MOLECULAR AND CELLULAR BIOLOGY, Jan. 1994, p. 77-860270-7306/94/$04.00+0

Vol. 14, No. 1

Exon Size Affects Competition between Splicing andCleavage-Polyadenylation in the Immunoglobulin ,uL Gene

MARTHA L. PETERSON,* MARY BETH BRYMAN, MICHELLE PEITER, AND CLARISSA COWAN

Department ofPathology and Laboratory Medicine, University ofKentuckyCollege of Medicine, Lexington, Kentucky 40536-0093

Received 5 August 1993/Returned for modification 17 September 1993/Accepted 22 September 1993

The alternative RNA processing of ,us and pm mRNAs from a single primary transcript depends oncompetition between a cleavage-polyadenylation reaction to produce ,us mRNA and a splicing reaction toproduce pm mRNA. The ratio of ps to pm mRNA is regulated during B-cell maturation; relatively morespliced p.m mRNA is made in B cells than in plasma cells. The balance between the efficiencies of splicing andcleavage-polyadenylation is critical to the regulation. The IL gene can be modified to either reduce or improvethe efficiency of each reaction and thus alter the ratio of the two RNAs produced. However, as long as neitherreaction is so strong that it totally dominates, expression of the modified ,u genes is regulated in B cells andplasma cells. The current experiments reveal a relationship between the CP4 exon size and the pus/pmexpression ratio. The shorter the distance between the Cp4 5' splice site and the nearest upstream 3' splice site,the more spliced pum mRNA was produced. Conversely, when this exon was expanded, more pus mRNA wasproduced. Expression from these p. genes with altered exon sizes were regulated between B cells and plasmacells. Since RNA processing in the pL gene can be considered a competition between defining the C,u4 exon asan internal exon (in p.m mRNA) versus a terminal exon (in ,us mRNA), exon size may affect the competitionamong factors interacting with this exon.

The immunoglobulin heavy-chain p. gene has been studiedfor a number of years as a model system for regulatedalternative RNA processing (12, 27). Alternative processingof the p. primary transcript results in two mRNAs that differat their 3' ends, a p.s mRNA that encodes the secreted formof immunoglobulin M and a p.m mRNA that encodes themembrane-associated form of immunoglobulin M. The p.primary transcript contains a poly(A) site within an intron; ifit is cleaved and polyadenylated at this p.s poly(A) site, p.smRNA is produced. If instead it undergoes splicing of theCp.4 and Ml exons to remove the p.s poly(A) site and iscleaved and polyadenylated at the p.m poly(A) site, it givesrise to p.m mRNA. The relative abundance of these twomRNAs is regulated during B-cell maturation; p.m mRNA ispredominant in pre-B and B cells, whereas p.s mRNAproduction is heavily favored in mature plasma cells.

Previous work has shown that developmentally regulatedproduction of p.s and p.m mRNA depends on the finely tunedefficiencies of the competing cleavage-polyadenylation andsplice reactions (26, 30). The strength of these reactions canbe altered individually or in concert, and this changes theratio of p.s to p.m mRNA production. However, as long as abalance between the efficiencies of the two reactions ismaintained, expression from these genes is regulated be-tween B and plasma cells. That is, there is a shift in theexpression ratio when the same construct is expressed in Bcells and plasma cells; B cells always make more spliced p.mmRNA than do plasma cells. The p. gene naturally containsbalanced suboptimal cleavage-polyadenylation and splicesignals; the suboptimal Cp.4 5' splice junction is evolution-arily conserved (30). But suboptimal signals are not required

* Corresponding author. Mailing address: Department of Pathol-ogy and Laboratory Medicine, University of Kentucky College ofMedicine, 800 Rose Street, Lexington, KY 40536-0093. Phone: (606)257-5478. Fax: (606) 257-7648. Electronic mail address: martha.peterson@ukwang.uky.edu.

for p. regulation because expression from p. genes withbalanced strong processing signals is also regulated (26).Studies with the p. gene have identified a number of param-eters that affect the overall efficiencies of the splice andcleavage-polyadenylation reactions. These include the sizeof the Cp,4-M1 intron (9, 29, 30, 39), the sequence of the Cp,45' splice junction (30), the p.s poly(A) site sequence (30), thesequence of the Ml 3' splice junction and p.m poly(A) site(26, 30, 41) and a general splice-activating sequence in M2that serves to activate C,u4-M1 splicing (42, 43). The p. gene,with competing splice and cleavage-polyadenylation reac-tions, provides a sensitive assay for subtle changes in theefficiencies of these reactions.To date, no p. gene-specific sequences have been shown to

be required for regulation. Much of the C,u4-M1 intronsequences have been replaced with no effect on regulation(10, 29, 39). Sequences within the p.s poly(A) site region canbe replaced with other poly(A) sites, and the p.m 3' splice-poly(A) site region can be replaced by the simian virus 40(SV40) t-antigen 3' splice and poly(A) site (26, 30). Theconserved suboptimal C,u4 5' splice junction can be mutatedto the consensus 5' splice junction sequence, and as long asthis splice is in competition with a strong poly(A) site, thisdoes not interfere with regulation (26). Therefore, none ofthese sequences are specifically required for regulation. Thissuggests that there may not be specific sites or specificfactors that direct p. mRNA processing. Rather, the balanceof the two competing reactions may be the critical parameterthat allows this gene to respond to subtle changes in theconcentration or activity of the general processing machin-ery.The initial observation that led to the current experiments

was that when a 1,165-bp fragment, 31 bp upstream from theCp4 5' splice junction, was removed from the p. gene, theratio of RNA cleaved and polyadenylated at the p.s poly(A)site (pA) to RNA that is spliced between the C,U4 and Mlexons and cleaved and polyadenylated at the p.m poly(A) site

77

78 PETERSON ET AL.

A

neo-Cgl Cp2II----------

#1.1.1.1...I,Bg BE

BI........,..

