Nucleic Acids Research, 2003, Vol. 31, No. 21 6264-6271
© 2003 Oxford University Press
U2AF modulates poly(A) length control by the poly(A)-limiting element
Department of Molecular and Cellular Biochemistry and the Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA
*To whom correspondence should be addressed at Department of Molecular and Cellular Biochemistry, The Ohio State University, 1645 Neil Avenue, Columbus, OH 43210-1218, USA. Tel: +1 614 688 3012; Fax: +1 614 292 4118; Email: schoenberg.3{at}osu.edu
Present address:
Haidong Gu, Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA
The poly(A)-limiting element (PLE) restricts the length of the poly(A) tail to <20 nt when present in the terminal exon of a pre-mRNA. We previously identified a 65 kDa protein that could be cross-linked to a functional PLE, but not to an inactive mutant element. This binding was competed by poly(U) and poly(C), but not poly(A) or poly(G). Selectivity for the pyrimidine-rich portion of the PLE was demonstrated by RNase footprinting of the binding activity in total nuclear extract. A 65 kDa protein that selectively cross-linked to the functional PLE was purified by conventional chromatography and identified as the large subunit of U2 snRNP auxiliary factor (U2AF). Overexpression of U2AF65 in cells transfected with a PLE-containing reporter construct resulted in the appearance of a population of mRNAs with heterogeneous poly(A) tails. However, this effect was lost following deletion of the C-terminal RNA recognition motifs (RRMs). A C
G mutation following the AG dinucleotide in the PLE resulted in mRNA with poly(A) ranging from 25–50 nt. This reverted to a discrete, <20 nt poly(A) tail in cells expressing U2AF65. Our results suggest that U2AF modulates the function of the PLE, perhaps by facilitating the binding of another protein to the element.
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