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 (YGUGUYN0-4UUYAYUGYGU) and element 2b (UUGYUN0-4AUUUACU(U/G)N0-2YCU). 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.
Beyer
K
K
Department of Cell Biology, Biozentrum of the University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Dandekar
T
T
Keller
W
W
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013950:Thymus Gland / Q000378:metabolism; D039221:mRNA Cleavage and Polyadenylation Factors
D008024:Ligands; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D039221:mRNA Cleavage and Polyadenylation Factors