Position and sequence requirements for poly(A) length regulation by the poly(A) limiting element

The poly(A)-limiting element (PLE) is a cis-acting sequence that acts to li mit poly(A) tail length on pre-mRNA to <20 nt, Functional PLEs are present in a number of genes, underscoring the generality of this control mechanism . The current study sought to define further the position requirements for poly(A) length regulation and the core sequence that comprises a PLE, Incre asing the spacing between the PLE and the upstream 3' splice site or betwee n the PLE and the downstream AAUAAA had no effect on poly(A) length control . However, moving the PLE from the terminal exon to either an upstream exon or intron eliminated poly(A) length control. Poly(A) length control was fu rther evaluated using a battery of constructs in which the PLE was maintain ed in the terminal exon, but where upstream introns were either deleted, mo dified, or replaced with a polypyrimidine tract. Poly(A) length control was retained in all cases, indicating that the key feature is the presence of the PLE in the terminal exon, A battery of mutations demonstrated the impor tance of the 5' pyrimidine-rich portion of the element. Finally, UV crossli nking experiments identified an <similar to>62-kDa protein in Hela nuclear extract that binds to a wild-type 23-nt PLE RNA oligonucleotides but not to a mutated nonfunctional form of the element.