Dataset Information

In vivo identification of ribonucleoprotein-RNA interactions.

ABSTRACT: To understand the role of RNA-binding proteins (RBPs) in the regulation of gene expression, methods are needed for the in vivo identification of RNA-protein interactions. We have developed the peptide nucleic acid (PNA)-assisted identification of RBP technology to enable the identification of proteins that complex with a target RNA in vivo. Specific regions of the 3' and 5' UTRs of ankylosis mRNA were targeted by antisense PNAs transported into cortical neurons by the cell-penetrating peptide transportan 10. An array of proteins was isolated in complex with or near the targeted regions of the ankylosis mRNA through UV-induced crosslinking of the annealed PNA-RNA-RBP complex. The first evidence for pharmacological modulation of these specific protein-RNA associations was observed. These data show that the PNA-assisted identification of the RBP technique is a reliable method to rapidly identify proteins interacting in vivo with the target RNA.

SUBMITTER: Zielinski J

PROVIDER: S-EPMC1345716 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

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In vivo identification of ribonucleoprotein-RNA interactions.

Zielinski Jennifer J Kilk Kalle K Peritz Tiina T Kannanayakal Theresa T Miyashiro Kevin Y KY Eiríksdóttir Emelía E Jochems Jeanine J Langel Ulo U Eberwine James J

Proceedings of the National Academy of Sciences of the United States of America 20060123 5

To understand the role of RNA-binding proteins (RBPs) in the regulation of gene expression, methods are needed for the in vivo identification of RNA-protein interactions. We have developed the peptide nucleic acid (PNA)-assisted identification of RBP technology to enable the identification of proteins that complex with a target RNA in vivo. Specific regions of the 3' and 5' UTRs of ankylosis mRNA were targeted by antisense PNAs transported into cortical neurons by the cell-penetrating peptide tr ...[more]

PMID: 16432185

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Project description:We report an in vitro RNA synthesis assay for the RNA-dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19 nucleotides (nt) of the rabies virus genome, we demonstrate that L alone initiates synthesis on naked RNA and that P serves to enhance the initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11 to 50 of P and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with those of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P protein in which the oligomerization domain (OD) and the CTD were replaced by those of VSV P stimulated RABV RdRP activity on naked RNA but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis, extending our knowledge of ribonucleoprotein interactions that are critical for gene expression. The current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L protein is lacking. This study reports the expression and purification of the full-length L protein of RABV and the characterization of its RdRP activity in vitro The study provides a new assay that has utility for screening inhibitors and understanding their mechanisms of action, as well as defining new interactions that are required for RdRP activity.

Dataset Information

In vivo identification of ribonucleoprotein-RNA interactions.

Publications

In vivo identification of ribonucleoprotein-RNA interactions.

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