At least half of all human pre-mRNAs are subject to alternative 3 processing that may modulate both the coding capacity of the message and the array of post-transcriptional regulatory elements embedded within the 3 UTR. Vertebrate poly(A) site selection appears to rely primarily on the binding of CPSF to an A(A/U)UAAA hexamer upstream of the cleavage site and CstF to a downstream GU-rich element. At least one-quarter of all human poly(A) sites, however, lack the A(A/U)UAAA motif. We report that sequence-specific RNA binding of the human 3 processing factor CFI m can function as a primary determinant of poly ( The process of mRNA 3Ј end formation is not simply a perfunctory step in eukaryotic gene expression. At least one-half of all human genes are subject to alternative 3Ј processing (Iseli et al. 2002), the consequences of which may impact the protein coding capacity of the message, as well as its localization, translation efficiency, and stability (Edwalds-Gilbert et al. 1997). Moreover, poly(A) site selection may be modulated in a developmental and tissue-specific manner. In addition, pre-mRNA 3Ј processing contributes directly to transcription termination (Zorio and Bentley 2004), pre-mRNA splicing , and mRNA export (Hammell et al. 2002;Lei and Silver 2002). While the processing of constitutive poly(A) sites has been examined in considerable detail, the fundamental mechanisms responsible for the regulation of alternative poly(A) site selection have yet to be fully elucidated (Barabino and Keller 1999).The processing of the majority of human poly(A) sites involves the recognition of an AAUAAA or AUUAAA hexamer by CPSF, coupled with the binding of CstF to a GU-rich downstream element (DSE) (Zhao et al. 1999). The binding of CPSF and CstF appears to be sufficient, at least in vitro, to direct the assembly of a 3Ј processing complex composed of at least 14 different proteins. In vivo, however, the hexamer and DSE alone are unlikely to suffice for poly(A) site definition. The recognition of an authentic poly(A) site within a nascent RNA in vivo appears to rely on the "biosynthetic context" provided by the transcription elongation complex (Proudfoot 2004). At least nine 3Ј processing proteins are recruited to the transcription complex, at least in part through interactions with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) (Calvo and Manley 2003). The colocalization of 3Ј processing factors, along with capping enzymes and spliceosome components, to the transcription elongation complex, allows for the cooperative interaction of these processing machineries within an "mRNA factory" (Zorio and Bentley 2004).Cotranscriptional recognition of a poly(A) site provides an elegant mechanism for the identification of a processing site demarcated by a limited set of sequence motifs. Yet the mechanisms that regulate the selection of alternative poly(A) sites within a pre-mRNA, or allow for the recognition of poly(A) sites that lack the canonical A(A/U)UAAA motif, are poorly understood. Seque...

Four HeLa cell nuclear factors that are required for specific pre-mRNA cleavage and polyadenylation have been extensively purified, thereby permitting an investigation of the role of each in the 3' processing reaction. Two factors, termed PF1 and PF2, are required for specific polyadenylation of the cleaved RNA. PFI is a poly(A) polymerase, and PF2 is a factor that confers AAUAAA specificity to the reaction. Both of these factors, along with two additional factors termed CF1 and CF2, are required for the endonucleolytic cleavage of the premRNA. The ability of each of these factors to form specific complexes with the pre-mRNA was assayed using native gel electrophoresis. Two distinct complexes were detected. PF2 forms an initial complex with the pre-RNA, dependent on the AAUAAA sequence element but independent of specific downstream sequences. Formation of the PF2-RNA complex permits the subsequent interaction of CF1 and the formation of a second, larger complex. CF1 binding requires the downstream sequence element in addition to PF2 binding. Whereas the PF2-RNA complex is unstable and dissociates rapidly, the ternary complex formed by CF1, PF2, and RNA is stable. Thus, the interaction of CFI, dependent on the downstream sequence element, can be viewed as a commitment of the poly(A) site for processing. On the addition of the poly(A) polymerase (PFI) and factor CF2, the pre-mRNA is specifically cleaved at the poly(A) site.

Human cleavage factor I(m) (CFI(m)) is a heterodimeric RNA binding protein complex that functions at an early step in the assembly of the pre-mRNA 3' processing complex. In this report we show that CFI(m) can stimulate both cleavage and poly(A) addition, and can act to suppress poly(A) site cleavage in a sequence-dependent manner. Elevated levels of CFI(m) suppressed cleavage at the primary poly(A) site of the pre-mRNA encoding the 68 kDa subunit of CFI(m). CFI(m)-mediated suppression of poly(A) site cleavage was dependent upon the presence of three copies of an RNA element initially identified by CFI(m)-SELEX. These data provide evidence for a mechanism for the regulation of poly(A) site selection by a basal pre-mRNA 3' processing factor.

