Progress 08/01/01 to 07/31/04
Outputs
The specific aims of this grant were: 1) To test whether DNA-binding protein B (DBPB) is required for selenoprotein translation, 2) To investigate the role of nucleolin in the translation of selenoproteins, and 3) to investigate protein-protein interactions in the translation complex. 1. Functional role of DBPB: To test the functional role of DBPB in selenoprotein translation, we have used RNA silencing to suppress DBPB expression in cells transfected with an artificial selenoprotein reporter construct. We developed and acquired from Dharmacon, Inc. (Boulder, CO) four double-stranded siRNA duplexes specifically designed for silencing DBPB expression. To measure the effect of the siRNA on selenoprotein expression, we cotransfected the duplexes with a gene fusion construct consisting of a LacZ cDNA; a firefly luciferase cDNA; in between, a DNA segment containing a TGA selenocysteine codon; and finally a selenocysteine insertion sequence (SECIS) in the 3'-untranslated
region. Translation of this reporter fusion gene as a selenoprotein has been demonstrated to be dependent on the function of the SECIS-recognition complex. We found that three of the four siRNA duplexes consistently diminished SECIS-dependent luciferase activity 50-70% in transfected cells, compared to negative controls with irrelevant or no siRNA transfected. For an independent approach, we obtained two mouse embryonic stem cell lines in which the DBPB gene is disrupted by a gene targeting vector. These lines have been injected into blastocytes and we have just obtained the resultant litters of chimeras, including 28 mice that are 50-95% chimeric for the transgenic strain. We are now back-crossing these chimeras to C57Black6 mice in order obtain knock-out heterozygotes. In view of the high degree of chimerism observed, we expect to find germline transmission with each construct. 2. Functional role of nucleolin: We have chosen to concentrate on DBPB and have not pursued this aim. 3.
Protein-protein interactions: By immunoprecipitation after reversible formalin cross-linking of RNA binding proteins, we have demonstrated close association of nucleolin and DBPB with mRNAs encoding the selenoproteins cytosolic glutathione peroxidase and phospholipid glutathione peroxidase in both human and rat cell lines. We are now examining interactions of DBPB with nucleolin, SeC-p43 (a SeC-tRNA-binding protein), SECIS-binding protein 2, and selB (an elongation factor specific for selenoprotein translation). Co-immunoprecipitation studies have already demonstrated association of DBPB with nucleolin and SeC-p43. 4. Other studies: We have also initiated a new, in vivo, aim: to examine the molecular mechanisms of the anti-carcinogenic effects of selenium. We have completed the first phase of an IACUC-approved study comparing the rates of gastric cancer development and prostate tissue oxidant damage in gastrin-overexpressing transgenic mice infected with Helicobacter and fed diets
deficient, replete, or supplemented with selenium. Tissues are now being scored by a mouse pathologist and samples have been stored for protein and RNA analysis.
Impacts
The analysis of the function and interaction of proteins involved in the biosynthesis of selenium-containing proteins have provided fundamental insight into the processes governing incorporation of the micronutrient into functional effector molecules. The development of in vivo models of carcinogenesis should provide a means to investigate the mechanisms of the element's anti-carcinogenic effects.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
Our specific aims remain the same as in the original application: 1) To test whether DNA-binding protein B (dbpB) is required for selenoprotein translation, 2) To investigate the role of nucleolin in the translation of selenoproteins, and 3) to investigate protein-protein interactions in the translation complex. To test the functional role of dbpB in selenoprotein translation, we have developed two approaches: DBPB gene knockout and RNA silencing. In collaboration with the UMass core, we have constructed a knockout vector and transfected murine embryonic stem cells, which we are now screening for gene disruption. As an alternative approach, we have also designed and synthesized four small interfering RNAs and are testing them in human and rat cells both as stabilized synthetic RNAs and as a construct using the pSilencer plasmid (Ambion). For both approaches, we have developed a readout system utilizing a reporter gene we have constructed by fusion of enhanced green
fluorescent protein to cytosolic glutathione peroxidase to create a selenium-dependent fluorescent product easily detectable by microscopy or flow cytometry. By immunoprecipitation after reversible formalin cross-linking of RNA binding proteins, we have demonstrated close association of nucleolin and dbpB with mRNAs encoding the selenoproteins cytosolic glutathione peroxidase and phospholipid glutathione peroxidase in both human (Hela) and rat (McArdle7777) cell lysates. We have also developed systems to examine the interaction of dbpB with nucleolin, SeC-p43 (a selenocysteinyl-tRNA binding protein), SBP2 (another known selenocysteine insertion sequence-binding protein) and selB (an elongation factor specific for selenoprotein translation). Co-immunoprecipitation demonstrated association of dbpB with nucleolin and SeC-p43. For more quantitative and structural studies, we have developed reagents to apply the yeast two-hybrid system to investigate protein-protein interaction. We have
inserted the cDNAs encoding each protein into bait or prey vectors, which are now being used to transform yeast cells and measure beta-glactosidase expression as a reporter for the strength of protein-protein interactions. In addition, we have initiated a new, in vivo, aim of the study: to examine the molecular mechanisms of the anti-carcinogenic effects of selenium. We have completed the first phase of an IACUC-approved study comparing the rates of gastric cancer development and prostate tissue oxidant damage in gastrin-overexpressing transgenic mice infected with Helicobacter felis and fed with diets deficient, replete, or supplemented with selenium. Tissues are now being scored by a consultant mouse pathologist and samples have been stored for protein and RNA analysis.
Impacts
The analysis of the function and interaction of proteins involved in the biosynthesis of selenium-containing proteins should provide fundamental insight into the processes governing incorporation of the micronutrient into functional effector molecules. The development of in vivo models of carcinogenesis will provide a means to investigate the mechanisms of the elements anti-carcinogenic effects.
Publications
- No publications reported this period
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