Abstract
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.
Highlights
The primary transcripts of a eukaryotic cell undergo several different maturation steps to become fully functional messenger RNAs
To investigate whether this 70-kDa subunit represents an alternative form of the 64-kDa polypeptide, calf thymus and HeLa cleavage stimulation factor (CstF) were separated on high resolution SDS-polyacrylamide gels and either stained with silver (Fig. 1B) or used for Western blot analysis and immunodetection with polyclonal antibodies raised against the human 64-kDa subunit (Fig. 1C)
SV-G/G containing two minimal GU motifs was processed as efficiently as SV-G/A and reached nearly wild type activity at higher CstF concentrations (Fig. 3, A and D, right panel). This indicates that the GU motif can substitute for the AY motif, but not vice versa, and suggests that the GU motif is the more important part of element 2a. These results demonstrate that the sequences selected by CstF as well as shortened versions are able to function as downstream elements in 3Ј-end processing of pre-messenger RNAs (mRNAs), to different extents
Summary
The primary transcripts (pre-mRNAs) of a eukaryotic cell undergo several different maturation steps to become fully functional messenger RNAs (mRNAs) One of these maturation events is the 3Ј-end processing reaction, during which the pre-mRNA receives a new 3Ј-end that is in almost all cases a poly(A) tail. In vivo and in vitro studies have revealed a requirement for distinct sequence elements for 3Ј-end processing of pre-mRNAs, a highly conserved AAUAAA sequence located upstream of the poly(A) site and so-called downstream elements. Several sequences located upstream of the AAUAAA signal have been shown to enhance the cleavage reaction It was shown that a UUUUU element located 6 –25 nucleotides downstream of the AAUAAA sequence is sufficient to confer cleavage activity to a substrate whose natural downstream region has been completely deleted [14]. Moving the downstream element further downstream can abolish cleavage but can shift the cleavage site [14, 19, 21,22,23,24,25]
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