Limits...
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.

Show MeSH

Related in: MedlinePlus

ZNF9 interacts with the translating human eukaryotic ribosome.A. Western blots of sucrose gradient fractions of HeLa cells extracts transfected with either ZNF9-myc or LacZ-myc and probed with the indicated antibodies. 10% of each fraction was analyzed by western blotting. WCL: whole cell extract, C: cytoplasmic, N: nuclear B. ZNF9 and GAPDH western blotting of supernatant and polysome fractions of HeLa cell lysates isolated in the absence or presence of EDTA. Quantification was normalized to the levels of ZNF9 in the supernatant fraction in the absence of EDTA C. ZNF9 western blotting of sucrose gradient fractions of HeLa cell lysates in the absence or presence of EDTA. Equal cell lysate amounts (10 O.D. units) were applied to each gradient (−/+50 mM EDTA), and each fraction was TCA precipitated. 50% of the total precipitated protein was analyzed by western blotting. D. ZNF9 western blotting analysis of supernatant and pellet fractions from ribosome salt wash experiments. Western blots were quantified and the data are reported as ratios of ZNF9 in the pellet fraction to the amount of ZNF9 in the supernatant, normalized to the values for 100 mM potassium acetate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2823779&req=5

pone-0009301-g002: ZNF9 interacts with the translating human eukaryotic ribosome.A. Western blots of sucrose gradient fractions of HeLa cells extracts transfected with either ZNF9-myc or LacZ-myc and probed with the indicated antibodies. 10% of each fraction was analyzed by western blotting. WCL: whole cell extract, C: cytoplasmic, N: nuclear B. ZNF9 and GAPDH western blotting of supernatant and polysome fractions of HeLa cell lysates isolated in the absence or presence of EDTA. Quantification was normalized to the levels of ZNF9 in the supernatant fraction in the absence of EDTA C. ZNF9 western blotting of sucrose gradient fractions of HeLa cell lysates in the absence or presence of EDTA. Equal cell lysate amounts (10 O.D. units) were applied to each gradient (−/+50 mM EDTA), and each fraction was TCA precipitated. 50% of the total precipitated protein was analyzed by western blotting. D. ZNF9 western blotting analysis of supernatant and pellet fractions from ribosome salt wash experiments. Western blots were quantified and the data are reported as ratios of ZNF9 in the pellet fraction to the amount of ZNF9 in the supernatant, normalized to the values for 100 mM potassium acetate.

Mentions: Based on its proposed role as an ITAF for cap-independent translation, we hypothesized that ZNF9 interacts with translating ribosomes. Previous work from our lab showed that ZNF9 co-sediments with the polyribosome fractions, suggesting an interaction with the ribosome and a role in translation [26]. Therefore, we sought to analyze the intracellular localization of ZNF9 and whether ZNF9 associates with the ribosome during active translation. Subcellular fractionation experiments revealed that the majority of ZNF9 protein is found in the cytoplasm (Fig. 2A). Both myc-tagged and endogenous ZNF9 localize to the cytoplasm (Fig. 2A). Western blotting for a known nuclear protein (c-myc) and a cytoplasmic protein (β-galactosidase) were used as controls for the selectivity of the fractionation technique. Both control proteins were found exclusively in the expected fraction. A small amount (∼5%) of the total ZNF9 found in the cell was localized to the nucleus. While this may be carryover from the cytoplasmic fraction, the experimental and control results suggest that a small fraction of ZNF9 is nuclear. The significance and function of nuclear ZNF9 are unknown, but ZNF9 has been shown previously to have a nuclear pool [24], [25]. The cytoplasmic localization of ZNF9 is consistent with its proposed role in translation.


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 interacts with the translating human eukaryotic ribosome.A. Western blots of sucrose gradient fractions of HeLa cells extracts transfected with either ZNF9-myc or LacZ-myc and probed with the indicated antibodies. 10% of each fraction was analyzed by western blotting. WCL: whole cell extract, C: cytoplasmic, N: nuclear B. ZNF9 and GAPDH western blotting of supernatant and polysome fractions of HeLa cell lysates isolated in the absence or presence of EDTA. Quantification was normalized to the levels of ZNF9 in the supernatant fraction in the absence of EDTA C. ZNF9 western blotting of sucrose gradient fractions of HeLa cell lysates in the absence or presence of EDTA. Equal cell lysate amounts (10 O.D. units) were applied to each gradient (−/+50 mM EDTA), and each fraction was TCA precipitated. 50% of the total precipitated protein was analyzed by western blotting. D. ZNF9 western blotting analysis of supernatant and pellet fractions from ribosome salt wash experiments. Western blots were quantified and the data are reported as ratios of ZNF9 in the pellet fraction to the amount of ZNF9 in the supernatant, normalized to the values for 100 mM potassium acetate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2823779&req=5

pone-0009301-g002: ZNF9 interacts with the translating human eukaryotic ribosome.A. Western blots of sucrose gradient fractions of HeLa cells extracts transfected with either ZNF9-myc or LacZ-myc and probed with the indicated antibodies. 10% of each fraction was analyzed by western blotting. WCL: whole cell extract, C: cytoplasmic, N: nuclear B. ZNF9 and GAPDH western blotting of supernatant and polysome fractions of HeLa cell lysates isolated in the absence or presence of EDTA. Quantification was normalized to the levels of ZNF9 in the supernatant fraction in the absence of EDTA C. ZNF9 western blotting of sucrose gradient fractions of HeLa cell lysates in the absence or presence of EDTA. Equal cell lysate amounts (10 O.D. units) were applied to each gradient (−/+50 mM EDTA), and each fraction was TCA precipitated. 50% of the total precipitated protein was analyzed by western blotting. D. ZNF9 western blotting analysis of supernatant and pellet fractions from ribosome salt wash experiments. Western blots were quantified and the data are reported as ratios of ZNF9 in the pellet fraction to the amount of ZNF9 in the supernatant, normalized to the values for 100 mM potassium acetate.
Mentions: Based on its proposed role as an ITAF for cap-independent translation, we hypothesized that ZNF9 interacts with translating ribosomes. Previous work from our lab showed that ZNF9 co-sediments with the polyribosome fractions, suggesting an interaction with the ribosome and a role in translation [26]. Therefore, we sought to analyze the intracellular localization of ZNF9 and whether ZNF9 associates with the ribosome during active translation. Subcellular fractionation experiments revealed that the majority of ZNF9 protein is found in the cytoplasm (Fig. 2A). Both myc-tagged and endogenous ZNF9 localize to the cytoplasm (Fig. 2A). Western blotting for a known nuclear protein (c-myc) and a cytoplasmic protein (β-galactosidase) were used as controls for the selectivity of the fractionation technique. Both control proteins were found exclusively in the expected fraction. A small amount (∼5%) of the total ZNF9 found in the cell was localized to the nucleus. While this may be carryover from the cytoplasmic fraction, the experimental and control results suggest that a small fraction of ZNF9 is nuclear. The significance and function of nuclear ZNF9 are unknown, but ZNF9 has been shown previously to have a nuclear pool [24], [25]. The cytoplasmic localization of ZNF9 is consistent with its proposed role in translation.

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