U2AF modulates poly(A) length control by the poly(A)-limiting element.

Abstract

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.