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Participation of the Nuclear Cap Binding Complex in Pre-Mrna 3 Processing

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Participation of the Nuclear Cap Binding Complex in Pre-Mrna 3 Processing Proc. Natl. Acad. Sci. USA Vol. 94, pp. 11893–11898, October 1997 Biochemistry Participation of the nuclear cap binding complex in pre-mRNA 3* processing SEAN M. FLAHERTY*, PURI FORTES†,ELISA IZAURRALDE†‡,IAIN W. MATTAJ†, AND GREGORY M. GILMARTIN*§ *Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, Vermont Cancer Center, University of Vermont, Burlington, VT 05405; †Gene Expression Programme, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; and ‡Department of Molecular Biology, Sciences II, 30, quai Ernest-Ansermet, 1211 Geneva 4, Switzerland Communicated by Christine Guthrie, University of California, San Francisco, CA, August 29, 1997 (received for review May 7, 1997) ABSTRACT Communication between the 5* and 3* ends is processing was not completely abolished (16, 18). Uncapped a common feature of several aspects of eukaryotic mRNA pre-mRNAs were found to be poorly processed in nuclear extract, metabolism. In the nucleus, the pre-mRNA 5* end is bound by and have been shown to compete less efficiently for 39 processing the nuclear cap binding complex (CBC). This RNA–protein factors than capped pre-mRNAs (17, 18). Although not essential complex plays an active role in both splicing and RNA export. for 39 end formation in vitro, the impact of the cap upon We provide evidence for participation of CBC in the process- processing in crude nuclear extract suggested that the cap may ing of the 3* end of the message. Depletion of CBC from HeLa indeed contribute to processing within the complex milieu of the cell nuclear extract strongly reduced the endonucleolytic nucleus. A similar picture has emerged from the investigation of cleavage step of the cleavage and polyadenylation process. pre-mRNA splicing, in which the 59 cap, although likewise not Cleavage was restored by addition of recombinant CBC. CBC essential for processing, does contribute to the removal of the cap depletion was found to reduce the stability of poly(A) site proximal intron (19–22). cleavage complexes formed in nuclear extract. We also provide In the nucleus, the 59 cap is bound by the nuclear cap-binding evidence that the communication between the 5* and 3* ends complex (CBC) that in human cells is comprised of two of the pre-mRNA during processing is mediated by the phys- proteins: CBP20 and CBP80 (23–27). This RNA–protein com- ical association of the CBCycap complex with 3* processing plex plays an active role in both splicing (19–21, 24) and RNA factors bound at the poly(A) site. These observations, along export (25, 28). We now show that CBC participates in a with previous data on the function of CBC in splicing, second mRNA processing reaction. Depletion of CBC from illustrate the key role played by CBC in pre-mRNA recognition HeLa cell nuclear extract diminished poly(A) site cleavage, and processing. The data provides further support for the and subsequent addition of recombinant CBC restored pro- hypothesis that pre-mRNAs and mRNAs may exist and be cessing. Poly(A) site cleavage complex stability was found to be functional in the form of ‘‘closed-loops,’’ due to interactions reduced in CBC-depleted nuclear extracts, and the physical between factors bound at their 5* and 3* ends. interaction of the CBCycap complex with the 39 processing complex was demonstrated by coimmunoprecipitation. The biosynthesis of most eukaryotic nuclear mRNAs requires The 59 and 39 ends of eukaryotic mRNAs communicate at the modification of the 59 end of the RNA by the cotranscrip- nearly every stage in the life of the message. Nuclear export tional addition of an m7G(59)ppp(59)N cap structure (1, 2), the (29), translation (30–32), and mRNA decay (33) all appear to removal of introns by splicing (3), and the modification of the involve the coordinate recognition of RNA–protein complexes 39 end by endonucleolytic cleavage and poly(A) addition (4, 5). at both termini. We now present evidence that CBC bound at Polyadenylation in vertebrates requires two cis-acting RNA the 59 end of the pre-mRNA participates in the actual biosyn- sequence elements which straddle the cleavage site and embody thesis of the mRNA 39 end. the core poly(A) site: the AAUAAA hexamer 10–30 nucleotides METHODS upstream of the cleavage site, and an amorphous U- or G1U-rich element downstream of the cleavage site. Six factors, comprised Preparation of HeLa Nuclear Extracts. HeLa cell nuclear of at least 13 proteins, are required for pre-mRNA cleavage and extracts were prepared as described (34), with the following polyadenylation (4, 5). Cleavage and polyadenylation specificity exceptions. All buffers contained Tris (pH 7.9) at the concen- factor (CPSF) (6, 7) binds the pre-mRNA upon recognition