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

Published online by Cambridge University Press:  06 July 2001

JAYDIP DAS GUPTA ,
HAIDONG GU  and
DANIEL R. SCHOENBERG
JAYDIP DAS GUPTA
Affiliation:
Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA Current address: Department of Immunology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195-0001.
HAIDONG GU
Affiliation:
Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA Ohio State Biochemistry Program and The Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA
DANIEL R. SCHOENBERG
Affiliation:
Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA
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Abstract

The poly(A)-limiting element (PLE) is a cis-acting sequence that acts to limit 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. Increasing the spacing between the PLE and the upstream 3′ splice site or between 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 further evaluated using a battery of constructs in which the PLE was maintained in the terminal exon, but where upstream introns were either deleted, modified, 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 importance of the 5′ pyrimidine-rich portion of the element. Finally, UV crosslinking experiments identified an ∼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.

Keywords

3′ processingpolyadenylationpoly(A)-limiting elementpre-mRNA processing
Type
Research Article
Information
RNA , Volume 7 , Issue 7 , July 2001 , pp. 1034 - 1042
Copyright
2001 RNA Society

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