The Cleavage/Polyadenylation Activity Triggered by a U-rich Motif Sequence Is Differently Required Depending on the Poly(A) Site Location at Either the First or Last 3′-Terminal Exon of the 2′-5′ Oligo(A) Synthetase Gene

Abstract

Production of the two mRNAs encoding distinct forms of 2-5-oligoadenylate synthetase depends on processing that involves the recognition of alternative poly(A) sites and an internal 5-splice site located within the first 3-terminal exon. The resulting 1.6-and 1.8-kb mRNAs are expressed in fibroblast cell lines, whereas lymphoblastoid B cells, such as Daudi, produce only the 1.8-kb mRNA. In the present study, we have shown that the 3-end processing at the last 3-terminal exon occurs independently of the core poly(A) site sequence or the presence of regulatory elements. In contrast, in Daudi cells, the recognition of the poly(A) site at the first 3-terminal exon is impaired because of an unfavorable sequence context. The 3-end processing at this particular location requires a strong stabilization of the cleavage/polyadenylation factors, which can be achieved by the insertion of a 25-nucleotide long U-rich motif identified upstream of the last poly(A) site. Consequently, we speculate that in cells expressing the 1.6-kb mRNA, such as fibroblasts, direct or indirect participation of a specific mechanism or cell type-specific factors are required for an efficient polyadenylation at the first 3-terminal exon.Polyadenylation of nearly all eukaryotic pre-mRNAs is an obligatory step in the maturation of transcripts (reviewed in Ref. 1). The 3Ј-end processing occurs by cleavage of the precursor RNA, generating the upstream cleavage product that is elongated by a poly(A) tail (for review, see Refs. 2 and 3). The core elements of a polyadenylation signal correspond to a highly conserved hexanucleotide AAUAAA found 10 -30 nucleotides upstream of the cleavage site and a less highly conserved GU-rich element located downstream of the cleavage site. Cleavage/polyadenylation is an intricate process requiring multiple cellular factors. At the first step of the polyadenylation reaction, the GU-rich sequence is recognized by one component of the cleavage stimulating factor (CstF), 1 the protein CstF-64, which stabilizes the binding of the cleavage/polyadenylation specificity factor (CPSF) to the AAUAAA hexamer (2). Other factors that are required are the cleavage factors (CFI m , CFII m ) (4, 5) and poly(A) polymerase. Finally, the Poly(A) binding protein II (PABII) specifies the correct length of the poly(A) tail and increases the efficiency of polyadenylation (6 -8), enhancing mRNA stability and translation (9, 10).Besides the core elements and secondary structures that influence cleavage/polyadenylation (11), auxiliary sequences located either upstream or downstream of poly(A) sites have been reported to influence 3Ј-end processing efficiency in a positive or negative manner. Upstream sequence enhancers (USEs) were primarily identified in viral poly(A) sites (12-16) and further in poly(A) sites of cellular genes, such as those encoding the complement factor C2 (17), lamin B2 (18, 19), and the secretory form of IgM (20). For these genes, the presence of USEs that are often U-rich increases polyadenylation efficiency. In contrast t...

Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition

“…Strikingly, most USEs identified to date encompass one or more UGUAN sequences. These include the USEs of the SV40 late (Schek et al 1992), GSHV (Russnak 1991), HIV-1 (Valsamakis et al 1991), Lamin B2 (Brackenridge and Proudfoot 2000), 2/5Ј-oligoadenylate synthase (Aissouni et al 2002), and the collagen 1A2 (Natalizio et al 2002) poly(A) sites. A preliminary analysis of the collagen 1A2 poly(A) site indicated that mutations within the USE that reduced the efficiency of 3Ј processing also reduced the binding of CFI m (K. Venkataraman, unpubl.).…”

Sequence determinants in human polyadenylation site selection

“…It is present in a large proportion of genes, and its deletion has been shown to suppress polyadenylation [6]. The presence of a USE has been described in fewer cases: in viruses [5] and in four human genes [7-9]. The position and sequence of the USE are poorly defined, although U-richness is also suspected.…”

The relationship between the prothrombin upstream sequence element and the G20210A polymorphism: the influence of a competitive environment for mRNA 3'-end formation

“…USEs are described as U-rich in sequence (12) and have been predominately characterized in viruses such as SV40, adenovirus, hepatitis B and HIV-1 pre-mRNAs (16–19). More recently, functional USEs have been described in cellular genes including complement factor C2, lamin B2, collagen and 2′-5′-oligoadenylate synthetase enzyme (20–23). …”