Single-stranded nucleic acid-binding protein, Pur alpha, interacts with RNA homologous to 18S ribosomal RNA and inhibits translation in vitro.
Pur alpha is a highly conserved, eukaryotic sequence-specific DNA- and RNA-binding protein involved in diverse cellular and viral functions including transcription, replication, and cell growth. Pur alpha exerts its activity in part by interacting with other viral and cellular proteins. One such protein is the human immunodeficiency virus (HIV) type I regulatory protein Tat. Earlier studies have demonstrated that this interaction is mediated by Pur alpha-associated RNA (PARNA) and that RNA immunopurified from mammalian expressed Pur alpha was capable of reconstituting the interaction between these two proteins. In the current study, we characterize four RNA species which were immunopurified with Pur alpha. Northern blot analysis with one of the PARNAs revealed a highly abundant signal of approximately 2.0 kilobases (kb) present in all cell lines tested. Sequence analysis of each of the four PARNA clones revealed a high homology to different regions of the human 18S ribosomal RNA sequence. Based on this homology, we investigated the influence of Pur alpha on translation. Luciferase assays were performed after coupled in vitro transcription/translation reactions with a vector containing a luciferase reporter construct and increasing concentrations of BSA, GST, and GST-Pur alpha. Inclusion of GST-Pur alpha in these reactions resulted in a dose-dependent inhibition of luciferase activity. Similar inhibition was observed with in vitro translation reactions performed with in vitro transcribed luciferase RNA and increasing concentrations of GST-Pur alpha. In control experiments, inclusion of increasing concentrations of GST-Pur alpha with luciferase protein resulted in no effect on luciferase activity. Taken together, these data demonstrate that Pur alpha inhibits translation reactions in vitro. Moreover, this Pur alpha-mediated inhibition of translation can be abrogated by HIV-1 Tat protein.
Gallia
G L
GL
Center for NeuroVirology and Cancer Biology, Laboratory of Molecular Neurovirology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122, USA.
Darbinian
N
N
Jaffe
N
N
Khalili
K
K
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D015696:Gene Products, tat / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011485:Protein Binding; D014176:Protein Biosynthesis; D012337:RNA, Ribosomal, 18S / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D015696:Gene Products, tat; C501130:PURA protein, human; D012337:RNA, Ribosomal, 18S; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D008156:Luciferases; D005982:Glutathione Transferase