B

Ps UrM

Ct3 Cp4 _M2AA

BE Bgs-m

A

E .......B

Cs-m AB

pA- _ *9 #A -pA

Cp4 A

s-m

640 probe412 pA

* - 223 splice

Asplice- -d -splice

BE~~~A...........H3

B PC B PC 594 probe395 pA

- 206 spliceFIG. 1. Structure and expression of s-m and AB plasmids in B cells and plasma cells. (A) Diagram of the chimeric SV40-neo-C,u gene s-m

and its AB derivative. The sequences deleted in the AB plasmid are omitted but are not shown joined. Open box, SV40-neo sequence; solidboxes, Clu sequence common to ,.s and pLm mRNA; hatched box, ps-specific sequence; cross-hatched boxes, pm-specific sequence. Theangled line above the map designates the Cp,4-M1 splice. The exons are labeled above the map; "A" designates the Ps and pmcleavage-polyadenylation signals as labeled. Restriction sites: Bg, BglII; BE, BstEII. (B) S1 nuclease protection analysis of cytoplasmic RNAfrom M12 B cells (B) and S194 plasmacytoma cells (PC) transfected with the plasmids shown above the lanes; the positions of the poly(A)(pA) and spliced RNAs are identified. For the B lanes, 25 p.g ofRNAwas used and a 6.5-h exposure is shown; for the PC lanes, 50 ,ug of RNAwas used and a 16-h exposure is shown. (C) Diagram of the probes used in the S1 assays and the protected fragments generated. Restrictionsites: P, PstI; H3, HindIII; Pvu, PvuII.

(splice) was altered; in both B cells and plasma cells, theamount of the spliced p.m RNA increased relative to theamount of ,us RNA. The deleted sequences are outside theregion expected to be directly involved in Cp,4-M1 splicingand p,s cleavage-polyadenylation. Therefore, it was possiblethat the deleted fragment contained a previously unidentifiedspecific cis-acting element affecting p. RNA metabolism.This element could potentially function at the level of RNAprocessing, affecting either splice or poly(A) site efficiency,or it could act at another level of RNA metabolism, such astransport. We thus took the approach of introducinggenomic and cDNA subfragments of the deleted region backinto the deleted p. gene (AB) in an attempt to restore theoriginal processing pattern. Although the inserted fragmentsaltered the AB expression ratio, the expression patterns arenot consistent with simply the presence or absence of aspecific cis-acting site. Instead, a relationship between thesize of the Cp,4 exon and the expression ratio has beenestablished.

MATERIALS AND METHODS

Plasmid constructions. Construction of the s-m plasmid,which contains a 6-kb BglII fragment from Cp, downstreamfrom the SV40 promoter of plasmid pSV5neo, has beendescribed elsewhere (29); this plasmid has been used instudies of p.s/p.m alternative RNA processing regulation (26,28, 30). All plasmids used in this study are derivatives of thes-m plasmid. AB was created by deleting the 1,165-bp BstEII

fragment from s-m, thus fusing exons Cp.2 and Cp.4 (Fig.1A).The series of plasmids containing replaced subfragments

of the deleted 1,165-bp BstEII fragment were constructed bystandard techniques (34). The AB plasmid was linearizedwith BstEII, and the ends were made blunt with Klenowenzyme. The subfragments, whose ends were made blunt,were introduced, and the plasmids were screened for thepresence and orientation of inserts. The following fragmentswere inserted: a 437-bp BstXI-ApaI fragment from Cp., a536-bp PstI fragment from Cp., a 586-bp BstEII-PstI frag-ment from Cp. cDNA, a 224-bp XmnI fragment from Cp., a118-bp XmnI fragment from Cp. cDNA, a 623-bp BstEII-BstXI fragment from Cp., and a 334-bp BstEII-BstXI frag-ment from Cp. cDNA. These fragments and their locationswithin the p. gene are diagrammed in Fig. 2.The AB1 plasmid, which retains only the BstEII site in the

Cp.4 exon of s-m, was made by partially digesting s-m withBstEII, filling in the ends with Klenow enzyme, and religat-ing. AB1 was then linearized with BstEII and made bluntwith Klenow enzyme, and the 224-bp XmnI fragment fromCp. and the 118-bp XmnI fragment from Cp. cDNA wereinserted and screened for insert orientation to create ABiXmn and ABi XmncDNA (see Fig. 6).

Plasmids AB m-m, AB m-m(A), AB SPm-m, and AB SPm(see Fig. 7) were made from m-m, m-m(A) (30), SPm-m, andSPm (26), respectively, by deleting the 1,165-bp BstEIIfragment as for the original AB plasmid.

MOL. CELL. BIOL.

EFFECT OF EXON SIZE ON GENE EXPRESSION 79

CeoC,C1CCIC A M2Ml A

A BE PBXX E A A

....l..- -B

r:_ 0AB BA

A Pat

}---jj-[ AB BPCDNA

mn

:I ABXmncDN

.I ABXmncDN

A

NA

NA (-)

AB BBX

AB BBXCDNA

FIG. 2. Diagram of the AB plasmid series containing replacedsubfragments of the deleted region. The inserted fragments are

shown aligned with their normal locations within the p gene; thename of each plasmid is shown to the right of each insert. The (-)indicates the fragment is inserted in the inverse orientation. Stippledboxes, exon sequences; thin lines, introns; dotted lines, introns areabsent as the fragment is derived from cDNA. All other symbols are

as described in the legend to Fig. 1. Restriction sites: Bg, BglII; BE,BstEII; P, PstI; BX, BstXI; X, XmnI; A, ApaI.

