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ZNF9 activation of IRES-mediated translation of the human ODC mRNA is decreased in myotonic dystrophy type 2.

Sammons MA, Antons AK, Bendjennat M, Udd B, Krahe R, Link AJ - PLoS ONE (2010)

Bottom Line: First, a gain-of-function disease model hypothesizes that the repeat expansions in the transcribed RNA do not directly affect ZNF9 function.In the alternative model, the repeat expansions impair ZNF9 function and lead to a decrease in the level of translation.ZNF9 can activate IRES-mediated translation of ODC within primary human myoblasts, and this activity is reduced in myoblasts derived from a DM2 patient.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
Myotonic dystrophy types 1 and 2 (DM1 and DM2) are forms of muscular dystrophy that share similar clinical and molecular manifestations, such as myotonia, muscle weakness, cardiac anomalies, cataracts, and the presence of defined RNA-containing foci in muscle nuclei. DM2 is caused by an expansion of the tetranucleotide CCTG repeat within the first intron of ZNF9, although the mechanism by which the expanded nucleotide repeat causes the debilitating symptoms of DM2 is unclear. Conflicting studies have led to two models for the mechanisms leading to the problems associated with DM2. First, a gain-of-function disease model hypothesizes that the repeat expansions in the transcribed RNA do not directly affect ZNF9 function. Instead repeat-containing RNAs are thought to sequester proteins in the nucleus, causing misregulation of normal cellular processes. In the alternative model, the repeat expansions impair ZNF9 function and lead to a decrease in the level of translation. Here we examine the normal in vivo function of ZNF9. We report that ZNF9 associates with actively translating ribosomes and functions as an activator of cap-independent translation of the human ODC mRNA. This activity is mediated by direct binding of ZNF9 to the internal ribosome entry site sequence (IRES) within the 5'UTR of ODC mRNA. ZNF9 can activate IRES-mediated translation of ODC within primary human myoblasts, and this activity is reduced in myoblasts derived from a DM2 patient. These data identify ZNF9 as a regulator of cap-independent translation and indicate that ZNF9 activity may contribute mechanistically to the myotonic dystrophy type 2 phenotype.

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ZNF9 activates cap independent-translation of the human ODC mRNA.A. Diagram of the ODC bicistronic vector for measuring cap-independent translation of human ODC. 5′meG: 5′-methyl-G cap structure. B. HeLa cells expressing the ODC bicistronic vector and either V5-ZNF9 or V5-LacZ were analyzed for luciferase activity. The level of cap-independent translation is reported as a ratio of cap-independent translation (firefly) to cap-dependent translation (Renilla). Values are averages of 6 independent experiments. The error bars are the standard error of the mean (S.E.M). The insert figure is a representative western blot analysis showing the V5-LacZ and V5-ZNF9 levels in the HeLa cells lysates. C. ZNF9 mRNA and protein levels after RNAi of ZNF9 in HeLa cells. HeLa cells expressing either a control shRNA or a ZNF9-specific shRNA were analyzed using quantitative RT-PCR and western blotting to quantify expression of ZNF9 RNA and protein, respectively. The RT-PCR values were computed using the ΔΔCt method and are normalized to the levels of GAPDH mRNA. Error bars represent the standard error. The insert figure is a western blot showing the ZNF9 proteins levels. Recombinant ZNF9 (rZNF9) was used as a positive control, and equal amounts of protein were loaded in each lane as assessed by BCA protein assays. D. HeLa cells expressing the ODC bicistronic vector and either a control or ZNF9-specific shRNA were assessed for luciferase activity. The level of cap-independent translation is reported as a ratio of cap-independent translation (firefly) to cap-dependent translation (Renilla). Values are the averages of 3 independent experiments and are normalized to the control shRNA. The error bars are the S.E.M.
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pone-0009301-g003: ZNF9 activates cap independent-translation of the human ODC mRNA.A. Diagram of the ODC bicistronic vector for measuring cap-independent translation of human ODC. 5′meG: 5′-methyl-G cap structure. B. HeLa cells expressing the ODC bicistronic vector and either V5-ZNF9 or V5-LacZ were analyzed for luciferase activity. The level of cap-independent translation is reported as a ratio of cap-independent translation (firefly) to cap-dependent translation (Renilla). Values are averages of 6 independent experiments. The error bars are the standard error of the mean (S.E.M). The insert figure is a representative western blot analysis showing the V5-LacZ and V5-ZNF9 levels in the HeLa cells lysates. C. ZNF9 mRNA and protein levels after RNAi of ZNF9 in HeLa cells. HeLa cells expressing either a control shRNA or a ZNF9-specific shRNA were analyzed using quantitative RT-PCR and western blotting to quantify expression of ZNF9 RNA and protein, respectively. The RT-PCR values were computed using the ΔΔCt method and are normalized to the levels of GAPDH mRNA. Error bars represent the standard error. The insert figure is a western blot showing the ZNF9 proteins levels. Recombinant ZNF9 (rZNF9) was used as a positive control, and equal amounts of protein were loaded in each lane as assessed by BCA protein assays. D. HeLa cells expressing the ODC bicistronic vector and either a control or ZNF9-specific shRNA were assessed for luciferase activity. The level of cap-independent translation is reported as a ratio of cap-independent translation (firefly) to cap-dependent translation (Renilla). Values are the averages of 3 independent experiments and are normalized to the control shRNA. The error bars are the S.E.M.

