Biochemical and Biophysical Research Communications 289, 513–518 (2001)

doi:10.1006/bbrc.2001.5992, available online at http://www.idealibrary.com on

Interaction of Poly(A) Polymerase with the 25-kDaSubunit of Cleavage Factor I

Hana Kim*,† and Younghoon Lee*,†,1

*Department of Chemistry and †Center for Molecular Design and Synthesis,Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea

Received October 25, 2001

nals (NLS1 and NLS2), and a serine/threonine-rich

Mammalian poly(A) polymerase (PAP), a key enzyme

in the pre-mRNA 3*-end processing reaction, carriesthe catalytic domain in the N-terminal region, an RNAbinding domain, two nuclear localization signals, anda serine/threonine-rich regulatory domain in theC-terminal region. Using LexA-based yeast two-hybridscreening, we identified a cDNA encoding the 25-kDasubunit of cleavage factor I (CFI-25) as a protein thatinteracts with the C-terminal region of mouse PAP.The glutathione S-transferase pull-down assay and theimmunoprecipitation experiment revealed that PAPdirectly interacts with CFI-25 and that the C-terminal69 residues of PAP and the N-terminal 60 residues ofCFI-25 are sufficient for the interaction betweenCFI-25 and PAP. Since CFI is known to function in theassembly of the pre-mRNA 3*-processing complex, thisinteraction may play an important role in the assem-bly of the processing complex and/or in the regulationof PAP activity within the complex. © 2001 Elsevier Science

Poly(A) polymerase (PAP) is a key protein in thepre-mRNA 39-end processing reaction in mammals be-cause PAP catalytically participates in the polyadenyl-ation reaction of pre-mRNA. The mammalian pre-mRNA 39-end processing machinery requires at leastsix factors: (i) cleavage- and polyadenylation-specificityfactor (CPSF), (ii) cleavage-stimulation factor (CstF),(iii) and (iv) two cleavage factors (CFI and CFII), (v)PAP, and (vi) poly(A)-binding protein II (PAB II) (re-viewed in Refs. 1–3). PAP adds the poly(A) tails of200–250 residues at the specific sites of pre-mRNAsintroduced by CPSF, CstF, CFI, and CFII. The cata-lytic function of PAP resides in its N-terminal region,which has the sequence similarity with a nucleotidyltransferase family (4). The C-terminal region carriesan RNA-binding region, two nuclear localization sig-

1 To whom correspondence and reprint requests should be ad-dressed. Fax: 182-42-869-2810. E-mail: ylee@mail.kaist.ac.kr.

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region (5, 6). The activity of PAP is regulated by phos-phorylation during the cell cycle (7, 8). The serine/threonine-rich domain of PAP is responsible for theregulation by phosphorylation. The C-terminal regionof PAP is also involved in the coupling of splicingand polyadenylation via protein–protein interactionswith splicing factors U1A and U2AF65 (9, 10). TheC-terminal region of PAP can serve as a target of otherregulatory proteins for protein–protein interactions.Thus, a search for partner proteins that interact withthe C-terminal region of PAP is essential in order tounderstand the functional significance of the regula-tion of the PAP functions in the pre-mRNA 39-processing.

In this study, we performed a LexA-based yeast two-hybrid screening to identify PAP-interaction partnerproteins, which would be involved in the regulation ofthe PAP functions. One clone was found to encode the25-kDa subunit of CFI (CFI-25). We show that theC-terminal region of PAP directly interacts with CFI-25, and we discuss the significance of this interactionin the mammalian polyadenylation machinery.

MATERIALS AND METHODS

Yeast two-hybrid techniques. The Matchmaker LexA two-hybridsystem was obtained from Clontech. The system consists of twoplasmids: (i) pEQ202 expresses a LexA-fused bait and (ii) pJG4-5expresses a B42 transactivation domain fused to interacting-partnerproteins. The yeast two-hybrid assay was performed using the yeaststrain EGY048, which was transformed with the appropriate plas-mids. Yeast cells were grown in SD media lacking histidine, trypto-phan, and uracil, and tested for b-galactosidase activity. Clonesexpressing b-galactosidase activity were screened on the plates byusing 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) as asubstrate. Alternately, the b-galactosidase activity of the liquid cul-ture was determined using the substrate o-nitrophenyl-b-D-galac-topyranoside (ONPG) as described (11).

