for Sec insertion, increased ribosome loading and translation of wild-type PHGPx mRNA, but had no effect on the mutant mRNAs. These results suggest that elongation is impeded at UGA/Sec, and that selenium and SBP2 alleviate this block by promoting Sec incorporation instead of termination.

3' Untranslated Regions↗
PubMed2000

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.

3' Untranslated Regions↗
PubMed2000

Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA.

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.

APOBEC-1 Deaminase↗
PubMed1999

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 approximately 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.

Animals↗
PubMed1999

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 approximately 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.

Amino Acid Sequence↗
PubMed1998

A sequence-specific RNA-binding protein complements apobec-1 To edit apolipoprotein B mRNA.

The editing of apolipoprotein B (apo-B) mRNA involves the site-specific deamination of cytidine to uracil. The specificity of editing is conferred by an 11-nucleotide mooring sequence located downstream from the editing site. Apobec-1, the catalytic subunit of the editing enzyme, requires additional proteins to edit apo-B mRNA in vitro, but the function of these additional factors, known as complementing activity, is not known. Using RNA affinity chromatography, we show that the complementing activity binds to a 280-nucleotide apo-B RNA in the absence of apobec-1. The activity did not bind to the antisense strand or to an RNA with three mutations in the mooring sequence. The eluate from the wild-type RNA column contained a 65-kDa protein that UV cross-linked to apo-B mRNA but not to the triple-mutant RNA. This protein was not detected in the eluates from the mutant or the antisense RNA columns. Introduction of the mooring sequence into luciferase RNA induced cross-linking of the 65-kDa protein. A 65-kDa protein that interacted with apobec-1 was also detected by far-Western analysis in the eluate from the wild-type RNA column but not from the mutant RNA column. For purification, proteins were precleared on the mutant RNA column prior to chromatography on the wild-type RNA column. Silver staining of the affinity-purified fraction detected a single prominent protein of 65 kDa. Our results suggest that the complementing activity may function as the RNA-binding subunit of the holoenzyme.

APOBEC-1 Deaminase↗
PubMed1997

Quantification of lumbar intradiscal deformation during flexion and extension, by mathematical analysis of magnetic resonance imaging pixel intensity profiles.

STUDY DESIGN: A magnetic resonance imaging study of the internal kinematic response of normal lumbar intervertebral discs to non-weight-bearing flexion and extension. OBJECTIVES: To quantify the pattern of magnetic resonance imaging pixel intensity variation across discs, and noninvasively monitor displacement of the nucleus pulposus during sagittal-plane movements. SUMMARY OF BACKGROUND DATA: Invasive techniques used to study intradiscal movements of the nucleus pulposus have suggested that it moves posteriorly during flexion and anteriorly during extension. A noninvasive study based on magnetic resonance images gave similar results for normal young women. Quantification has been problematic, and the invasive procedures may have altered disc dynamics. METHODS: Ten male subjects (age, 21-38 years) with healthy backs were positioned in a magnetic resonance imaging portal with their lumbar spine stabilized in flexion and extension by supporting pads. For each disc, a T2-weighted image was obtained, as was a computer-generated profile of pixel intensities along a horizontal mid-discal transect. Mathematical curve-fitting regression analysis was used to characterize the shape of the intensity profile and to compute the point of maximum pixel intensity. RESULTS: A single equation fitted the profile for all normal discs. The intensity peak shifted posteriorly during flexion, anteriorly during extension. CONCLUSIONS: Automated mathematical modeling of magnetic resonance imaging pixel data can be used to describe the fundamental shape of the pixel intensity profile across a normal lumbar disc, to determine the precise location of the site of maximum pixel intensity, and to measure the movement of this peak with flexion and extension. This technique may be of value in recognizing incipient degenerative changes in lumbar discs.

Adult↗
PubMed1997

An RNA-binding protein recognizes a mammalian selenocysteine insertion sequence element required for cotranslational incorporation of selenocysteine.

In mammalian selenoprotein mRNAs, the recognition of UGA as selenocysteine requires selenocysteine insertion sequence (SECIS) elements that are contained in a stable stem-loop structure in the 3' untranslated region (UTR). In this study, we investigated the SECIS elements and cellular proteins required for selenocysteine insertion in rat phospholipid hydroperoxide glutathione peroxidase (PhGPx). We developed a translational readthrough assay for selenoprotein biosynthesis by using the gene for luciferase as a reporter. Insertion of a UGA or UAA codon into the coding region of luciferase abolished luciferase activity. However, activity was restored to the UGA mutant, but not to the UAA mutant, upon insertion of the PhGPx 3' UTR. The 3' UTR of rat glutathione peroxidase (GPx) also allowed translational readthrough, whereas the PhGPx and GPx antisense 3' UTRs did not. Deletion of two conserved SECIS elements in the PhGPx 3' UTR (AUGA in the 5' stem or AAAAC in the terminal loop) abolished readthrough activity. UV cross-linking studies identified a 120-kDa protein in rat testis that binds specifically to the sense strands of the PhGPx and GPx 3' UTRs. Direct cross-linking and competition experiments with deletion mutant RNAs demonstrated that binding of the 120-kDa protein requires the AUGA SECIS element but not AAAAC. Point mutations in the AUGA motif that abolished protein binding also prevented readthrough of the UGA codon. Our results suggest that the 120-kDa protein is a significant component of the mechanism of selenocysteine incorporation in mammalian cells.