Cell culture and DNA transfections. The M12 B-cell linewas maintained in RPMI 1640 medium supplemented 10%fetal bovine serum and 50 p.M 2-mercaptoethanol. The S194plasmacytoma cell line was maintained in Dulbecco modifiedEagle medium supplemented with 10% horse serum. All cellculture reagents were from GIBCO-BRL. The DEAE-dex-tran transfection protocol was used for the transient expres-sion assays (11).RNA preparation and Si analysis. Cytoplasmic RNA was

prepared from transfected cells (35); poly(A)+ RNA wasisolated by passing total cytoplasmic RNA over oligo(dT)columns. For S1 nuclease analysis, 100 p.g of RNA, acombination of specific and nonspecific carrier RNA, washybridized in a 50-p.l volume as described previously (30).Between 10 and 100 p.g of specific RNA was assayed; whenpoly(A)+ RNA was analyzed, 0.5 to 1 p.g of RNA was used.The amount of specific RNA used for each gel lane shown isreported in the figure legends and was chosen, along with thespecific autoradiographic exposure, to best illustrate therelative amounts of the protected fragments. A unique probethat distinguishes mRNA cleaved at the poly(A) site frommRNA spliced within the Cp4 exon was isolated for each p.

gene; the structure of each probe is diagrammed in thefigures. The probes were 3' end labeled with Klenow enzymeand [a-32P]dCTP and were present in excess over the spe-cific RNA. Hybridization was carried out at 50°C for allprobes. The protected fragments were separated on either 4or 6% acrylamide-7 M urea gels. The gels were dried, andthe protected fragments were quantitated with an AmbisRadioanalytic imaging system.

Northern (RNA) blot analysis. Poly(A)+ RNA (0.25 to 1.25p.g) from transfected cells was analyzed by Northern blotanalysis after treatment with oligo(dT) and RNase H (bothfrom GIBCO-BRL) as described previously (1). TreatedRNA was separated on a 1.5% agarose-0.22 M formalde-hyde gel run in buffer containing 0.22 M formaldehyde. Aftercapillary transfer of the RNA to Nytran (Schleicher &Schuell, Inc.), the RNA was UV irradiated as instructed by

TABLE 1. Expression of p. genes in B cells and plasma cells

Exon pA/splice0 Reglation,Plasmid size Rlo

(nt) B PC PC/B"

AB Xmn 126 0.68 + 0.1 7.8 ± 0.7 11AB1 Xmn 126 0.69 + 0.1 15 ± 0.3 22AB Pst 136 0.82 ± 0.1 7.8 ± 0.2 10AB XmncDNA(-) 197 0.37 + 0.05 6.4 ± 0.6 17AB 200 0.36 ± 0.03 1.0 ± 0.1 3AB BBX 205 1.4 ± 0.2 11 2 8AB BA 217 1.1 ± 0.1 9.4 ± 0.7 9AB XmncDNA 318 2.7 ± 0.3 27 + 3 10s-m 333 2.0 ± 0.2 9.8 ± 0.8 5AB1 XmncDNA 448 5.9 + 0.6 >100 >15AB BBXcDNA 544 9.5 ± 1 >100 >10AB BPcDNA 786 17 ± 1 >100 >6

a Expression ratios are derived from quantitation of Si nuclease protectionassays of non-poly(A)-selected cytoplasmic RNA with an Ambis Radioana-lytic imaging system and are averages + standard deviations of 3 to 14independent determinations. B, B cells; PC, plasma cells.

b The estimated standard deviation for each ratio is less than 25%.

the manufacturer and hybridized to random-prime-labeled(Boehringer Mannheim) SVneo probe, a 384-bp BglI-BglIIfragment from pSV2neo. RNA size markers (RNA Ladder;GIBCO-BRL) were also run on the gels, which were thentransferred and stained with methylene blue (34).

RESULTSA deletion upstream from the C,u4 5' splice junction affects

the ,us/,um expression ratio. The plasmid that initiated thisstudy was originally constructed to identify RNA fromtransfected p. genes in cell lines producing endogenous p.transcripts. A 1,165-bp BstEII fragment, located 31 nucle-otides (nt) upstream from the Cp,4 5' splice junction, wasremoved from the p. gene (Fig. 1A, AB). An Si nucleaseprobe spanning the deletion junction easily distinguishestransfected from endogenous p. mRNA. The sequencesdeleted, from mid-exon Cp.2 to mid-exon Cp.4, are outsidethe region expected to be directly involved in Cp.4-M1splicing or ,us poly(A) site use. In addition, the deletion joinstwo exons so there are no unpaired splice junctions thatmight complicate the RNA processing pattern of this tran-script. Also, an upstream intron, which is required for theaccumulation of a number of different transcripts (2, 5, 16,20, 33), is retained. However, as shown by Si nucleaseprotection analysis of RNA from M12 B cells and S194plasmacytoma cells transiently transfected with the unde-leted p. gene s-m and AB (Fig. 1B), the pA/splice ratio isaltered by the deletion in the AB p gene. The pA/splice ratiois lower in both B cells and plasma cells by about 6- and10-fold, respectively (Table 1). The deletion in AB hasaltered a feature of the p. primary transcript that is indepen-dent of the developmental regulation, since the pA/spliceratio remains higher in plasma cells than in B cells. The ABp gene was also expressed in other pre-B- and B-cell lines,including 3-1, 70Z/3, and A20, and a similar enhancement ofp.m mRNA relative to p.s mRNA was observed (data notshown).

