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Molecular and Cellular Biology, April 2004, p. 2789-2796, Vol. 24, No. 7
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.7.2789-2796.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Secondary Structure as a Functional Feature in the Downstream Region of Mammalian Polyadenylation Signals

Chunxiao Wu and James C. Alwine^*

Department of Cancer Biology, Abramson Family Cancer Research Institute School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6142

Received 17 November 2003/ Returned for modification 12 December 2003/ Accepted 5 January 2004

Secondary structure within the downstream region of mammalian polyadenylation signals has been proposed to perform important functions. The simian virus 40 late polyadenylation signal (SVLPA) forms alternate secondary structures in equilibrium. Their formation correlates with cleavage-polyadenylation efficiency (H. Hans and J. C. Alwine, Mol. Cell. Biol. 20:2926-2932, 2000; M. I. Zarudnaya, I. M. Kolomiets, A. L. Potyahaylo, and D. M. Hovorun, Nucleic Acids Res. 3:1375-1386, 2003), and oligonucleotides that disrupt the secondary structure inhibit in vitro cleavage. To define the important features of downstream secondary structure, we first minimized the SVLPA by deletion, forming a downstream region with fewer, and more stable, stem-loop structures. Specific mutagenesis showed that both stem stability and loop size are important functional features of the downstream region. Stabilization of the stem, thus minimizing alternative structures, decreased cleavage efficiency both in vitro and in vivo. This was most deleterious when the stem was stabilized at the base of the loop, constraining loop size by inhibiting breathing of the stem. The significance of loop size was supported by mutants that showed increased cleavage efficiency with increased loop size and vice versa. A loop of at least 12 nucleotides promoted cleavage; U richness in the loop also promoted cleavage and was particularly important when the stem was stabilized. A mutation designed to eliminate downstream secondary structure still formed many relatively weak alternative structures in equilibrium and retained function. The data suggest that although the downstream region is very important, its structure is quite malleable and is able to tolerate significant mutation within a wide range of primary and secondary structural features. We propose that this malleability is due to the enhanced ability of GU- and U-rich downstream elements to easily form secondary structures with surrounding sequences.

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* Corresponding author. Mailing address: 314 Biomedical Research Building, 421 Curie Blvd., University of Pennsylvania, Philadelphia, PA 19104-6142. Phone: (215) 898-3256. Fax: (215) 573-3888. E-mail: alwine{at}mail.med.upenn.edu.

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Molecular and Cellular Biology, April 2004, p. 2789-2796, Vol. 24, No. 7
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.7.2789-2796.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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