Yeast two-hybrid screening. The C-terminal region of PAP, whichconsists of 268 amino acids, was used as a bait for yeast two-hybridscreening. For the bait construction, the cDNA encoding the

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C-terminal region of PAP was amplified by a polymerase chain

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reaction (PCR) and cloned into the BamHI–XhoI site of the pEQ202LexA fusion-plasmid vector. The human HeLa cDNA library, whichwas cloned into vector pJG4-5, was screened from 2 3 106 primarytransformants of yeast strain EGY48. The insert DNA from one ofthe selected clones was completely sequenced. BLAST search iden-tified this sequence as cDNA coding for the 25-kDa-subunit of cleav-age factor I (CFI-25). The coding sequence of mouse CFI-25 wasobtained by PCR amplification from a mouse kidney cDNA libraryusing the sequence information for mouse CFI-25 cDNA (GenBankAccession No. NM007006).

Preparation of recombinant cDNA constructs. Different LexA–PAP fusion constructs encoding full-length or defined regions ofmouse PAP were made by PCR amplification and cloned into theBamHI–XhoI site of pEQ202. Full-length mouse CFI-25 fused to theB42 transactivating domain was constructed by PCR amplificationand cloning into BamHI–XhoI of pJG4-5. Different B42–CFI fusionconstructs encoding defined regions of CFI-25 were also made byPCR amplification and cloned into the BamHI–XhoI site of pJG4-5.For expression of PAP fused to glutathione S-transferase (GST) inmammalian cells, the full-length or the defined regions were PCR-amplified and cloned into the BamHI–ClaI site of pEBG. Full-lengthCFI-25 fused to hemagglutinin (HA) and its truncated derivativeswere constructed by PCR amplification and cloning into the SalI–SacI site of pSRa as well.

Expression and purification of GST–CFI-25 fusion proteins. Thecoding sequence of full-length mouse CFI-25 was amplified by PCRand cloned into the BamHI–XhoI site of the pGEX4T-1 vector(Amersham–Pharmacia Biotech) for the expression of full-lengthCFI-25 fused to GST in Escherichia coli. The truncated derivatives ofCFI-25 were also constructed by PCR amplification and cloning intopGEX4T-1. The E. coli JM109 strain and the GST purification sys-tem (Amersham–Pharmacia Biotech) were used for expression andpurification of GST-fusion proteins.

In vitro binding assay. A GST pull-down experiment was per-formed for an in vitro binding assay. [35S]Methionine-labeled full-length PAP was obtained by a TNT Quick Coupled Transcription/Translation kit (Promega) according to the manufacturer’sinstructions. Purified GST or GST-fusion proteins were bound toglutathione beads (Amersham–Pharmacia Biotech). The 35S-labeledtranslation mixture and glutathione beads were incubated in a 500ml binding buffer (20 mM Tris–HCl, pH 7.4, 50 mM NaCl, 2 mMMgCl2, 1 mM DTT, and 0.1% Nonidet-P 40) on ice for 2 h. The beadswere recovered by centrifugation and were washed four times withthe same fresh buffer. To elute bound proteins, we boiled the beadsin an SDS sample buffer, and we analyzed them by SDS–PAGE andautoradiography. For a control experiment to rule out the possibilityof the RNA involvement in protein–protein interactions, RNase A (20mg/ml) was added in the binding buffer.

Coimmunoprecipitation. NIH/3T3 cells were maintained in Dul-becco’s modified Eagle’s medium supplemented with 10% fetal calfserum. NIH/3T3 cells were transfected with 1 mg of mammalianexpression plasmid DNA per 100-mm dish by using the calciumphosphate method (11). NIH/3T3 cells (1 3 108) were lysed at 4°C for1 h in 1 ml EBC buffer (50 mM Tris–HCl, pH 7.5, 120 mM NaCl, 0.5%Nonidet P-40, 50 mM NaF, 200 mM sodium orthovanadate, and 1mM PMSF), and the lysate was centrifuged at 14,000g for 10 min.Proteins from the supernatant were incubated at 4°C for 1 h with 25ml of a 50% slurry solution of either glutathione–Sepharose, oranti-HA antibody (2 mg)-treated protein G–agarose. The immunopre-cipitated materials were collected on either the glutathione–Sepharose or protein G–agarose beads, and washed five times withEBC buffer. We eluted the bound proteins by boiling them in an SDSsample buffer. Finally the proteins were resolved on a 5% SDS–PAGE gel and subjected to Western blot analysis as described (11).

