The upstream sequence element of the C2 complement poly(A) signal activates mRNA 3′ end formation by two distinct mechanisms

Abstract

The poly(A) signal of the C2 complement gene is unusual in that it possesses an upstream sequence element (USE) required for full activity in vivo. We describe here in vitro experiments demonstrating that this USE enhances both the cleavage and poly(A) addition reactions. We also show that the C2 USE can be cross-linked efficiently to a 55-kD protein that we identify as the polypyrimidine tract-binding protein (PTB), implicated previously in modulation of pre-mRNA splicing. Mutation of the PTB-binding site significantly reduces the efficiency of the C2 poly(A) site both in vivo and in vitro. Furthermore, addition of PTB to reconstituted processing reactions enhances cleavage at the C2 poly(A) site, indicating that PTB has a direct role in recognition of this signal. The C2 USE, however, also increases the affinity of general polyadenylation factors independently for the C2 poly(A) signal as detected by enhanced binding of cleavage-stimulaton factor (CstF). Strikingly, this leads to a novel CstF-dependant enhancement of the poly(A) synthesis phase of the reaction. These studies both emphasize the interconnection between splicing and polyadenylation and indicate an unexpected flexibility in the organization of mammalian poly(A) sites.

A Complex Containing Polypyrimidine Tract-Binding Protein Is Involved in Regulating the Stability of CD40 Ligand (CD154) mRNA

“…49). PTB is also implicated in the control of polyadenylation (56,57), mRNA localization (58), and activates internal ribosome entry site-driven translation in picornaviruses (59,60). That PTB may also be involved in RNA stability is supported by results demonstrating that PTB binds to an element in the 3Ј UTR of GTPase-activating protein 43 mRNA identified as an instability element (61).…”

Protein factors in pre-mRNA 3′-end processing

“…1A), does not have a consensus sequence, but often consists of a U-rich element (UUUU) [2] or similar sequences (UGUA, UAUA) [54]. The efficiency of cleavage and polyadenylation is enhanced by the presence of this auxiliary element, as it promotes the binding of other polyadenylation factors to the cleavage site [55][56][57][58]. Most auxiliary upstream elements have been identified with the enhanced expression of intronless genes, which are normally expressed at lower levels than transcripts that contain introns [59].…”

Identification of DeltaN Isoform and Polyadenylation Site Choice Variants in Molluscan p63/p73-Like Homologues

“…The choice of a particular polyadenylation site is thought to involve 3' signaling elements, such as the polyadenylation signals (PAS) hexamers with the sequence AATAAA (or close variants ATTAAA, TTTAAA) that are located within 40 nucleotides of the cleavage site. In addition, phylogenetically conserved elements upstream of PAS, known as upstream efficiency elements (USEs) are known to enhance polyadenylation efficiency and mRNA processing (Moreira et al, 1998). Figure 6 shows that both site choice variants contain potential PAS signals within 30 nts of the poly-A tail, suggesting that both variants are in use, possibly under different cell conditions.…”