Regulation of TNF mRNA stability by an RNA-binding protein implicated in myotonic dystrophy Libin Zhang, Jerome E. Lee * , Karen C. Moraes, Jeffrey Wilusz * and Carol J. Wilusz * Department of Microbiology, Immunology & Pathology, and * Cell & Molecular Biology Program, Colorado State University, Fort Collins, CO 80523 Summary Myotonic dystrophy (DM1) is caused by expression of a toxic RNA containing an expanded CUG repeat. The RNA-binding protein CUG-BP is over-expressed and aberrantly confined to the nucleus in DM1 cells resulting in well-characterized changes in splicing of various clinically relevant genes. Expression of the toxic expanded-repeat RNA in C2C12 mouse myoblast cells, which induces similar changes in CUG-BP expression as seen in DM1, results in stabilization of TNF RNA. We have also mimicked the cytoplasmic depletion of CUG-BP seen in DM1 by expressing shRNAs against CUG-BP in C2C12 cells. This also resulted in stabilization of the TNF mRNA. These data are consistent with the observation that TNF levels are routinely elevated in DM patients. We find that CUG-BP binds to the 3’UTR of the TNF mRNA, recognizing both the AU-rich element and flanking UG-rich tetramers. Moreover, binding of CUG-BP results in rapid poly(A) shortening in vitro. Finally, we find that CUG-BP interacts directly with a deadenylase, PARN. Taken together our results suggest that CUG-BP is required to modulate levels of TNF mRNA in muscle cells. Aberrant expression of TNF in DM1 appears to result directly from insufficient cytoplasmic CUG-BP levels. As excess TNF has been linked with muscle wasting, cardiac conduction defects and insulin resistance, it may well cause or exacerbate aspects of DM pathogenesis. Introduction Myotonic dystrophy (DM1) is an autosomal dominant, late onset, inherited disease caused by a triplet repeat expansion in the 3’UTR of the dystrophia myotonica protein kinase (DMPK) gene. Symptoms include myotonia, cardiac problems and insulin resistance(1). DM1 is unusual in that the majority of the pathology is not caused by insufficiency of the affected gene, but rather by toxic effects of the mRNA expressed from it. The DMPK mRNA bearing the expanded repeat accumulates in nuclear foci where it sequesters various proteins including splicing factors(2), and transcription factors(3). Much of the pathogenesis has in fact been attributed to altered splicing patterns(4;5). One protein whose expression is affected by the toxic RNA is CUG-BP. In DM1 patients, CUG- BP is over-expressed and confined to the nucleus resulting in an overall reduction in its cytoplasmic concentration(6). CUG-BP is an RNA-binding protein that has been previously implicated in regulation of splicing, translation and mRNA stability, thus alterations in its expression pattern have broad implications for gene expression. Here we have examined the role of CUG-BP in regulating decay of TNF mRNA in muscle cells. TNF undergoes a large amount of post- transcriptional regulation, most of which is mediated by an AU-rich element (ARE) in its 3’ UTR(7). TNF levels are elevated in DM1 patients although it is presently unclear whether this is a direct cause of pathogenesis or merely reflects the disease state(8). In addition, TNF levels are regulated during muscle differentiation and aberrant levels of TNF can prevent this process(9). Results In order to determine whether changes in CUG- BP expression seen in DM1 have effects on TNF mRNA stability, we expressed an RNA consisting of the last three exons of DMPK, including the 3’UTR, in C2C12 mouse myoblasts. Two constructs were used, one with the normal 3’UTR, the other containing 960 CUG repeats in the 3’UTR. Following transfection of the expression plasmids, transcription was inhibited by addition of actinomycin D and cells were harvested for analysis of RNA abundance at several time points. Levels of TNF and GAPDH mRNAs at each time were assessed by qRT-PCR. The results showed that in cells expressing the normal DMPK 3’UTR, the TNF mRNA decayed very rapidly with a half life of 8 min. However, transfection of the expanded repeat 3’UTR, which is known to induce formation of nuclear foci and disrupt CUG-BP expression, resulted in stabilization of the TNF mRNA such that its half life increased to 20 min. As the DMPK toxic RNA has a wide range of effects on cellular gene expression, we needed to ascertain whether the paucity of CUG-BP was