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RESULTS

Identification of the 25-kDa Subunit of CleavageFactor I as an Interacting Partnerof Poly(A) Polymerase

We performed a yeast two-hybrid screen using theC-terminal 268 residues of mouse PAP as a bait toscreen a human HeLa cDNA library. Approximatelytwo million colonies were screened to find clones codingfor proteins that would interact with the C-terminalregion of PAP. A sequence analysis revealed that one ofthe clones encodes the 25-kDa subunit of pre-mRNAcleavage factor I (CFI-25). We obtained the coding se-quence of mouse CFI-25 using PCR amplification froma mouse kidney cDNA library (Fig. 1).

CFI-25 Interacts with PAP in Vitro

Interaction between the mouse CFI-25 and PAP wasexamined in vitro. A GST–CFI-25 fusion protein waspurified from E. coli and used for in vitro protein–protein interaction assays with in vitro translated PAP(Fig. 2). A significant fraction of the input PAP wasbound to GST–CFI-25 (Fig. 2, lanes 2 and 3), but not tothe GST control protein (Fig. 2, lane 6). To exclude thepossibility that the interaction between CFI-25 andPAP would occur through RNA-mediated binding, weincluded RNase A in the assay buffer. This RNase Atreatment did not affect the interaction, indicating thatCFI-25 interacts with PAP not through the RNA me-diation. The interaction was also dependent on the saltconcentration.

The C-Terminal Region of PAP Bindsto CFI-25 in Vivo

To determine whether or not the interaction ob-served in vitro occurs in vivo, we performed a coimmu-

FIG. 1. Schematic diagram presenting significant features ofPAP. The catalytic domain, the S/T-rich region, two nuclear localiza-tion signal sequences (NSL), and an RNA-binding region are indi-cated. The C-terminal region of mouse PAP composed of 268 aminoacids was used for a bait for yeast two-hybrid screening.

the N-terminal 60 residues was sufficient for the inter-

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noprecipitation experiment using NIH/3T3 cells co-transfected with both the GST–PAP fusion plasmidand the HA–CFI-25 fusion plasmid. Protein lysatesderived from the cotransfected cells were immunopre-cipitated with anti-HA antibody, and the coimmuno-precipitated materials were immunoblotted with anti-GST antibody (Fig. 3A). This immunoblot revealed thepresence of PAP in the pull-down of CFI-25 withanti-HA antibody. Alternatively, the protein lysateswere immunoprecipitated with anti-GST antibody, andthe coimmunoprecipitated materials were immuno-blotted with anti-HA antibody (Fig. 3B). This immuno-blot again showed that the immunoprecipitate of PAPcontained CFI-25. These in vivo results suggest thatthe PAP-CFI-25 interaction occurs in the mammaliancells and that this interaction is likely to be physiolog-ically relevant. The coimmunoprecipitation experi-ment was also performed with the C-terminal region orthe N-terminal region of PAP instead of the intact PAP(Fig. 3, lanes 3 and 4). CFI-25 binds to the C-terminal268 residues of PAP, but not to the remainingN-terminal region. This result confirms that theC-terminal region of PAP indeed represents a domainfor interaction with CFI-25.