Introducing subfragments of the deleted region into AB.While it was not expected that deleting the BstEII fragmentwould alter the pA/splice ratio, several models could ac-count for this observation. It was possible that a novelcis-acting sequence affecting the efficiency of either theCp.4-M1 splice or usage of the p.s poly(A) site had been

VOL. 14, 1994

80 PETERSON ET AL.

BA B

-pA

!4w -splice

Pst BPcDNA

b..-

* w-m-pA

-splice

B PC B PC

B PC

A

Li-[> ;AB BA

Xmn515 probe316 pA127 splice

A

Li---- AB PstBX

654 probe455 pA266 splice

C DXmn XmncDNA

- _mqpi -pA

so -K. -splice

B PC B PC

A

_u_n1Z B XmncDNA

Pvtj

BBX BBXcDNA

4W * -pA

_b -A- -splice

B PC B PC

AI

1 2 AB BBXcNZPvuE----1IBAB BBX

Pvu

717 probe 942 probe

518 pA 743 pA329 splice 554 splice

FIG. 3. Expression of the AB series plasmids in B cells and plasma cells. Shown is S1 nuclease protection analysis of cytoplasmic RNAfrom M12 B cells (B) and S194 plasmacytoma cells (PC) transfected with the plasmids shown above the lanes; the positions of the poly(A)(pA) and spliced RNAs are identified. RNA from each plasmid was assayed with a unique probe that is diagrammed below theautoradiographs. Restriction sites: X, XmnI; H3, HindIII; BX, BstXI; Pvu, PvuII. (A) AB BA, assayed with 50 Lg of B-cell RNA (an 8-hexposure is shown) and 100 ,g of PC RNA (a 17-h exposure is shown). (B) AB Pst and AB BPcDNA, assayed with 50 pg of B-cell RNA and100 p,g of PC RNA (an 8.5-h exposure is shown). (C) AB Xmn and AB XmncDNA, assayed with 25 ,.g of B-cell RNA (a 6-h exposure is shown)and 100 p,g of PC RNA (a 22-h exposure is shown). (D) AB BBX and AB BBXcDNA, assayed with 0.5 ,ug of poly(A)+ B-cell RNA and 1.0Lg of poly(A)+ PC RNA (a 15.5-h exposure is shown).

deleted. Another possibility was that sequences involved inother aspects of RNA metabolism, such as RNA stability(14) or RNA transport (3), were affected. Alternatively, asequence brought closer to the splice and poly(A) signals bythe deletion could have altered the efficiency of eitherreaction. To investigate the mechanism by which the dele-tion affected the pA/splice mRNA expression ratio, subfrag-ments of the deleted region were placed back into the AB ,ugene. Fragments of various sizes that spanned the 1,165-bpdeletion and that either did or did not contain an intron werechosen for this set of plasmid constructions (Fig. 2). If aspecific cis-acting element had been deleted from the AB p,transcript, then replacing that sequence should restore theoriginal pA/splice expression ratio. If the spacing betweenupstream sequences and the processing signals was respon-sible for the deletion effect, then no specific sequence wouldrestore the original pA/splice ratio but rather an effectrelated to fragment size would be seen. In several cases,fragments with the same endpoints, either with or without an

intron, were inserted to distinguish between contributions of

exon and intron sequences or between specific sequencesand the presence of a spliceable intron; each of the twointrons in the BstEII fragment was replaced in the AB gene(Fig. 2, AB Xmn and AB XmncDNA, AB BBX, and ABBBXcDNA).Each of the AB replacement plasmids was transiently

expressed in the M12 B-cell line and the S194 plasmacytomacell line, and non-poly(A)-selected or poly(A)+ cytoplasmicRNA was analyzed for pA/splice RNA expression by Sinuclease protection analysis (Fig. 3; Table 1). A unique Siprobe was designed for each gene or cDNA-genomic DNApair; these are diagrammed below the autoradiographs inFig. 3. Several observations can be made from these data.First, the RNA from all of the plasmids showed the regulatedpA/splice ratio shift between B cells and plasma cells.Second, within the region covered by the S1 probes, noaberrant splicing was detected. Third, the pA/splice ratiovaried dramatically among the different fragment-containingconstructs, and the expression ratios do not support eithermodel for the AB deletion effect. For example, both the BA

A

MOL. CELL. BIOL.

EFFECT OF EXON SIZE ON ,u GENE EXPRESSION 81

A A

*b 4bll b It, el >- s&k>v exw?,

AV do X

-pA

* -cryptic splice

B PC

B A

LiIIIH IJ~t AB XmncDNA (-3Pvu

717 probe518 pA329 predicted splice

n- 199 cryptic splice

Crypticsplice AGAGTGACT7/8 match toconsensus

FIG. 4. Expression of the AB XmncDNA(-) plasmid in B cellsand plasma cells. (A) S1 nuclease protection analysis of cytoplasmicRNA from M12 B cells (B) and S194 plasmacytoma cells (PC)transfected with AB XmncDNA(-); the positions of the probe andthe poly(A) (pA) and spliced (cryptic splice) RNAs are identified. Alane of carrier (nonspecific) RNA is included to show that theunmarked band is not a specific protected fragment. The assay wasperformed with 15 jig of B-cell RNA and 75 jig of PC RNA; a 17-hexposure is shown. (B) Diagram of the probe used in the S1 assayand the protected fragments generated. The cryptic splice sequenceis shown, with its match to the consensus 5' splice junctionsequence designated by asterisks.

and BBX fragments restored the expression ratio to near thatof s-m, but these fragments do not contain overlappingsequences. Also, plasmids with the cDNA versions of theinserted fragments always had a higher expression ratio thandid plasmids with the corresponding genomic fragments. Infact, the plasmids containing the BBXcDNA and BPcDNAinserts, when expressed in plasma cells, produced only ,usmRNA; no spliced RNA was detected. Together, the datafrom this series of AB sequence replacement plasmids do notpoint to a specific cis-acting sequence or to a simple distanceeffect as the cause of the expression ratio change observedwith the AB p. gene. They do, however, suggest that thepresence of an intron may in some way be affecting thepA/splice expression ratio.

