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J. Biol. Chem., Vol. 283, Issue 42, 28010-28019, October 17, 2008

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Species-specific cis-Regulatory Elements in the 3'-Untranslated Region Direct Alternative Polyadenylation of Bone Morphogenetic Protein 2 mRNA^*

Donglin Liu, David T. Fritz, Melissa B. Rogers¹, and Aaron J. Shatkin²

From the Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854 and Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, New Jersey 07101

BMP2 (bone morphogenetic protein 2) is a multifunctional member of the transforming growth factor-β family of growth factors. Disruption of BMP2 signaling results in developmental defects, cancers, and other diseases. BMP2 mRNAs are alternatively polyadenylated, resulting in mRNAs with distinct 3'-untranslated regions. The longer mRNA contains additional putative binding sites for post-transcriptional regulatory factors, including micro-RNAs. We combined functional assays with computational analyses of emerging genome data to define site- and species-specific polyadenylation determinants. In all mouse and human cell lines tested, shorter mRNAs resulting from using the first polyadenylation signal (PA1) were more abundant than mRNAs from the second signal (PA2). However, the PA1/PA2 usage ratios were 2–3-fold higher in human than in mouse cells. Expression of human BMP2 constructs in mouse cells and mouse constructs in human cells showed that cis-regulatory elements direct species-specific 3' processing of BMP2 transcripts. A 72-nucleotide region downstream of PA2 in the mouse sequence contains two novel cis-acting elements previously hypothesized to regulate polyadenylation in a bioinformatics analysis. Mutations that humanized the mouse-specific elements lowered the affinity for cleavage stimulation factor CstF64 and significantly weakened the PA2 signal relative to the PA1 signal. Thus, we have experimentally defined for the first time cis-regulatory elements that control a species-specific difference in the 3'-end processing of BMP2 and potentially of other genes.

Received for publication, June 26, 2008 , and in revised form, July 30, 2008.

^* This work was supported by New Jersey Commission on Cancer Research Postdoctoral Fellowship 05-2413-CCR-EO (to D. L.), by a grant from the Foundation of UMDNJ (to M. B. R.), and by the Center for Advanced Biotechnology and Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence may be addressed: Dept. of Biochemistry and Molecular Biology (MSB E627), UMDNJ-New Jersey Medical School, 185 South Orange Ave., P.O. Box 1709, Newark, NJ 07101. Tel.: 973-972-2984; Fax: 973-972-5594; E-mail: rogersmb{at}umdnj.edu. ² To whom correspondence may be addressed: Center for Advanced Biotechnology and Medicine, 679 Hoes Ln., Piscataway, NJ 08854. Tel.: 732-235-5311; Fax: 732-235-5318; E-mail: shatkin{at}cabm.rutgers.edu.

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