Communication between the 5 and 3 ends is a common feature of several aspects of eukaryotic mRNA metabolism. In the nucleus, the pre-mRNA 5 end is bound by the nuclear cap binding complex (CBC). This RNA-protein complex plays an active role in both splicing and RNA export. We provide evidence for participation of CBC in the processing of the 3 end of the message. Depletion of CBC from HeLa cell nuclear extract strongly reduced the endonucleolytic cleavage step of the cleavage and polyadenylation process. Cleavage was restored by addition of recombinant CBC. CBC depletion was found to reduce the stability of poly(A) site cleavage complexes formed in nuclear extract. We also provide evidence that the communication between the 5 and 3 ends of the pre-mRNA during processing is mediated by the physical association of the CBC͞cap complex with 3 processing factors bound at the poly(A) site. These observations, along with previous data on the function of CBC in splicing, illustrate the key role played by CBC in pre-mRNA recognition and processing. The data provides further support for the hypothesis that pre-mRNAs and mRNAs may exist and be functional in the form of ''closed-loops,'' due to interactions between factors bound at their 5 and 3 ends.The biosynthesis of most eukaryotic nuclear mRNAs requires the modification of the 5Ј end of the RNA by the cotranscriptional addition of an m 7 G(5Ј)ppp(5Ј)N cap structure (1, 2), the removal of introns by splicing (3), and the modification of the 3Ј end by endonucleolytic cleavage and poly(A) addition (4, 5).Polyadenylation in vertebrates requires two cis-acting RNA sequence elements which straddle the cleavage site and embody the core poly(A) site: the AAUAAA hexamer 10-30 nucleotides upstream of the cleavage site, and an amorphous U-or GϩU-rich element downstream of the cleavage site. Six factors, comprised of at least 13 proteins, are required for pre-mRNA cleavage and polyadenylation (4, 5). Cleavage and polyadenylation specificity factor (CPSF) (6, 7) binds the pre-mRNA upon recognition of the AAUAAA hexamer, while cleavage stimulatory factor (CstF) (8) binds the downstream element. Together, CPSF and CstF form a relatively stable pre-mRNA-protein complex (9) that allows for the recruitment of cleavage factors I m and II m (10), and poly(A) polymerase (11,12). Following the endonucleolytic cleavage of the pre-mRNA, poly(A) addition requires both CPSF and poly(A) polymerase (7, 13). Poly(A) binding protein II, however, confers both processivity and tail length control to the poly(A) addition reaction (14, 15).The 5Ј cap structure has been shown to influence the efficiency of 3Ј processing in vitro (16)(17)(18). The addition of the cap analog m 7 GpppG to HeLa cell nuclear extract resulted in a reduction in poly(A) site cleavage, although even at high levels of the analog, processing was not completely abolished (16,18). Uncapped pre-mRNAs were found to be poorly processed in nuclear extract, and have been shown to compete less efficiently for 3Ј processing factors...

Summary Cleavage Factor Im (CFIm) is a highly conserved component of the eukaryotic mRNA 3′ processing machinery that functions in sequence-specific poly(A) site recognition through the collaboration of two protein subunits, a 25 kDa subunit containing a nudix domain and a larger subunit of 59, 68, or 72 kDa containing an RNA recognition motif (RRM). Our previous work demonstrated that CFIm25 is both necessary and sufficient for sequence-specific binding of the poly(A) site upstream element UGUA. Here we report the crystal structure of CFIm25 complexed with the RRM domain of CFIm68 and RNA. The CFIm25 dimer is clasped on opposite sides by two CFIm68 RRM domains. Each CFIm25 subunit binds one UGUA element specifically. Biochemical analysis indicates that the CFIm68 RRMs serve to enhance RNA binding and facilitate RNA looping. The intrinsic ability of CFIm to direct RNA looping may provide a mechanism for its function in the regulation of alternative poly(A) site selection.