Expression from the AB XmncDNA(-) p. gene, whichcontains the same fragment as AB XmncDNA but in theopposite orientation, is shown in Fig. 4. The RNA from thisgene was spliced at a cryptic 5' splice junction instead of thenormal C,u4 5' splice junction. This splice was mapped byresolving the Si nuclease-protected fragments on a sequenc-ing gel next to Maxam and Gilbert sequencing reactions ofthe S1 probe (data not shown). The sequence of the crypticsplice, shown in Fig. 4B, has a 7/8 match to the consensus5' splice junction sequence (36). Expression from ABXmncDNA(-) also showed a regulated shift between B cellsand plasma cells. However, the pA/splice expression ratiofor AB XmncDNA(-) was five- to sevenfold lower than thatfor AB XmncDNA, which contains the same fragment in theopposite orientation (Table 1). Therefore, since the insertsize is the same in these two genes, spacing between

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1617

B

g'urb>' b uewb:b

de

a.*

4S

'.. m0p

1 2 3 4 5 6 7 8 9 10 11 12FIG. 5. Expression of s-m and the AB series plasmids in B cells

and plasma cells, assayed by Northern blot analysis. Poly(A)+ RNAfrom cells transfected with the plasmids shown above the lanes washybridized with oligo(dT), treated with RNase H to remove thepoly(A) tails, and then separated on 1.5% formaldehyde gels asdescribed in Materials and Methods. The blots were hybridized witha probe specific for the SVneo sequences at the 5' end of thechimeric RNA. The larger band in each lane is the jim form of RNA,and the smaller band is the pLs form; the predicted size of each RNAis given in Table 2. (A) M12 B cell mRNA. Lanes: 1, s-m, 1.25 ,ug ofRNA, 16-h exposure; 2, AB, 0.25 jig of RNA, 3-h exposure; 3, ABBA, 0.25 jig of RNA, 7-h exposure; 4, AB Pst, 0.25 iLg of RNA, 7-hexposure; 5, AB BPcDNA, 0.25 jig of RNA, 7-h exposure; 6, ABXmn, 0.25 jig of RNA, 7-h exposure; 7, AB XmncDNA, 0.25 jig ofRNA, 7-h exposure; 8, AB XmncDNA(-), 0.25 jig of RNA, 7-hexposure; 9 to 11, same samples as lanes 6 to 8, but 3-h exposure;12, AB BBX, 0.25 jig of RNA, 64-h exposure; 13, AB BBXcDNA, 1jig of RNA, 64-h exposure; 14 and 15, same samples as lanes 12 and13, but 16.5-h exposure; 16, AB1 Xmn, 0.5 jig of RNA, 46-hexposure; 17, AB1 XmncDNA, 0.5 jig of RNA, 46-h exposure. (B)S194 plasmacytoma cell mRNA Lanes: 1, s-m, 0.5 jLg of RNA,16.5-h exposure; 2, AB, 1 jig of RNA, 16.5-h exposure; 3, AB BA,1 ,ug of RNA, 63-h exposure; 4, AB Pst, 0.5 jLg of RNA, 24-hexposure; 5, AB BPcDNA, 0.5 jig of RNA, 24-h exposure; 6, ABXmn, 0.5 jig of RNA, 24-h exposure; 7, AB XmncDNA, 0.5 jig ofRNA, 24-h exposure; 8, AB XmncDNA(-), 0.5 jig of RNA, 24-hexposure; 9, AB BBX, 0.5 jig of RNA, 24-h exposure; 10, ABBBXcDNA, 0.5 jig of RNA, 24-h exposure; 11, AB1 Xmn, 1 jig ofRNA, 63-h exposure; 12, AB1 XmncDNA, 1 jig of RNA, 63-hexposure.

elements in the primary transcript is not likely to be anexplanation for the deletion effect.To ensure that the RNA processed from this set of genes

was of the predicted size, thus implying that no unexpectedprocessing was occurring, poly(A)+ RNA from transfectedB cells and plasma cells was analyzed by Northern blotting(Fig. 5). All of the RNA detected by a probe specific for the5' end of the transfected p. gene was of the predicted size(Table 2); no other RNA bands were observed. The relativedifferences in pA/splice mRNA ratios among this set of

VOL. 14, 1994

82 PETERSON ET AL.

TABLE 2. Expected sizes of p.s and p.m mRNA transcripts froms-m wild-type and the AB plasmid series

Size (nt)Plasmid

,us mRNA ,um mRNA

s-m 1,556 1,758AB 788 980AB BA 1,112 1,304AB Pst 1,213 1,405AB BPcDNA 1,373 1,565AB Xmn 905 1,097AB XmncDNA 905 1,097AB XmncDNA(-) 905 967AB BBX 1,130 1,322AB BBXcDNA 1,130 1,322AB Xmn 1,673 1,875AB XmncDNA 1,673 1,875

plasmids seen by Si nuclease analysis were also seen byNorthern blot analysis. In addition, the regulated shift inexpression ratio between B cells (Fig. 5A) and plasma cells(Fig. SB) was observed.

Inserting sequences at the BstEII site of the normal s-m ,ugene. The pA/splice mRNA ratios obtained from the ABreplacement plasmids argued against the proposals that acis-acting sequence had been deleted or that upstream se-quences had been brought close to the p. RNA processingsignals and thereby changed the pA/splice expression ratiofrom that of AB. In fact, the inserted fragments had a widerange of effects on the pA/splice ratio, some returning it tonear that of s-m and others increasing it dramatically beyondthat of s-m. To investigate these effects further and to provethat a critical sequence had not been deleted, the Xmn andXmncDNA fragments were inserted into the 3'-most BstEIIsite of the s-m p. gene (Fig. 6A). This puts these two

A us urm

neo-Cpl Cp2 Ci3 COA Ml M2\B1i.......... ~~~~~~~~~~~~~Bg

Bg A BE

E[j Xmn

-o XmncDNA

B ?

