Cellular Protein hnRNP-A1 Interacts with the 3’-end and the Intergenic Sequence of Mouse Hepatitis Virus Negative-Strand RNA to Form a Ribonucleoprotein Complex
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We previously showed that several cellular proteins specifically bind to the 3’-end and the intergenic sequences of the negative-strand RNA of mouse hepatitis virus (MHV), and proposed that these distant RNA sequences can be brought together by cellular and vi-ral proteins (Furuya and Lai, 1993; Zhang et al., 1994; Zhang and Lai, 1995). The cellular protein p35 has been identified as a heterogeneous nuclear ribonucleoprotein (hnRNP) Al. We have now expressed hnRNP-Al as a glutathione-S-transferase (GST) fusion protein and demonstrated that the amino terminal two-thirds of hnRNP-Al interacted with the two MHV regulatory RNA sequences (3’-end and intergenic sequences) through protein-RNA interaction while its carboxy-terminal glycine-rich domain mediated homomeric (protein-protein) interactions. In a partially reconstituted reaction, in which the two MHV RNA fragments and the purified GST-hnRNP-Al fusion protein were mixed, an RNP complex was formed. Depletion of either hnRNP-Al or one of the RNA components abolished the complex formation. These results indicate that hnRNP-Al can mediate the formation of an MHV RNP complex, which includes both the negative-strand leader and intergenic se-quences. Site-directed mutagenesis revealed that mutations in the MHV intergenic se-quences, which inhibiterd MHV RNA transcription, also inhibited the RNP complex forma-tion. Deletion analysis showed that the amino terminal RNA-binding domains of hnRNP-A1 is essential for the RNP complex formation while the carboxy-terminal protein-binding domain enhanced the complex formation by 90-fold. These findings provide direct evidence demonstrating that the negative-strand leader RNA and intergenic sequences can form an RNP complex mediated by cellular protein hnRNP-A1.
KeywordsIntergenic Sequence Heterogeneous Nuclear Ribonucleoprotein Mouse Hepatitis Virus Reconstitution Reaction Vitro Formation
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