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

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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