" -pA

, -splice

CCKL4 A

_1g_VAB1 XmncDNAABI Xmn

734 probe535 pA346 splice

B PC B Pl

FIG. 6. Structure and expression of AB1 plasmids in B cells andplasma cells. (A) Diagram of the chimeric SV40-neo-Cp gene AB1and the derivatives that contain the inserted fragments shownbelow. All symbols are as in Fig. 1 and 2. Restriction sites: Bg,BglII; BE, BstEII. (B) Si nuclease protection analysis of cytoplas-mic RNA from M12 B cells (B) and S194 plasmacytoma cells (PC)transfected with the plasmids shown above the lanes; the positionsof the poly(A) (pA) and spliced RNAs are identified. One microgramof poly(A)+ RNA was used in each lane, and a 17.5-h exposure isshown. (C) Diagram of the probe used in the S1 assays andthe protected fragments generated. Restriction sites: P, PstI; H3,HindIII.

neo-C4l Cp2 CtZ3 C94 MlI2 A

-~~~~~1Ae~~ 4 RV4Bg BE BE

SP gm SPm-mA

SP rm SPm

A A

.....:voW AB

zm AB m-mSP pm AB SPm-m

Am̂ AB mr-rnK)SP M A ABSPm

FIG. 7. Diagram of s-m and AB plasmids containing improvedC,u4-Ml splice and poly(A) site efficiencies. Below each map isshown the structure of the plasmid named to the right; the samealterations are present in both the s-m and the AB plasmids. pm, thepLm poly(A) site is substituted for the p.s poly(A) site; SP, the Cp,4 5'splice site is mutated to the consensus sequence; bars designatedwith A, sequences that have been deleted from the plasmid. Restric-tion sites: Bg, BglII; BE, BstEII; Ac, AccI; Rv, EcoRV.

fragments in the same location as in the AB p. gene, but nowthe 1,165-bp BstEII fragment is retained. To facilitate thecloning, the BstEII site in the C,u2 exon was destroyed (ABl;Fig. 6A); this had no effect on pA/splice mRNA expression(data not shown). The AB1 Xmn and AB1 XmncDNAplasmids were transiently expressed in the M12 B-cell andthe S194 plasmacytoma cell lines, and cytoplasmic RNA wasanalyzed by S1 nuclease protection analysis (Fig. 6B; Table1); the unique S1 probe is diagrammed in Fig. 6C. Again,expression from these constructs showed a regulated shiftbetween B cells and plasma cells. Also, insertion of the Xmnand XmncDNA fragments altered the pA/splice mRNAexpression from that of the s-m gene; AB1 Xmn has a lowerratio, and AB1 XmncDNA has a higher ratio (Table 1). Inplasma cells, RNA from AB1 XmncDNA is totally cleavedand polyadenylated. The AB1 Xmn and AB Xmn plasmidsare identical except for the presence or absence of the1,165-bp BstEII fragment, and the expression ratios fromthese two plasmids are very similar. Thus, the BstEIIfragment has no effect on expression in this context. Theexpression ratio from AB1 XmncDNA is severalfold higherthan from AB XmncDNA, and both are higher than ratiosfrom the plasmids containing their corresponding genomicfragments. Northern blot analysis showed that the RNAsproduced from the AB1 plasmids were of the predicted size(Fig. 5; Table 2).The AB effect is independent of splice and poly(A) site

efficiencies. The effect of the deletion in AB was to increaselevels of p.m mRNA relative to p.s mRNA. To determinewhether this effect was dependent on the suboptimal naturesof the Cp.4-M1 splice or of the p.s poly(A) site (30), the ABdeletion was placed in the context of p. genes containingimproved splice and cleavage-polyadenylation efficiencies(Fig. 7). In this series of constructs, the more efficient p.mpoly(A) site was substituted for the p.s site (m-m) and theCp.4-M1 splice was improved either by mutating the Cp.4 5'splice junction to the consensus sequence (SP), by decreas-ing the intron size [m-m(A)], or by replacing the Ml 3' spliceand p.m poly(A) site with similar sequences from SV40(SPm) (26, 30). An Si nuclease analysis of the RNA isolatedfrom transiently transfected M12 B cells and S194 plasma-cytoma cells is shown in Fig. 8, and the data are quantitated

MOL. CELL. BIOL.

VOL.14,1994~~~~~~EFFECTOF EXON SIZE ON p. GENE EXPRESSION 83

A

- - - - - Ee- -probe

"'- .8 m - -pA

- Oie mm -splice

B PC

B

C A

&

derivatives

TBE640 probe293subpA223 splice

FIG. 8. Expression of s-rn and AEand poly(A) site efficiencies in B cell.,nuclease protection analysis of cytol(B) and S194 plasmacytoma cells (PCshown above the lanes; the position

(pA) and spliced RNAs are identified

of the probes used in the Si assay~

generated; sub pA refers to RNA cle~

p.m poly(A) site and represents the e.

probe (from p.s-containing DNA) andPMt; H3, HindIll; Pvu, Pvull; BE,assayed with 50 (lanes 1 and 2), 25 (Ito 8) p.g of RNA. An 8-h exposuriderivatives, assayed with 15 (lane 1),

4), 70 (lane 5), 100 (lane 6), 80 (lane~An 8-h exposure is shown.

in Table 3. The data for the AR'i

shown in comparison with data:of the same plasmids. In all

fragment decreased the pA/spliceeffect of the deletion is independtion with other parameters affec

efficiency. Also, the RNA from e

regulated shift in pA/splice expr,

plasma cells.

