TY - JOUR
T1 - Simian retrovirus serogroup 2 constitutive transport element recognizes the ribosomal L10-like protein and translocon gamma subunit-like protein in a yeast three-hybrid assay
AU - Li, Biao
AU - Li, Xiaorong
AU - Bai, Ying
AU - Hou, Jing Wen
AU - Ma, Mark
AU - Machida, Curtis A.
N1 - Funding Information:
The authors thank Xiaorong Li for his critical reading of the manuscript and for helpful suggestions advice, and assistance in the screening of the Hela cell cDNA activation domain library, in the analyses of the interactive clones, and for the provision and description of the β-galactosidase filter screening assay. We thank Drs. Robert R. Traut and Olga Griaznova (University of California, Davis) for providing monoclonal antibodies recognizing Escherichia coli L10 protein and Dr. Toshio Uchiumi (Shinshu University) for providing anti-P monoclonal antibodies that react with P0 (corresponding to eukaryotic L10-like protein). We also thank Yibing Jia for his assistance in the synthesis of oligonucleotide primers and for automated sequencing support, provided as a service from the ONPRC Molecular and Cell Biology Core Facility, and also for sequencing support from the University of Nebraska Medical College. We thank Matthew Palmer and Susan Nguyen for technical assistance during early phases of this research project, and C. Elisabeth Campbell for technical assistance in latter phases of this research project. We also thank current and former laboratory members, Philbert Kirigiti, Huafei Lu, and C. Elisabeth Campbell for technical advice and support. Special thanks are extended to Drs. Tom Shearer and Sharon Turner for their overall support of the research program. LB was a 1997 Leukemia Research Foundation Postdoctoral Fellow and was an Assistant Professor at the University of Nebraska Medical College. This research has been supported by grants awarded to CAM from NIH DK53462 and the Collins Medical Trust, by core grant projects awarded to CAM from NCRR 00163, and by the Dean’s Award from the Oregon Health & Science University School of Dentistry. CAM is currently supported by NIH MH63137 and AHA 0250017N, and was a former recipient of an American Heart Association Established Investigatorship.
PY - 2004/1
Y1 - 2004/1
N2 - The simian retrovirus (SRV) serogroup 2 genome contains a constitutive transport element (CTE) within its 3′ intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. In a previous report [Virology 264 (1999) 37], CTE RNA-protein complexes were detected using UV-crosslinking/sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To identify these CTE-interacting cellular proteins, we utilized yeast three-hybrid interaction approaches using the complete IR as bait, modified to eliminate transcriptional termination signals recognized by RNA polymerase III, and identified several interactive clones from a Hela cell cDNA activation domain (AD) library. UV-crosslinking of RNA-protein complexes, using Hela cell extracts and the modified IR bait, were conducted prior to library screening, to verify appropriate interaction of CTE RNA-protein complexes. Over one million recombinants were screened, and our yeast hybrid results indicate that the CTE interacts with several molecules involved in cellular translational and translocation machinery, including the ribosomal L10-like protein and the tranlocon protein gamma subunit-like protein. UV-crosslinking/immunoblot assays have verified the interaction of the CTE region with molecules immunologically reactive to antibodies recognizing the ribosomal L10-like protein.
AB - The simian retrovirus (SRV) serogroup 2 genome contains a constitutive transport element (CTE) within its 3′ intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. In a previous report [Virology 264 (1999) 37], CTE RNA-protein complexes were detected using UV-crosslinking/sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To identify these CTE-interacting cellular proteins, we utilized yeast three-hybrid interaction approaches using the complete IR as bait, modified to eliminate transcriptional termination signals recognized by RNA polymerase III, and identified several interactive clones from a Hela cell cDNA activation domain (AD) library. UV-crosslinking of RNA-protein complexes, using Hela cell extracts and the modified IR bait, were conducted prior to library screening, to verify appropriate interaction of CTE RNA-protein complexes. Over one million recombinants were screened, and our yeast hybrid results indicate that the CTE interacts with several molecules involved in cellular translational and translocation machinery, including the ribosomal L10-like protein and the tranlocon protein gamma subunit-like protein. UV-crosslinking/immunoblot assays have verified the interaction of the CTE region with molecules immunologically reactive to antibodies recognizing the ribosomal L10-like protein.
KW - Constitutive transport element
KW - Ribosomal proteins
KW - Secondary structural analyses
KW - Simian retrovirus
KW - Translation
KW - UV-crosslinking/immunoblot experiments
KW - Yeast three-hybrid interaction
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U2 - 10.1016/j.virusres.2003.10.008
DO - 10.1016/j.virusres.2003.10.008
M3 - Article
C2 - 14687949
AN - SCOPUS:0348110312
SN - 0168-1702
VL - 99
SP - 69
EP - 80
JO - Virus Research
JF - Virus Research
IS - 1
ER -