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The 3' untranslated region of the rabies virus glycoprotein mRNA specifically interacts with cellular PCBP2 protein and promotes transcript stability.

Palusa S, Ndaluka C, Bowen RA, Wilusz CJ, Wilusz J - PLoS ONE (2012)

Bottom Line: Viral polymerase entry and pausing at intergenic junctions is predicted to lead to a defined polarity in the levels of rhabdovirus gene expression.PCBP2 specifically interacts with reporter transcripts containing this 72 base 3' UTR sequence.Furthermore, the PCBP2 interaction is directly associated with the stability of reporter transcripts.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.

ABSTRACT
Viral polymerase entry and pausing at intergenic junctions is predicted to lead to a defined polarity in the levels of rhabdovirus gene expression. Interestingly, we observed that the rabies virus glycoprotein mRNA is differentially over-expressed based on this model relative to other transcripts during infection of 293T cells. During infection, the rabies virus glycoprotein mRNA also selectively interacts with the cellular poly(rC)-binding protein 2 (PCBP2), a factor known to influence mRNA stability. Reporter assays performed both in electroporated cells and in a cell-free RNA decay system indicate that the conserved portion of the 3' UTR of the rabies virus glycoprotein mRNA contains an RNA stability element. PCBP2 specifically interacts with reporter transcripts containing this 72 base 3' UTR sequence. Furthermore, the PCBP2 interaction is directly associated with the stability of reporter transcripts. Therefore, we conclude that PCBP2 specifically and selectively interacts with the rabies virus glycoprotein mRNA and that this interaction may contribute to the post-transcriptional regulation of glycoprotein expression.

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The cellular PCBP2 protein selectively interacts with the G mRNA during rabies virus infection.Rabies virus infected 293T cells were treated with formaldehyde to stabilize RNA-protein complexes and cell lysates were immunoprecipitated using either control IgG or a PCBP2-specific antisera. Co-precipitating rabies viral mRNAs were analyzed by RT-PCR after reversal of the formaldehyde cross links and products run on a 1% agarose gel and visualized with ethidium bromide. The 10% input lane represents total RNA present in the lysate prior to immunoprecipitation. The numbers below the lanes represent quantification and standard deviation from three independent experiments.
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pone-0033561-g001: The cellular PCBP2 protein selectively interacts with the G mRNA during rabies virus infection.Rabies virus infected 293T cells were treated with formaldehyde to stabilize RNA-protein complexes and cell lysates were immunoprecipitated using either control IgG or a PCBP2-specific antisera. Co-precipitating rabies viral mRNAs were analyzed by RT-PCR after reversal of the formaldehyde cross links and products run on a 1% agarose gel and visualized with ethidium bromide. The 10% input lane represents total RNA present in the lysate prior to immunoprecipitation. The numbers below the lanes represent quantification and standard deviation from three independent experiments.

Mentions: The interaction of individual rabies virus mRNAs with select cellular RNA binding proteins was pursued using a co-immunoprecipitation approach. Human embryonic kidney (293T) cells were infected with rabies virus and RNA-protein complexes were stabilized using formaldehyde prior to cell lysis. Protein-RNA complexes were immunoprecipitated under stringent conditions and associated RNAs were detected by PCR. While most of the RNA binding proteins tested gave negative or inconclusive results, we made an interesting observation with the cellular protein PCBP2. As seen in Figure 1, the rabies virus G mRNA was selectively associated with PCBP2. All four of the other rabies virus mRNAs did not co-immunoprecipitate with PCBP2 significantly above background. Therefore we conclude that the rabies virus G mRNA selectively associates with the cellular PCBP2 protein during infection.


The 3' untranslated region of the rabies virus glycoprotein mRNA specifically interacts with cellular PCBP2 protein and promotes transcript stability.

Palusa S, Ndaluka C, Bowen RA, Wilusz CJ, Wilusz J - PLoS ONE (2012)

The cellular PCBP2 protein selectively interacts with the G mRNA during rabies virus infection.Rabies virus infected 293T cells were treated with formaldehyde to stabilize RNA-protein complexes and cell lysates were immunoprecipitated using either control IgG or a PCBP2-specific antisera. Co-precipitating rabies viral mRNAs were analyzed by RT-PCR after reversal of the formaldehyde cross links and products run on a 1% agarose gel and visualized with ethidium bromide. The 10% input lane represents total RNA present in the lysate prior to immunoprecipitation. The numbers below the lanes represent quantification and standard deviation from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3306424&req=5

pone-0033561-g001: The cellular PCBP2 protein selectively interacts with the G mRNA during rabies virus infection.Rabies virus infected 293T cells were treated with formaldehyde to stabilize RNA-protein complexes and cell lysates were immunoprecipitated using either control IgG or a PCBP2-specific antisera. Co-precipitating rabies viral mRNAs were analyzed by RT-PCR after reversal of the formaldehyde cross links and products run on a 1% agarose gel and visualized with ethidium bromide. The 10% input lane represents total RNA present in the lysate prior to immunoprecipitation. The numbers below the lanes represent quantification and standard deviation from three independent experiments.
Mentions: The interaction of individual rabies virus mRNAs with select cellular RNA binding proteins was pursued using a co-immunoprecipitation approach. Human embryonic kidney (293T) cells were infected with rabies virus and RNA-protein complexes were stabilized using formaldehyde prior to cell lysis. Protein-RNA complexes were immunoprecipitated under stringent conditions and associated RNAs were detected by PCR. While most of the RNA binding proteins tested gave negative or inconclusive results, we made an interesting observation with the cellular protein PCBP2. As seen in Figure 1, the rabies virus G mRNA was selectively associated with PCBP2. All four of the other rabies virus mRNAs did not co-immunoprecipitate with PCBP2 significantly above background. Therefore we conclude that the rabies virus G mRNA selectively associates with the cellular PCBP2 protein during infection.

Bottom Line: Viral polymerase entry and pausing at intergenic junctions is predicted to lead to a defined polarity in the levels of rhabdovirus gene expression.PCBP2 specifically interacts with reporter transcripts containing this 72 base 3' UTR sequence.Furthermore, the PCBP2 interaction is directly associated with the stability of reporter transcripts.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.

ABSTRACT
Viral polymerase entry and pausing at intergenic junctions is predicted to lead to a defined polarity in the levels of rhabdovirus gene expression. Interestingly, we observed that the rabies virus glycoprotein mRNA is differentially over-expressed based on this model relative to other transcripts during infection of 293T cells. During infection, the rabies virus glycoprotein mRNA also selectively interacts with the cellular poly(rC)-binding protein 2 (PCBP2), a factor known to influence mRNA stability. Reporter assays performed both in electroporated cells and in a cell-free RNA decay system indicate that the conserved portion of the 3' UTR of the rabies virus glycoprotein mRNA contains an RNA stability element. PCBP2 specifically interacts with reporter transcripts containing this 72 base 3' UTR sequence. Furthermore, the PCBP2 interaction is directly associated with the stability of reporter transcripts. Therefore, we conclude that PCBP2 specifically and selectively interacts with the rabies virus glycoprotein mRNA and that this interaction may contribute to the post-transcriptional regulation of glycoprotein expression.

Show MeSH
Related in: MedlinePlus