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.
Gu
Haidong
H
Department of Molecular and Cellular Biochemistry and the Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.
Schoenberg
Daniel R
DR
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