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Volume 272, Number 42, Issue of October 17, 1997 pp. 26769-26779
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

RNA Ligands Selected by Cleavage Stimulation Factor Contain Distinct Sequence Motifs That Function as Downstream Elements in 3-End Processing of Pre-mRNA

(Received for publication, May 16, 1997, and in revised form, August 13, 1997)

Katrin Beyer , Thomas Dandekar and Walter Keller

From the Department of Cell Biology, Biozentrum of the University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland and the  European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Federal Republic of Germany

Critical events in 3-end processing of pre-mRNA are the recognition of the AAUAAA polyadenylation signal by cleavage and polyadenylation specificity factor (CPSF) and the binding of cleavage stimulation factor (CstF) via its 64-kDa subunit to the downstream element. The stability of this CPSF·CstF·RNA complex is thought to determine the efficiency of 3-end processing. Since downstream elements reveal high sequence variability, in vitro selection experiments with highly purified CstF were performed to investigate the sequence requirements for CstF-RNA interaction. CstF was purified from calf thymus and from HeLa cells. Surprisingly, calf thymus CstF contained an additional, novel form of the 64-kDa subunit with a molecular mass of 70 kDa. RNA ligands selected by HeLa and calf thymus CstF contained three highly conserved sequence elements as follows: element 1 (AUGCGUUCCUCGUCC) and two closely related elements, element 2a (YGUGUYN_0-4UUYAYUGYGU) and element 2b (UUGYUN_0-4AUUUACU(U/G)N_0-2YCU). All selected sequences tested functioned as downstream elements in 3-end processing in vitro. A computer survey of the EMBL data library revealed significant homologies to all selected elements in naturally occurring 3-untranslated regions. The majority of element 2a homologies was found downstream of coding sequences. Therefore, we postulate that this element represents a novel consensus sequence for downstream elements in 3-end processing of pre-mRNA.

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