The N-Terminal 60 Residues of CFI-25 Are Sufficientfor Interaction with the C-Terminal Region of PAP

To identify regions of CFI-25 required for interactionwith PAP, several deletion plasmid constructs thatcould express truncated derivatives of CFI-25 weregenerated. The interaction abilities of the truncatedderivatives with PAP were assessed by both the yeasttwo-hybrid system and the coimmunoprecipitation ex-periment. Figure 4 shows that the truncation of theN-terminal 60 residues abolished the interaction abil-ity in both assays. Furthermore, a domain composed of

FIG. 2. In vitro interaction between PAP and GST–CFI-25 fusionproteins. The in vitro translated PAP labeled with [35S]methionineand the GST–CFI-25 fusion protein were mixed with glutathionebeads in the binding buffer. NaCl concentrations in the buffer areindicated above each lane. The pull-down materials were loaded on aSDS–PAGE gel and detected by autoradiography. Lane 1,[35S]methionine-labeled PAP alone; lanes 2 to 6, pull-down products;and lane 6, GST used as a negative control. To confirm that theprotein–protein interaction was not RNA-mediated, RNase A (20mg/ml) was included in the buffer (lane 3).

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action with PAP.

The C-Terminal End of PAP Is Sufficientfor Interaction with CFI-25

To delimit the binding domain in the C-terminalregion of PAP for interaction with CFI-25, we gener-ated several truncated derivatives of PAP. The trun-cated derivatives were constructed in such a way thatthey contained NLS1, NLS2, NLS1 1 NLS2, NSL2 1the C-terminal 69 residues, and the C-terminal 69residues alone, respectively (6). The truncated deriva-tives were tested for their ability to bind to CFI-25 byboth the yeast two-hybrid and the coimmunoprecipita-tion analyses (Fig. 5). Strong interactions were ob-served only when PAP contained the C-terminal 69residues, indicating that this C-terminal end is essen-tial and sufficient for the interaction. We also observedsmall b-galactosidase activities by PAP (472–661) orPAP (472–611), and a marginal b-galactosidase activ-ity by PAP (630–671) over the background in the two-hybrid analysis. The immunoprecipitation analysisalso showed a weak interaction by PAP (472–661) al-though no detectable interactions were observed with

FIG. 3. Association of CFI-25 with PAP in vivo. NIH/3T3 cellswere transfected with indicated clones. The cell extracts were immu-noprecipitated with anti-HA or anti-GST antibody. The immunopre-cipitates were resolved on a 10% SDS–PAGE and analyzed by recip-rocal immunoblot. Relative molecular mass markers are indicated onthe right. NIH/3T3 cells transfected with clones expressing GST andHA-CFI-25 (lane 1); GST–PAP and HA-CFI-25 (lane 2); GST–PAP(C268) and HA-CFI-25 (lane 3); GST–PAP (N471), and HA-CFI-25 (lane 4). The cell extracts were immunoprecipitated withanti-HA antibody and blotted with anti-GST antibody (A) or immu-noprecipitated with anti-GST antibody and blotted with anti-HAantibody (B).

appears to be a dimer of 25-kDa subunit (CFI-25) and

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PAP (472–611) and PAP (630–671). These results maysuggest the presence of a weak CFI-25-binding site inthe region of residues 472 to 672 of PAP besides thestrong binding site of the C-terminal 69 residues.

DISCUSSION

PAP is a key enzyme in the pre-mRNA 39-end pro-cessing machinery. It is known that the C-terminalregion of the mammalian PAP functions as a target forprotein–protein interactions that are involved in thecoupling of spicing and polyadenylation (9, 10). In thisstudy, we performed a LexA-based yeast two-hybridscreening to identify PAP-interaction partners thatwould be involved in regulation of functions of PAP inthe mouse, and we identified the 25-kDa subunit ofmouse CFI (CFI-25) as a partner protein.

CFs are factors that are required for the cleavagereaction only in the mammalian system because theyhave no counterparts in the yeast system (12, 13).While components of CFII are not known yet, CFI

FIG. 4. Regions of CFI-25 required for interaction with PAP. (A)Analysis of interaction between CFI-25 derivatives and full-lengthPAP using the yeast two-hybrid system. b-Galactosidase activitieswere determined at least in triplicate. (B) In vivo analysis. Lysates ofNIH/3T3 cells transfected with clones encoding GST–PAP and HA-CFI-25 derivatives were immunoprecipitated with anti-GST anti-body, and the resulting immunoprecipitates were blotted withanti-HA antibody. Relative molecular mass markers are indicated onthe left.