Animals↗
PubMed1996

Apobec-1 interacts with a 65-kDa complementing protein to edit apolipoprotein-B mRNA in vitro.

The editing of apolipoprotein-B (apoB) mRNA involves the deamination of cytidine at nucleotide 6666 to uridine. The catalytic subunit of the editing enzyme, apobec-1, is a cytidine deaminase that requires other unidentified proteins to edit apoB mRNA in vitro. We partially purified an activity from baboon kidney that functionally complements apobec-1. The complementing activity was protease-sensitive and micrococcal nuclease-resistant, had a native molecular mass of 65 +/- 10 kDa on size exclusion chromatography, and sedimented at 4.5 S in glycerol gradients. Purified recombinant His6-tagged apobec-1 immobilized on beads depleted >90% of the complementing activity from partially purified extracts. These beads edited apoB mRNA in vitro in the absence of exogenous apobec-1 or complementing activity. A functional holoenzyme containing apobec-1 and the complementing activity was eluted from the apobec-1-affinity resin using 0.5 M imidazole, whereas buffer containing 0.4 M KCl eluted only the complementing activity. The carboxyl-terminal 59 amino acids of apobec-1 were not required for interaction with the complementing activity in vitro. Our results demonstrate that the complementing protein interacts directly with apobec-1 in the absence of apoB mRNA.

APOBEC-1 Deaminase↗
PubMed1996

Hyperediting of multiple cytidines of apolipoprotein B mRNA by APOBEC-1 requires auxiliary protein(s) but not a mooring sequence motif.

An RNA-binding cytidine deaminase (APOBEC-1) and unidentified auxiliary protein(s) are required for apolipoprotein (apo) B mRNA editing. A sequence motif on apoB mRNA ("mooring sequence," nucleotides 6671-6681) is obligatory for the editing of cytidine 6666 (C6666), the only cytidine on apoB mRNA converted to uridine in normal animals. Transgenic animals with hepatic overexpression of APOBEC-1 develop liver tumors, and other non-apoB mRNAs are edited, suggesting a loss of the normally precise specificity. In this study, we examined apoB mRNA from these transgenic animals to determine if cytidines aside from C6666 are edited. Multiple cytidines downstream from C6666 in apoB mRNA were edited extensively by the overexpressed APOBEC-1. This pathophysiological "hyperediting" could be mimicked in vitro by incubating a synthetic apoB RNA substrate with the transgenic mouse liver extracts. Multiple cytidines in the synthetic apoB RNA were edited by recombinant APOBEC-1 but only with supplementation of the auxiliary protein(s). Mutations in the mooring sequence markedly decreased the normal editing of C6666 but, surprisingly, increased the hyperediting of downstream cytidines. Furthermore, cytidines in an apoB RNA substrate lacking the mooring sequence were also edited in vitro. These results indicate that the hyperediting of apoB mRNA by overexpressed APOBEC-1 depends upon auxiliary protein(s) but is independent of the mooring sequence motif. These results suggest that hyperediting may represent the first step in a two-step recognition model for normal apoB mRNA editing.

APOBEC-1 Deaminase↗
PubMed1996

Insulin inhibits changes in the phospholipid profiles in sciatic nerves from streptozocin-induced diabetic rats: a phosphorus-31 magnetic resonance study.

Sciatic nerve phospholipids obtained from insulin-treated streptozocin-induced diabetic, non-treated streptozocin-induced diabetic, and healthy, control male Sprague-Dawley rats after eighteen weeks of diabetes were studied by 31P NMR spectrometry. Eleven phospholipids resonances were identified as follows: Phosphatidic acid (Chemical shift, 0.30 ppm), dihydrosphingomyelin (0.13 ppm), ethanolamine plasmalogen (0.07 ppm), phosphatidylethanolamine (0.03 ppm), phosphatidylserine (-0.05 ppm), sphingomyelin (-0.09 ppm), lysophosphatidylcholine (-0.28 ppm), phosphatidylinositol (-0.30 ppm), alkylacylglycerophosphorylcholine (-0.78 ppm), choline plasmalogen (-0.80 ppm), and phosphatidylcholine (-0.84 ppm). Diabetic rats showed that phosphatidylcholine was significantly elevated (p < 0.05), and ethanolamine plasmalogen and choline plasmalogen were significantly lower when compared with both control and insulin treated rats. The choline ratio (choline-containing phospholipids over noncholine phospholipids) was significantly elevated in the diabetic group, when compared with both control and insulin-treated groups. The ethanolamine ratio (ethanolamine-containing phospholipids over nonethanolamine phospholipids) and the ratio of the ethanolamine ratio over the choline ratio, was significantly elevated in the control and the insulin-treated groups when compared with the diabetic group. The presence of phosphatidic acid and the significance in phosphatidylcholine and ethanolamine plasmalogen, suggested that insulin had a role in the phosphatidylcholine metabolism in the rat nerve.

Animals↗
PubMed1995

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 approximately 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 AUG61. 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 AUG61. However, translation initiated at AUG141 when AUG61 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 AUG61 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.

Amino Acid Sequence↗