TABLE 3. Expression of p. genepoly(A) efficiencies alone and in cor

pA/splice'Plasmid B

WT AB

s-rn 2.0 0.36rn-rn >100 2.3 ±0.3 >rn-rn(A) 1.7 0.71 ±0.05SPrn-rn 0.04 0.013± 0.003SPrn 0.08 0.043± 0.007

a Expression ratios are averages -+ stEindependent experiments. Expression ratifrorn previously reported data and areireferences 26 and 30). B, B cells; PC, plab The estimnated standard deviation for

DISCUSSION

f,1b,Ci~~' The experiments presented in this paper were designed to>0 ~~~~identify the mechanism by which a deletion of p. gene- - -- -probe sequences altered the pA/splice expression ratio. The dele-

tion did not infringe on sequences known to be required forwe -pA either splicing or cleavage-polyadenylation, the deletion

endpoint being 31 nt upstream from the Cp.4 5' splicea,40 - -splice junction. Therefore, by understanding the mechanism be-

1 2 3 45 6 7 s hind the altered expression ratio of the AR p. gene, insights.I ~~~into splice site choice and/or parameters related to compe-B PC tition between splicing and cleavage-polyadenylation might

be gained. We originally attempted to return the pA/spliceA expression ratio of the AB p. gene to that of s-in by replacingABrn-rn & sbrtmns tesqecs facs

derivatives sufaget of tedeleted sqec;ifaspecific csPV BE 4prb acting sequence had been deleted, this approach should have

276 sub pA revealed its identity. If instead the deletion had altered the206 splice distance between the Cp.4 5' splice junction and/or p.s

3 plasmids with irnproved splice poly(A) site and an upstream sequence, then replacing,sand plasma cells. Shown is 51 fragments of different sizes would have resulted in pA/splicepilasmic RNA from M12 B cells expression ratios whose values reflected the insert size. In~) transfected with the plasmids fact, the results did not support either of these models. Twoks of the probe and the poly(A) fragments that do not contain any overlapping sequences,1. Below each panel is a diagram BAadBX ohrsoe h Aslc xrsinrtoo,s and the protected fragments AndtBBX,abothato rstore theo pA/splice expressontratioe ofe,aved and polyadenylated at the A ona hto -n lo .gnsta otie h,xtent of homology between the same Xmn subfragment with or without the AB deletion (ARI the RNA. Restriction sites: P, Xmn and ABi Xmn) produced similar p.s/p.m expression~stEIL. (A) in-rn and derivatives, ratios. Both of these results suggest that a cis-acting elementlanes 3 and 4), and 100 (lanes 5 was not located within the 1,165-bp BstEII fragment. The-e is shown. (B) AB rn-rn and expression ratios also were not in proportion to insert size,,10 (lane 2), 30 (lane 3), 10 (lane as the plasmids containing smaller cDNA subfragments had7), and 100 (lane 8) p.g of RNA. higher expression ratios than did the plasmids with the

corresponding genomic subfragments (Xmn versus XmncDNA and BBX versus BBXcDNA). In addition, the plasmidswith the same fragmnent in the forward and reverse orienta-tions [XmncDNA versus XmncDNA(-)J did not produce the

versions of the plasmids are same expression ratios. A number of plasmids with insertedfor the nondeleted versions subfragmnents had much higher pA/splice expression ratioscases, deleting the BstEII than did s-in, which would not be predicted by either of the-1expression ratio. Thus, the above explanations for the deletion effect.lent of and acts in combina- A consistent pattern to the pA/splice expression data:ting splice and poly(A) site emerged when it was related to the size of the Cp.4 exon, that-lach construct displayed the is, the distance between the Cp.4 5' splice junction and the.ession between B cells and nearest upstream 3' splice junction (Fig. 9). When the

expression ratios (Table 1) are plotted against the exon size,the data show a striking trend; the smaller the exon, thelower the pA/splice expression ratio, and the larger the exon,the higher the expression ratio. Also, the expression ratiosare always higher in plasma cells than in B cells, as indicated

r,s with improved splice and by the distinct sets of data for the two cell lines. Expression.nbination with the AB deletion ratios from AB XmncDNA(-), which utilized a cryptic 5'

Regulation, splice junction, and from the ABi plasmids are also relatedPC/Bb to the exon size. The single point that deviates from this

PC~~~~~trend is the original AB plasmid expressed in plasma cells; its

CWIr AB expression ratio is lower than several other genes with aWT AB similar exon size (Table 1). However, the AB expression9.81.0 5 3

~ratio in B cells is not abnormally low, and most of the AR9.8 1.0 2 9

plasmids with improved splice and poly(A) site efficiencies100 2.0± 0.81 do not show low expression ratios in plasma cells, as

0.2 0.08 ±0.01 5 6 indicated by plasma cell/B-cell regulation indices of 6 to 91.0 0.24± 0.03 13 6 (Table 3). Thus, it is not clear why AR expression is low in

plasma cells, but it may be a result of the specific combina-andard deviations of at least three tion of parameters contributing to overall splice and cleav-:ios without standard deviations are aeplaeyainefcece nti eeincluded for cornparison (Table 1; age-polya Idenlation defficinciesi thislcag ene. c cnisrna cells; WT, wild type.ThBsEIdltodoscagtelclsquneo-each ratio is less than 35%. text of the splice junction, a variable known to affect splice

VOL. 14, 1994

54 10 .. ,P

84 PETERSON ET AL.

100

0

crC

0

01).-

xw

10

0 0 .

0.1

0 200 400 600 800

Exon Size

ps

gmAA

IR3' 5'

Exon Size

FIG. 9. Relationship between the p.s/p.m expression ratio and thesize of the C,u4 exon. The expression ratio (pA/splice; Table 1) foreach plasmid is plotted against the size, in nucleotides, of the C,u4exon. The exon size is diagrammed below and is the distancebetween the C,u4 5' splice junction and the nearest upstream 3'splice junction.

