Participation of the nuclear cap binding complex in pre-mRNA 3′ processing

Abstract

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...

Subnuclear Localization and Dynamics of the Pre-mRNA 3′ End Processing Factor Mammalian Cleavage Factor I 68-kDa Subunit

“…Polyadenylation requires in addition to CPSF, poly(A) polymerase, and the nuclear poly(A) binding protein 1 (PABPN1, previously called PAB II, for review, see Wahle and Rü egsegger, 1999). Other proteins involved in either transcription, such as the carboxy-terminal domain of RNA polymerase II, or capping (nuclear cap-binding complex) and splicing (U2AF65) have been shown to greatly enhance the efficiency of the first step of the reaction (Flaherty et al, 1997;Hirose and Manley, 1998;Millevoi et al, 2002).…”

“…Polyadenylation requires in addition to CPSF, poly(A) polymerase, and the nuclear poly(A) binding protein 1 (PABPN1, previously called PAB II, for review, see Wahle and Rü egsegger, 1999). Other proteins involved in either transcription, such as the carboxy-terminal domain of RNA polymerase II, or capping (nuclear cap-binding complex) and splicing (U2AF65) have been shown to greatly enhance the efficiency of the first step of the reaction (Flaherty et al, 1997;Hirose and Manley, 1998;Millevoi et al, 2002).The mammalian cell nucleus is a highly structured and dynamic compartment. Many nuclear factors are localized in morphologically well-defined structural units that include the nucleolus and several "nuclear bodies," such as the Cajal body (Gall, 2000), and the promyelocytic leukemia body (Zhong et al, 2000).…”

Transcriptional termination in the Balbiani ring 1 gene is closely coupled to 3′-end formation and excision of the 3′-terminal intron

“…The nuclear cap binding complex, CBC, stimulates excision of the most 5Ј-located intron in vitro (Ohno et al 1987;Inoue et al 1989;Izaurralde et al 1994) and has been shown to facilitate U1 snRNP binding to the cap-proximal 5Ј-splice site (Lewis et al 1996). It has also been shown that CBC enhances endonucleolytic cleavage at the polyadenylation site (Flaherty et al 1997).…”

Synergie entre les complexes de transcription et de maturation des ARN messagers

“…(Figure 4). Toutefois, il existe encore peu de données susceptibles d'éclairer la relation entre la structure coiffe et le processus de maturation de l'extrémité 3' de l'ARN pré-m. Malgré tout, on constate bien une association entre le complexe CBP20-CBP80 et certains facteurs participant au clivage endonucléolytique [14]. Cette affinité a pour objectif premier d'augmenter l'efficacité du clivage par la stabilisation de CPSF et de CstF à l'ARN.…”

“…Cette affinité a pour objectif premier d'augmenter l'efficacité du clivage par la stabilisation de CPSF et de CstF à l'ARN. On retrouve toutefois une influence positive sur l'élongation de la queue de poly(A) qui demeure modeste (Figure 4) [4,14]. En effet, la délétion du complexe CBP20-CBP80 a La position exacte de ce dernier site est déterminée par la distance existant entre l'hexanucléotide et la séquence riche en U ou en G/U.…”