Mentions: We have determined that ZNF9 binds specifically to the 5′UTR of the human ODC mRNA and interacts with translating ribosomes. Previous data from our lab revealed that ZNF9 can activate cap-independent translation of the rat ODC mRNA, which is similar, but not identical, to the human ODC mRNA. These data suggest a direct role for ZNF9 in regulating translation of the ODC mRNA. To test if ZNF9 promotes human ODC IRES activity, a bicistronic reporter plasmid with the full-length human ODC 5′UTR was used to test the ability of ZNF9 to activate cap-independent translation (Fig. 3A). Overexpression of ZNF9 in HeLa cells resulted in an ∼3-fold increase (p-value = 0.0031) in translation of the second cistron of the reporter compared to control experiments with β-galactosidase overexpression (Fig. 3B). These data, along with a demonstration of direct ZNF9 binding to the 5′UTR of ODC, strongly suggests that ZNF9 acts as a translation factor for cap-independent translation of the human ODC mRNA.


ZNF9 activation of IRES-mediated translation of the human ODC mRNA is decreased in myotonic dystrophy type 2.

Sammons MA, Antons AK, Bendjennat M, Udd B, Krahe R, Link AJ - PLoS ONE (2010)

ZNF9 activates cap independent-translation of the human ODC mRNA.A. Diagram of the ODC bicistronic vector for measuring cap-independent translation of human ODC. 5′meG: 5′-methyl-G cap structure. B. HeLa cells expressing the ODC bicistronic vector and either V5-ZNF9 or V5-LacZ were analyzed for luciferase activity. The level of cap-independent translation is reported as a ratio of cap-independent translation (firefly) to cap-dependent translation (Renilla). Values are averages of 6 independent experiments. The error bars are the standard error of the mean (S.E.M). The insert figure is a representative western blot analysis showing the V5-LacZ and V5-ZNF9 levels in the HeLa cells lysates. C. ZNF9 mRNA and protein levels after RNAi of ZNF9 in HeLa cells. HeLa cells expressing either a control shRNA or a ZNF9-specific shRNA were analyzed using quantitative RT-PCR and western blotting to quantify expression of ZNF9 RNA and protein, respectively. The RT-PCR values were computed using the ΔΔCt method and are normalized to the levels of GAPDH mRNA. Error bars represent the standard error. The insert figure is a western blot showing the ZNF9 proteins levels. Recombinant ZNF9 (rZNF9) was used as a positive control, and equal amounts of protein were loaded in each lane as assessed by BCA protein assays. D. HeLa cells expressing the ODC bicistronic vector and either a control or ZNF9-specific shRNA were assessed for luciferase activity. The level of cap-independent translation is reported as a ratio of cap-independent translation (firefly) to cap-dependent translation (Renilla). Values are the averages of 3 independent experiments and are normalized to the control shRNA. The error bars are the S.E.M.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2823779&req=5

