U2AF modulates poly(A) length control by the poly(A)-limiting element.
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.
Department of Molecular and Cellular Biochemistry and the Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.
Haidong
H
Gu
Daniel R
DR
Schoenberg
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D006367:HeLa Cells; D006801:Humans; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011061:Poly A / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D026723:Polyadenylation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics*; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D000072668:Splicing Factor U2AF
D009687:Nuclear Proteins; D021122:Protein Subunits; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D011061:Poly A