Single-stranded nucleic acid-binding protein, Purα, interacts with RNA homologous to 18S ribosomal RNA and inhibits translation in vitro

✍ Scribed by Gary L. Gallia; Nune Darbinian; Neil Jaffe; Kamel Khalili

• Publisher: John Wiley and Sons
• Year: 2001
• Tongue: English
• Weight: 233 KB
• Volume: 83
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/jcb.1247

✦ Synopsis

Abstract

Purα 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. Purα 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 Purα‐associated RNA (PARNA) and that RNA immunopurified from mammalian expressed Purα was capable of reconstituting the interaction between these two proteins. In the current study, we characterize four RNA species which were immunopurified with Purα. Northern blot analysis with one of the PARNAs revealed a highly abundant signal of ∼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 Purα 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‐Purα. Inclusion of GST‐Purα 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‐Purα. In control experiments, inclusion of increasing concentrations of GST‐Purα with luciferase protein resulted in no effect on luciferase activity. Taken together, these data demonstrate that Purα inhibits translation reactions in vitro. Moreover, this Purα‐mediated inhibition of translation can be abrogated by HIV‐1 Tat protein. J. Cell. Biochem. 83: 355–363, 2001. © 2001 Wiley‐Liss, Inc.