The 64-kilodalton subunit of the CstF polyadenylation factor binds to pre-mRNAs downstream of the cleavage site and influences cleavage site location.

Abstract

The CstF polyadenylation factor is a multisubunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. Using an RNase H-mediated mapping technique, we show that the 64-kDa subunit of CstF can be photo cross-linked to pre-mRNAs at U-rich regions located downstream of the cleavage site of the simian virus 40 late and adenovirus L3 pre-mRNAs. This positional specificity of cross-linking is a consequence of CstF interaction with the polyadenylation complex, since the 64-kDa protein by itself is cross-linked at multiple positions on a pre-mRNA template. During polyadenylation, four consecutive U residues can substitute for the native downstream U-rich sequence on the simian virus 40 pre-mRNA, mediating efficient 64-kDa protein cross-linking at the downstream position. Furthermore, the position of the U stretch not only enables the 64-kDa polypeptide to be cross-linked to the pre-mRNA but also influences the site of cleavage. A search of the GenBank database revealed that a substantial portion of mammalian polyadenylation sites carried four or more consecutive U residues positioned so that they should function as sites for interaction with the 64-kDa protein downstream of the cleavage site. Our results indicate that the polyadenylation machinery physically spans the cleavage site, directing cleavage factors to a position located between the upstream AAUAAA motif, where the cleavage and polyadenylation specificity factor is thought to interact, and the downstream U-rich binding site for the 64-kDa subunit of CstF.Polyadenylation of precursor mRNAs involves a series of distinct steps: recognition of pre-mRNA substrates which contain the hexanucleotide sequence AAUAAA (19) by polyadenylation factors; endonucleolytic cleavage of the premRNA at a distinct site five to 40 nucleotides (nt) 3' of the hexanucleotide; and addition of approximately 200 adenosine residues at the 3' end (11,22,29). In vivo, these processes are tightly coupled, but the steps can be readily uncoupled in vitro. Previous studies have identified U-rich or GU-rich regions, located as far as 50 nt downstream of the cleavage site, which influence polyadenylation both in vivo and in vitro (6, 13-15, 20, 21, 23, 24, 34). To date, the mechanism by which these downstream sequence elements influence polyadenylation has not been elucidated.A 64-kDa (64K) polypeptide can be efficiently photo crosslinked to AAUAAA-containing RNA substrates that are undergoing polyadenylation in nuclear extracts of HeLa cells (31,16). The 64K polypeptide is a subunit of cleavage and polyadenylation stimulation factor (CstF [27,28]; also termed CF1 [7,8]). A role for the factor in poly(A) addition was confirmed by the ability of antibodies specific for the 64K polypeptide to deplete CstF and block in vitro polyadenylation (27). The 64K polypeptide will specifically cross-link to AAUAAA-containing mRNAs as part of the CstF complex only in the presence of a second multisubunit factor (33) termed the cleavage and polyadenylation specificity factor (CPSF; formerly...

Protein factors in pre-mRNA 3′-end processing

“…The nucleotide sequence surrounding the cleavage site is not strictly conserved [51,52]. In vertebrate pre-mRNAs, 70% of the cleavage sites are located at the 3′ side of an adenosine residue [53], with a nucleotide preference of A > U > C ≫ G. The nucleotide preceding the cleavage site is cytosine in 59% of 269 pre-mRNA sequences examined [53], making the optimal cleavage site CA (Fig.…”

Cleavage Factor II of Saccharomyces cerevisiaeContains Homologues to Subunits of the Mammalian Cleavage/ Polyadenylation Specificity Factor and Exhibits Sequence-specific, ATP-dependent Interaction with Precursor RNA

“…However, subsequent experiments demonstrated that p64 was in contact with the GU-or U-rich downstream element (8), an interaction stabilized by CPSF acting at the AAUAAA site (39,40). The Rna15 subunit of yeast CF IA, whose ribonucleoprotein-type RNA binding domain closely ressembles that of CstF p64 (41), can be cross-linked to polyadenylation substrate, but this interaction does not show a sequence dependence like that of Cft2 (22).…”

“…4). The largest subunit (160 kDa) of CPSF binds specifically to the AAUAAA signal located upstream of the poly(A) site (6,7), and the CstF binds to the GUor U-rich downstream element via its 64-kDa subunit (8).…”

Chimeric human CstF-77/ Drosophila Suppressor of forked proteins rescue suppressor of forked mutant lethality and mRNA 3′ end processing in Drosophila

“…CstF-77 bridges the other two subunits of the complex (21). CstF-64 is responsible for the binding of CstF to RNA (12), as it interacts through a ribonucleoprotein-type RNA binding domain with the U͞GU-rich element downstream of poly(A) sites. CstF-77 and CstF-50 interact with the carboxyl-terminal domain of RNA polymerase II (14).…”

“…Assembly of the cleavage complex on the pre-mRNA results from multiple RNA-protein and protein-protein interactions. CPSF binds to a highly conserved AAUAAA element (11), the polyadenylation signal, located upstream of the cleavage site, whereas CstF recognizes a U͞GU-rich element (12), located downstream of the cleavage site. Binding of CPSF and CstF to the pre-mRNA is cooperative and allows the recognition of the poly(A) site.…”