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.
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.
Li
Biao
B
Center for Human Molecular Genetics, Munroe-Meyer Institute, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Li
Xiaorong
X
Bai
Ying
Y
Hou
Jing-Jen
JJ
Ma
Mark
M
Machida
Curtis A
CA
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002457:Cell Extracts; D021901:DNA, Intergenic; D006367:HeLa Cells; D006801:Humans; D016093:Mason-Pfizer monkey virus / Q000235:genetics* / Q000502:physiology*; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D029543:Nucleocytoplasmic Transport Proteins / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism*; D000080882:Ribosomal Protein L10; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D002135:Calcium-Binding Proteins; D002457:Cell Extracts; D021901:DNA, Intergenic; D008562:Membrane Glycoproteins; D029543:Nucleocytoplasmic Transport Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C000630686:RPL10 protein, human; D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D012269:Ribosomal Proteins; C066973:signal sequence receptor