Human Cleavage Factor Im (CFI m ) is an essential component of the pre-mRNA 3′ processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFI m 25) of the CFI m complex possesses a characteristic α/β/α Nudix fold, CFI m 25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFI m 25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFI m 25 is the first Nudix protein to be reported to bind RNA in a sequencespecific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson-Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap 4 A (diadenosine tetraphosphate) by CFI m 25 suggests a potential role for small molecules in the regulation of mRNA 3′ processing.T he transcriptome complexity of higher eukaryotes requires the coordinate recognition of an array of alternative pre-mRNA processing signals in a developmental and tissue-specific manner (1, 2). The sequences that direct pre-mRNA splicing and 3′ processing are initially recognized within the nascent transcript in a process that is intimately coupled to transcription (3, 4). While the recognition of exons within the pre-mRNA is mediated by both RNA:RNA and protein:RNA interactions (5), the 3′ processing of polyadenylated mRNAs appears to rely solely on the interaction of protein factors (6) with unstructured RNA sequences (7) within the nascent transcript.Vertebrate pre-mRNA 3′ processing signals are recognized by a tripartite mechanism through which a set of short RNA sequences direct the cooperative binding of three multimeric 3′ processing factors, cleavage factor I m (CFI m ), cleavage and polyadenylation specificity factor (CPSF), and cleavage stimulation factor (CstF) (8). CPSF and CstF bind the AAUAAA hexamer and downstream GU-rich elements that flank the poly(A) site, respectively, whereas CFI m interacts with upstream sequences that may function in the regulation of alternative polyadenylation (9-11). SELEX and biochemical analyses have identified the sequence UGUAN (N ¼ A > U > G, C) as the preferred binding site of CFI m (11). In this report we have taken a structural approach to determine the mechanism of sequence-specific RNA binding by CFI m .CFI m is composed of a large subunit of 59, 68, or 72 kDa and a small subunit of 25 kDa (CFI m 25, also referred to as CPSF5 or NUDT21) (12, 13), both of which contribute to RNA binding (14). The large subunit, encoded by either of two paralogs (CPSF6 and CPSF7), contains an N-terminal RNA Recognition Motif (RRM), an internal polyproline-rich region, and a C-termina...

The endonucleolytic cleavage and polyadenylation of a pre-mRNA in mammalian cells requires two c/s-acting elements, a highly conserved AAUAAA hexamer and an amorphous U-or GU-rich downstream element, that together constitute the "core" poly(A) site. The terminal redundancy of the HIV-1 pre-mRNA requires that the processing machinery disregard a core poly(A) site at the 5' end of the transcript, and efficiently utilize an identical signal that resides near the 3' end. Efficient processing at the 3' core poly(A) site, both in vivo and in vitro, has been shown to require sequences 76 nucleotides upstream of the AAUAAA hexamer. In this report we demonstrate that this HIV-1 upstream element interacts directly with the 160-kD subunit of CPSF (cleavage polyadenylation specificity factor), the factor responsible for the recognition of the AAUAAA hexamer. The presence of the upstream element in the context of the AAUAAA hexamer directs the stable binding of CPSF to the pre-mRNA and enhances the efficiency of poly(A) addition in reactions reconstituted with purified CPSF and recombinant poly(A) polymerase. Our results indicate that the dependence of HIV-1 3' processing on upstream sequences is a consequence of the suboptimal sequence context of the AAUAAA hexamer. We suggest that poly(A) site definition involves the recognition of multiple heterogeneous sequence elements in the context of the AAUAAA hexamer.

Poly(A) site processing of a pre-mRNA requires the participation of multiple nuclear factors. Two of these factors recognize specific sequences in the pre-mRNA and form a stable processing complex. Since these initial interactions are likely critical for the recognition of the poly(A) site and the efficiency of poly(A) site use, we have characterized these factors and the nature of their interaction with the pre-mRNA. The AAUAAA specificity factor PF2 is a large, multicomponent complex composed of at least five distinct polypeptides ranging in molecular size from 170 to 42 kDa. The 170-kDa polypeptide appears to mediate interaction with the pre-mRNA. Factor CF1, which provides specificity for the downstream G+U-rich element and stabilizes the PF2 interaction on the RNA, is also a multicomponent complex but is less complex than PF2. CF1 is composed of three polypeptides of molecular sizes 76, 64, and 48 kDa. UV cross-linking assays demonstrate that the 64-kDa polypeptide makes direct contact with the RNA, dependent on the G+U-rich downstream sequence element. Moreover, it is clear that these RNA-protein interactions are influenced by the apparent cooperative interaction involving PF2 and CF1, interactions that contribute to the efficiency of poly(A) site processing.