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the ;70-kDa subunit (CFI-70) (14). CFI-70 contains anribonucleoprotein (RNP)-type RNA-binding domain(14, 15). On the other hand, CFI-25 does not have anyknown functional domain (14). Figure 6 shows a modelof the mammalian processing complex for 39 end for-mation of mRNA. The 160-kDa, and perhaps the 30-kDa subunit of CPSF, bind to AAUAAA signal of thepre-mRNA (16, 17). The 160-kDa subunit is also in-volved in protein–protein interactions with PAP andwith the 77-kDa subunit of CsfF (18). The 64-kDasubunit of CafF binds to the GU-rich downstream ele-ment of the pre-mRNA and interacts with the 77-kDasubunit of CsfF (19, 20). However, it is not yet knownhow CFI and CFII are involved in the assembly for theprocessing complex. Since we demonstrated thatCFI-25 interacts with PAP in this study, CFI may beintroduced into the complex through this protein–protein interaction. If this is the case, protein–proteininteractions of CsfF–CPSF–PAP–CFI in the processingcomplex can be established. Alternatively, CFI may beinitially included in the cleavage complex through

FIG. 5. Regions of PAP required for interaction with CFI-25. (A)Analysis of interaction between PAP derivatives and full-lengthCFI-25 using the yeast two-hybrid system. b-Galactosidase activitieswere determined at least in triplicate. (B) In vivo analysis. Lysates ofNIH/3T3 cells transfected with clones encoding HA-CFI-25 andGST–PAP derivatives were immunoprecipitated with anti-GST an-tibody, and the resulting immunoprecipitates were blotted withanti-HA antibody. Relative molecular mass markers are indicated onthe left.

plex (13). Therefore, this region seems to be responsible

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RNA binding, because both CFI-25 and CFI-70 can becross-linked to RNA and because CFI-70 contains aribonucleoprotein (RNP)-type RNA-binding domain(14).

It was previously suggested that the 160-kDa sub-unit of CPSF is responsible for recruitment of PAPbecause it was the only known protein that binds toPAP (18). Since CFI-25 also interacts with PAP,CFI-25 may be involved in the recruitment of PAP.

We showed that the N-terminal 60 residues ofCFI-25 and the C-terminal 69 residues of PAP aresufficient for the interaction between CFI and PAP.Since CFI-25 has no similarity with any known proteinmotifs, its function has been unpredictable. Our resultsindicate that the N-terminal region of CFI-25 serves asa motif for interaction with PAP. Therefore, this regionacts at least as a link between CFI and other compo-nents of the processing complex through interactionwith PAP. The C-terminal region of PAP is known to beresponsible for regulation of the PAP activity. Thisregulation can be achieved in the following ways: (i)through phosphorylation in a serine/threonine-rich do-main (6–8) and (ii) protein–protein interactions, asexemplified in the interaction with spicing factor U1A(7) or U2AF65 (8). Therefore, the interaction betweenCFI-25 and PAP may play a role in regulation of thePAP activity and/or in assembly of the 39-end process-ing complex rather than serving as a simple link. Sincethe C-terminal 61 residues of PAP overlap with theregion that interacts with U1A and U2AF65 (7, 8),CFI-25 may be involved in regulation of the couplingbetween splicing and polyadenylation through compe-tition with the splicing factors for interaction withPAP. It is noteworthy that this C-terminal region of themammalian PAP lacks in the yeast PAP (21) and thatCFI has no counterpart in the yeast processing com-

FIG. 6. Schematic representation of the mammalian pre-mRNA39-end processing complex. The factors required for mammalian pre-mRNA 39-end processing and their components are shown. The ar-rangement of CFII is not yet known. The arrow indicates the site ofendonucleolytic cleavage. Known protein–protein interactions be-tween polypeptide of different factors are indicated by double-headedarrows. The interaction between PAP and CFI-25 is identified in thisstudy and highlighted by a thick double-headed arrow.

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for providing the pre-mRNA 39-end processing with amammalian specificity.

ACKNOWLEDGMENTS

We are grateful to Professor Cheol O. Joe for provision of cellculture facilities and Professor Joonho Choe for helpful discussions.This work was supported by grants of the Molecular MedicineResearch Program (00-J03-01-01-A-05, M10106000116-01A2000-02910) from the Ministry of Science and Technology and in part bythe Brain Korea 21 project in 2001.

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