efficiency by poorly defined mechanisms (references 31, 37,43, and 44 and references therein). However, if sequencecontext had a major effect on the expression from the AB andAB replacement plasmids, it is unlikely that the data wouldshow the consistent trend between exon size and expressionratio. It may have a minor effect and contribute to the scatterin the expression ratio data. mRNA stability is unlikely toaffect the differential accumulation between the p.s and p.mforms of the AB mRNA because p.s and p.m mRNAs havebeen shown to be equally stable (45).The results presented here identify the Cp.4 exon size as a

parameter affecting the competition between Cp.4-M1 splic-ing and cleavage-polyadenylation at the p.s poly(A) site.Previous studies of exon size and splicing efficiency havemainly focused on the processing of very small exons (6-8,37, 44). A maximum exon size has been suggested by studiesthat have shown that exons expanded to .300 nt do notsplice in vitro (32). This is consistent with the observationthat vertebrate internal exons have a mean length of 137 ntand are rarely larger than 300 nt (13). Our experiments arethe first to show that the size of an exon that is notconstrained by being too small affects the balance betweentwo competing RNA processing reactions. Also, since threedifferent 3' splice junctions, those at the 5' end of exonsCp.2, Cp.3, and Cp.4, and three different 5' splice junctions,the wild-type Cp.4 5' splice, the consensus Cp.4 5' splice, andthe cryptic 5' splice in AB XmncDNA(-), are represented inthis set of genes, the exon size effect must be independentof the specific sequence of the splice junctions.The effect of exon size on the competition between

splicing and cleavage-polyadenylation in the gene that weobserve can be interpreted in terms of the exon definitionmodel for splice site recognition (15, 32). This model pro-poses that exons are recognized as a unit by factors inter-acting at the 3' and 5' splice junctions during early spliceo-some assembly. Also, 5'- and 3'-terminal exons requireinteractions between a splice junction and the mRNA 5' cap

structure or poly(A) site, respectively. The p.s versus p.mRNA processing choice can be viewed as a competitionbetween defining Cp.4 as an internal exon (3' and 5' splicesites interact) or as a terminal exon [3' splice site and poly(A)site interact]. p. RNAs with C,u4 exons of <333 nt (the sizeof the s-m C,u4 exon) produce both p.m (spliced) and p.s (pA)RNA. Smaller exons may enhance the 3'-5' splice siteinteractions and increase splicing relative to cleavage-poly-adenylation. In plasma cells, p. RNAs with Cp,4 exons of2448 nt are 100% cleaved and polyadenylated. In thissituation, the 3' and 5' splice sites may be unable to interact,and the 3' splice site-poly(A) interaction dominates. Surpris-ingly, B cells still splice these same RNAs; a 786-nt exon is6% spliced, and a 544-nt exon is 10% spliced. This mayreflect some feature of the regulatory mechanism acting onthe p. gene (see below). It is interesting to note that the C,u4exon, at 333 nt, is near the upper size of natural vertebrateinternal exons. This size is evolutionarily conserved amongp. genes from species as diverse as humans and sharks andalso among other mouse immunoglobulin isotypes (personalobservation). This might suggest that exon size contributes,along with the evolutionarily conserved suboptimal C,u4 5'splice junction (30), to the overall splice and cleavage-polyadenylation efficiencies in this gene.Good evidence is accumulating to suggest that 3' splice

sites and poly(A) sites interact across 3'-terminal exons (4,16, 18, 20, 22-25, 40). Also, experiments with polyomavirus(19) and model adenovirus substrates (24) have shown that a5' splice site placed between a 3' splice site and poly(A) siteabolished cleavage-polyadenylation, presumably becausethe 3' splice site-poly(A) site interaction was interrupted.However, the p. gene naturally contains a 3' splice site-5'splice site-poly(A) site order of processing signals, and boththe 5' splice site and the poly(A) site function as RNAprocessing signals. The relative frequency with which eachis recognized and chosen is dictated by the cell type (B cellsversus plasma cells) as well as by other parameters thataffect the overall efficiency of each reaction (26, 30; thisreport). Therefore, a 5' splice site located between a 3' splicesite and a poly(A) site does not always eliminate poly(A) siterecognition. In fact, all experiments with the p. gene can beinterpreted in terms of simple competition between mutuallyexclusive RNA processing reactions. Intron size, 5' and 3'splice junction sequences, p.s poly(A) site sequences, andnow exon size are all parameters that affect the splice andcleavage-polyadenylation efficiencies in the p. gene. Theseparameters can be changed individually or in combination toalter the overall balance between the competing splice andcleavage-polyadenylation reactions.

Expression from all of the p. gene-derived plasmids pre-sented here is regulated when pA/splice ratios are comparedbetween B cells and plasma cells. Thus, the observedchanges in expression ratios among the p. genes reflectgeneral aspects of RNA processing efficiencies and notspecific aspects of processing regulation. How the change inpA/splice mRNA ratio is mediated and which cells regulate p.processing is still an open question. General cleavage-poly-adenylation efficiency is higher in plasma cells than in Bcells, as measured by the relative use of tandem poly(A) sites(17, 28). Most nonlymphoid cells tested to date process a p.pre-mRNA in a plasma cell-like pattern (21, 27a, 38), thussuggesting that the B cells regulate p. processing and theplasma cells process p. along the default pathway. Theseresults together may suggest that cleavage-polyadenylationis down-regulated in B cells. But given the observation thatexon size affects p.s/p.m expression, RNA processing in the

MOL. CELL. BIOL.

EFFECT OF EXON SIZE ON p. GENE EXPRESSION 85

p. gene also might be thought of as a competition betweendefining the CR4 exon as an internal versus a terminal exon.Therefore, any factors that influence the balance betweenthese two events could potentially be involved in the regu-lation. For example, B cells could contain a factor thatdecreases 3' splice site-poly(A) site communication or, al-ternatively, a factor that enhances 3'-5' splice site commu-nication. With respect to the latter possibility, it would beinteresting to determine whether B cells generally splicelarge internal exons more efficiently than plasma cells do.

ACKNOWLEDGMENTS

We thank Ed Gimmi and Bob Perry for the gift of the AB plasmid,Gina Russell for the Northern blot analyses in Fig. 5, and BrettSpear, Brian Rymond, and Bob Perry for helpful comments on themanuscript.

This research was supported by American Cancer Society insti-tutional research grant 163 and the National Science Foundation.

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