ZNF9 Activation of IRES-Mediated Translation of the Human ODC mRNA Is Decreased in Myotonic Dystrophy Type 2

Morgan A Sammons,Ralf Krahe,Bjarne Udd,Andrew J Link,Mourad Bendjennat,Amanda K Antons,Stefan Maas

doi:10.1371/journal.pone.0009301

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.

Highlights

Myotonic dystrophy types 1 and 2 are forms of muscular dystrophy resulting from large expansions of nucleotide repeats within noncoding regions of DMPK and ZNF9, respectively [1]
Our data show that ZNF9 activity is reduced in myoblasts from a patient affected with DM2, providing further evidence that a loss-of-function mechanism contributes to myotonic dystrophy type 2 disease
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 b-galactosidase overexpression (Fig. 3B). These data, along with a demonstration of direct ZNF9 binding to the 59UTR of ornithine decarboxylase (ODC), strongly suggests that ZNF9 acts as a translation factor for cap-independent translation of the human ODC mRNA

Summary

Introduction

Myotonic dystrophy types 1 and 2 are forms of muscular dystrophy resulting from large expansions of nucleotide repeats within noncoding regions of DMPK and ZNF9, respectively [1]. Two studies of ZNF9 mRNA and protein expression levels reported no change in diseased myoblasts or lymphoblastoid cell lines compared to unaffected cells [9,10] These results indicate that the repeat RNA does not affect normal ZNF9 function and have led to a disease model in which the expanded repeats somehow dominantly interfere with other cellular processes [11,12,13,14]. A second study has reported that the expression of the CCTG repeats in mouse and human myoblasts alters translation and protein degradation [25] These data support the notion that the ZNF9 protein may play a role in DM2 and that the RNA-mediated dominant gain-of-function model for myotonic dystrophy is not the only pathomechanism for the disease. Our data show that ZNF9 activity is reduced in myoblasts from a patient affected with DM2, providing further evidence that a loss-of-function mechanism contributes to myotonic dystrophy type 2 disease

Materials and Methods

Findings

Discussion