pone-0009301-g003: ZNF9 activates cap independent-translation of the human ODC mRNA.A. Diagram of the ODC bicistronic vector for measuring cap-independent translation of human ODC. 5′meG: 5′-methyl-G cap structure. B. HeLa cells expressing the ODC bicistronic vector and either V5-ZNF9 or V5-LacZ were analyzed for luciferase activity. The level of cap-independent translation is reported as a ratio of cap-independent translation (firefly) to cap-dependent translation (Renilla). Values are averages of 6 independent experiments. The error bars are the standard error of the mean (S.E.M). The insert figure is a representative western blot analysis showing the V5-LacZ and V5-ZNF9 levels in the HeLa cells lysates. C. ZNF9 mRNA and protein levels after RNAi of ZNF9 in HeLa cells. HeLa cells expressing either a control shRNA or a ZNF9-specific shRNA were analyzed using quantitative RT-PCR and western blotting to quantify expression of ZNF9 RNA and protein, respectively. The RT-PCR values were computed using the ΔΔCt method and are normalized to the levels of GAPDH mRNA. Error bars represent the standard error. The insert figure is a western blot showing the ZNF9 proteins levels. Recombinant ZNF9 (rZNF9) was used as a positive control, and equal amounts of protein were loaded in each lane as assessed by BCA protein assays. D. HeLa cells expressing the ODC bicistronic vector and either a control or ZNF9-specific shRNA were assessed for luciferase activity. The level of cap-independent translation is reported as a ratio of cap-independent translation (firefly) to cap-dependent translation (Renilla). Values are the averages of 3 independent experiments and are normalized to the control shRNA. The error bars are the S.E.M.
Mentions: We have determined that ZNF9 binds specifically to the 5′UTR of the human ODC mRNA and interacts with translating ribosomes. Previous data from our lab revealed that ZNF9 can activate cap-independent translation of the rat ODC mRNA, which is similar, but not identical, to the human ODC mRNA. These data suggest a direct role for ZNF9 in regulating translation of the ODC mRNA. To test if ZNF9 promotes human ODC IRES activity, a bicistronic reporter plasmid with the full-length human ODC 5′UTR was used to test the ability of ZNF9 to activate cap-independent translation (Fig. 3A). Overexpression of ZNF9 in HeLa cells resulted in an ∼3-fold increase (p-value = 0.0031) in translation of the second cistron of the reporter compared to control experiments with β-galactosidase overexpression (Fig. 3B). These data, along with a demonstration of direct ZNF9 binding to the 5′UTR of ODC, strongly suggests that ZNF9 acts as a translation factor for cap-independent translation of the human ODC mRNA.

Bottom Line: First, a gain-of-function disease model hypothesizes that the repeat expansions in the transcribed RNA do not directly affect ZNF9 function.In the alternative model, the repeat expansions impair ZNF9 function and lead to a decrease in the level of translation.ZNF9 can activate IRES-mediated translation of ODC within primary human myoblasts, and this activity is reduced in myoblasts derived from a DM2 patient.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
Myotonic dystrophy types 1 and 2 (DM1 and DM2) are forms of muscular dystrophy that share similar clinical and molecular manifestations, such as myotonia, muscle weakness, cardiac anomalies, cataracts, and the presence of defined RNA-containing foci in muscle nuclei. DM2 is caused by an expansion of the tetranucleotide CCTG repeat within the first intron of ZNF9, although the mechanism by which the expanded nucleotide repeat causes the debilitating symptoms of DM2 is unclear. Conflicting studies have led to two models for the mechanisms leading to the problems associated with DM2. First, a gain-of-function disease model hypothesizes that the repeat expansions in the transcribed RNA do not directly affect ZNF9 function. Instead repeat-containing RNAs are thought to sequester proteins in the nucleus, causing misregulation of normal cellular processes. In the alternative model, the repeat expansions impair ZNF9 function and lead to a decrease in the level of translation. Here we examine the normal in vivo function of ZNF9. We report that ZNF9 associates with actively translating ribosomes and functions as an activator of cap-independent translation of the human ODC mRNA. This activity is mediated by direct binding of ZNF9 to the internal ribosome entry site sequence (IRES) within the 5'UTR of ODC mRNA. ZNF9 can activate IRES-mediated translation of ODC within primary human myoblasts, and this activity is reduced in myoblasts derived from a DM2 patient. These data identify ZNF9 as a regulator of cap-independent translation and indicate that ZNF9 activity may contribute mechanistically to the myotonic dystrophy type 2 phenotype.

Show MeSH
Related in: MedlinePlus