A+U-Rich Instability Elements Differentially Activate 5′-3′ and 3′-5′ mRNA Decay

Abstract

The A+U-rich elements (or AREs) are cis-acting sequences that activate rapid mRNA decay, yet the overall polarity of this process is unknown. The current study describes an unbiased approach to this using the Invader RNA assay (Third Wave Technologies, Inc.) to quantify the decay of each of the three exons of human β-globin mRNA without added instability elements or with the AREs from c-fos or granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA in the 3′ untranslated region. Each of these genes under tetracycline operator control was stably transfected into cells, and β-globin mRNA was quantified with exon-specific probes following transcription termination. There was little overall evidence for polarity in stable mRNA decay. Adding the c-fos ARE activated rapid and simultaneous decay from both ends of the mRNA. In contrast, the GM-CSF ARE activated decay primarily from the mRNA 5′ end. These data were supported by reciprocal RNA interference knockdowns, and we present evidence that the 5′-3′ and 3′-5′ decay pathways are functionally linked.

This work was supported by PHS grant R01 GM38277 to D.R.S. E.L.M. was supported in part by PHS grant T32 CA09338, and support for core facilities was provided by PHS center grant P30 CA16058 from the National Cancer Institute to the Ohio State University Comprehensive Cancer Center.

We thank Yan Chen, Yong Peng, and Yuichi Otsuka for help with some of these experiments; Tsetka Takova for help with probe design, troubleshooting, and data analysis; and Peggy Eis and David Fritz for help with design of the primary and secondary oligonucleotide probe sets. We also thank Mike Kiledjian for antibody to Dcp2, Reinout Raijmakers and Ger Pruijn for antibodies to PM/Scl-100 and Rrp41, and Jim Dahlberg and members of the Schoenberg lab for helpful discussions.

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