of the trations indicated for HepeszKOH. Buffer C contains pepstatin at AAUAAA hexamer, while cleavage stimulatory factor (CstF) (8) 0.7 mgyml and leupeptin at 0.4 mgyml. The high salt buffer C binds the downstream element. Together, CPSF and CstF form contained 800 mM (NH4)2SO4 instead of NaCl, and the low salt a relatively stable pre-mRNA–protein complex (9) that allows for buffer C contained KCl instead of NaCl. The nuclear extract was centrifuged for 60 min at 95,000 3 g and used undialyzed for in the recruitment of cleavage factors Im and IIm (10), and poly(A) polymerase (11, 12). Following the endonucleolytic cleavage of vitro assays. the pre-mRNA, poly(A) addition requires both CPSF and Depleted Nuclear Extracts. Depletions were conducted as poly(A) polymerase (7, 13). Poly(A) binding protein II, however, described (24), with the following exceptions. The HeLa nuclear confers both processivity and tail length control to the poly(A) extract was adjusted to 500 mM (NH4)2SO4 and then clarified by addition reaction (14, 15). centrifugation at 14,000 rpm in a microfuge for 5 min at 4°C prior The 59 cap structure has been shown to influence the efficiency to filtration through a 0.45 mm syringe filter. Eluates from of 39 processing in vitro (16–18). The addition of the cap analog filtration were recycled over protein-A Sepharose antibody col- m7GpppG to HeLa cell nuclear extract resulted in a reduction in umns (either the rabbit preimmune or the polyclonal anti- poly(A) site cleavage, although even at high levels of the analog, CBP80), which had been crosslinked as described (24). CBC The publication costs of this article were defrayed in part by page charge Abbreviations: CBC, cap binding complex; CPSF, cleavage and poly- adenylation specificity factor; CstF, cleavage stimulatory factor; rCBC, payment. This article must therefore be hereby marked ‘‘advertisement’’ in recombinant CBC; TMG, trimethylguanosine; RNP, ribonucleopro- accordance with 18 U.S.C. §1734 solely to indicate this fact. tein; snRNP, small nuclear RNP. © 1997 by The National Academy of Sciences 0027-8424y97y9411893-6$2.00y0 §To whom reprint requests should be addressed. e-mail: ggilmart@ PNAS is available online at http:yywww.pnas.org. zoo.uvm.edu. 11893 Downloaded by guest on September 29, 2021 11894 Biochemistry: Flaherty et al. Proc. Natl. Acad. Sci. USA 94 (1997) levels were monitored by enhanced chemiluminescence (Amer- reaction, 4.0 3 10212 mol of unlabeled capped L3 pre-mRNA sham) Western blot analysis, using a polyclonal CBP80 antiserum. was added. Incubation of the reaction was continued at 30°C Depleted extracts were dialyzed into buffer D (100 mM KCly20 and 5 ml aliquots were removed from each reaction at 5, 10, 20, mM Hepes, pH 7.9y20% glyceroly0.2 mM EDTAy0.5 mM and 40 min, placed on ice and treated with 5 mgyml of heparin. DTTy0.1 mM phenylmethylsulfonyl fluoride). Reactions were electrophoresed on a nondenaturing 3% poly- Preparation of Recombinant CBC. rCBC was prepared as acrylamide (80:1) gel in 25 mM Tris, 25 mM boric acid, and 1 described (25). Briefly, human CBP20 was expressed in Esch- mM EDTA at 300 V for 1.5 hr at 4°C. The gel was preelec- erichia coli (1-l culture) as a histidine-tagged protein, and trophoresed for 30 min prior to loading. human CBP80 was expressed in E. coli (6-l culture) as an RNase HyImmunoprecipitations. RNA–protein complexes untagged protein. Histidine-tagged CBP20 was purified on a were assembled as follows: 125 ml reactions contained 12.5% nickel-nitrilotriacetic acid column as previously described (volyvol) of nuclear extract (preadsorbed with protein A- (25). 2 ml of purified CBP20 along with 0.5 ml nickel- Sepharose to remove nonspecific binding proteins), 1% polyvinyl nitrilotriacetic acid-agarose was added to a CBP80 expressing alcohol, 2.5 mg tRNA, 30 mg total yeast RNA, 75 mM KCl, 15 mM lysate and incubated at room temperature for 1 hr. The Tris (pH 7.9), 7.5% glycerol, 20 mM EDTA, 50 mM DTT, 10 mM 213 heterodimeric CBC complex was then purified via the histidine phenylmethylsulfonyl fluoride, 1 mM MgCl2, and 2.2 3 10 mol tag resident on CBP20. Purification of CBC was monitored by (500,000 dpm) of 32P-labeled HIV-1 pre-mRNA (useyCPS full- gel mobility shift assays using capped (m7GpppG) or uncapped length; ref. 35). The HIV-1 pre-mRNA containing a wild-type (pppG) 32P-labeled RNAs. Gel shift assays were conducted as hexamer (AAUAAA) was either capped (m7GpppG) or un- described (23). capped (pppG), and an HIV-1 pre-mRNA containing a mutant Generation of 32P-Labeled RNA Substrates. L3 RNA sub- hexamer (AAUAAA 3 CCACCC) was capped. Reactions were strates for in vitro cleavage reactions were prepared as described incubated for 10 min at 30°C. For RNase H digestion, 1.3 mM of (9). The RNA previously termed AAU 1 ds (9) is referred to the DNA oligo (59-GTACAGGCAAAA-39), complementary to here as L3.
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