Global analysis of mRNA decay rates and RNA-binding specificity reveals novel roles for CUGBP1 and PARN deadenylase in muscle cells

Lee, Jerome Edward, author
Wilusz, Carol J., advisor
Wilusz, Jeffrey, advisor
Garrity, Deborah M., committee member
Curthoys, Norman P., committee member
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Type I Myotonic Dystrophy (DM1) is characterized by myotonia, cardiac conduction defects, muscle wasting, and insulin resistance. In patient muscle cells expression and function of the RNA-binding proteins CUGBP1 and MBNL1 are disrupted, resulting in altered mRNA metabolism at the levels of splicing and translation. Intriguingly, despite strong evidence for CUGBP1 being a regulator of mRNA turnover in humans and other organisms, the possibility that defects in mRNA decay contribute to DM1 pathogenesis has not been investigated to date. As such, we sought to further characterize the roles of CUGBP1 and its partner, the deadenylase PARN, in mRNA decay in mouse C2C12 muscle cells. The TNF message, which encodes a cytokine known to cause muscle wasting and insulin resistance when over-expressed, was stabilized by depletion of CUGBP1. The normally rapid decay of the TNF mRNA was also disrupted in cells treated with phorbol ester and this coincided with phosphorylation of CUGBP1. These findings provided impetus to undertake a global analysis of mRNA decay rates in muscle cells. Our investigation revealed that GU- and AU-rich sequence elements are enriched in labile transcripts, which encode cell cycle regulators, transcription factors, and RNA-processing proteins. Transcripts specifically bound to CUGBP1 in myoblasts are linked with processes such as mRNA metabolism, protein targeting to the endoplasmic reticulum, cytoskeletal organization, and transcriptional regulation, all of which have implications for muscle cell biology. Consistent with this, CUGBP1 depletion profoundly altered the formation of myotubes during differentiation. Finally we investigated whether PARN, which interacts with CUGBP1 and mediates rapid deadenylation of TNF in HeLa cell extracts, also plays a role in mediating mRNA decay in muscle. We identified 64 mRNA targets whose decay was dependent on PARN. Moreover, deadenylation of the Brf2 mRNA was impaired in PARN knock-down cells supporting that this mRNA is directly and specifically targeted for decay by PARN. Taken together our findings demonstrate that CUGBP1 and PARN are critical regulators of decay for specific sets of transcripts in muscle cells. It seems likely that some or all of the CUGBP1 targets we have identified may be affected in myotonic dystrophy. Defective mRNA turnover could be linked with defects in myogenesis, TNF over-expression, muscle wasting and/or ER stress, all of which have been documented in DM1.
2011 Summer.
Includes bibliographical references.
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mRNA decay
gene expression
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