A Set of Highly Conserved RNA-binding Proteins, aCP-1 andaCP-2, Implicated in mRNA Stabilization, Are Coexpressed froman Intronless Gene and Its Intron-containing Paralog*

(Received for publication, April 22, 1999)

Aleksandr V. Makeyev, Alexander N. Chkheidze, and Stephen A. Liebhaber‡

From the Howard Hughes Medical Institute and the Departments of Genetics and Medicine,University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Gene families normally expand by segmental genomicduplication and subsequent sequence divergence. Al-though copies of partially or fully processed mRNA tran-scripts are occasionally retrotransposed into the ge-nome, they are usually nonfunctional (“processedpseudogenes”). The two major cytoplasmic poly(C)-bind-ing proteins in mammalian cells, aCP-1 and aCP-2, areimplicated in a spectrum of post-transcriptional con-trols. These proteins are highly similar in structure andare encoded by closely related mRNAs. Based on thisclose relationship, we were surprised to find that one ofthese proteins, aCP-2, was encoded by a multiexon gene,whereas the second gene, aCP-1, was identical to andcolinear with its mRNA. The aCP-1 and aCP-2 geneswere shown to be single copy and were mapped to sep-arate chromosomes. The linkage groups encompassingeach of the two loci were concordant between mice andhumans. These data suggested that the aCP-1 gene wasgenerated by retrotransposition of a fully processedaCP-2 mRNA and that this event occurred well beforethe mammalian radiation. The stringent structural con-servation of aCP-1 and its ubiquitous tissue distributionsuggested that the retrotransposed aCP-1 gene was rap-idly recruited to a function critical to the cell and dis-tinct from that of its aCP-2 progenitor.

Two highly similar human proteins, aCP-1 and aCP-2 (alsoknown as heterogeneous nuclear ribonucleoprotein E1/PCBP1and heterogeneous nuclear ribonucleoprotein E2/PCBP2), be-long to a growing family of K homology (KH)1 domain contain-ing RNA-binding proteins that includes Nova-1, an autoanti-gen in paraneoplastic opsoclonus myoclonus ataxia (1); andFMR1, which is associated with fragile X mental retardationsyndrome (2). The aCP proteins appear to be multifunctional.Both aCP-1 and aCP-2 have been identified as components of aribonucleoprotein complex that assembles on the 39-UTR of a

subset of long-lived mRNAs (3–5) and have been linked tostabilization of human a-globin, rat collagen, and rat tyrosinehydroxylase mRNAs (4, 6, 7). aCP-1 and aCP-2 also function astranslational coactivators of poliovirus RNA via a sequence-specific interaction with stem-loop IV of the IRES and promotepoliovirus RNA replication by binding to its 59-terminal clover-leaf structure (8). Finally, these proteins have been implicatedin translational control of the 15-lipoxygenase mRNA (9), hu-man Papillomavirus type 16 L2 mRNA (10), and hepatitis Avirus RNA (11). Thus, the aCP proteins appear to mediate avariety of functions relating to mRNA stability and expression.

The sequences of the human (h) aCP-1 and haCP-2 mRNAshave been previously established (12, 13), as has that of amurine (m) aCP-2 mRNA (heterogeneous nuclear ribonucleo-protein X (14), murine CBP (15)). With the aim of better un-derstanding the potential roles of these related and highlyabundant RNA-binding proteins as well as to establish a foun-dation for delineation of related genetic defects, we extendedthese analyses by establishing the structures of murine andhuman aCP-1 mRNAs and genes. The data were remarkable inthat aCP-1 mRNA in both species was an exact colinear copy ofthe aCP-1 gene. This intronless structure of the human aCP-1gene contrasted with the multiexon structure of the aCP-2genes in both species. We have concluded from these and otherdata that the aCP-1 gene was most likely generated by retro-transposition of a fully processed aCP-2 mRNA prior to themammalian radiation and that this processed gene was imme-diately recruited to serve a critical function distinct from thatof its aCP-2 progenitor.

EXPERIMENTAL PROCEDURES

Isolation and Characterization of P1 Genomic Clones—Genomicclones containing the maCP-2 gene (clones 4716 and 4717) and thehaCP-1 gene (clone 19548) were identified (Genome Systems, Inc., St.Louis, MO) in 129SV mouse ES and human P1 libraries, respectively,by PCR screening with primers derived from the maCP-2 cDNA (Gen-BankTM accession number L19661: 59-GAC AGG TAC AGC ACAGGC-39 (nucleotides 712–729 of the (1)-strand) and 59-GTG GTC CCTCCA GGT CAG-39 (nucleotides 773–796 of the (2)-strand)) and thehaCP-1 cDNA (GenBankTM accession X78137: 59-GCG ACG CTG TGGGCT AC-39 (nucleotides 635–651 of the (1)-strand) and 59-GGA ATGGTG AGT TCA TGG GT-39 (nucleotides 871–890 of the (2)-strand)).Genomic clones containing the maCP-1 gene (clones 12172 and 12173)were identified in 129SV mouse ES P1 libraries by PCR screening withthe same primer set that had been used for isolation of haCP-1 genomicclones. Clone inserts were analyzed by Southern hybridization withgene-specific probes amplified from the 39-UTRs with primers INV2 andMH14 (see Table I and Fig. 2).

Isolation and Cloning of the haCP-1 Gene—Human genomic DNAwas isolated from the K562 cell line by the method of Blin and Stafford(16). The coding region of the haCP-1 gene was amplified from this DNAby PCR using sets of nested primers: INV6 and INV7 (first PCR) andINV6 and CP17 (second nested PCR). Sequences of the gene-specificprimers and their positions on cDNAs are shown in Table I and Fig. 1.Polymerase chain reactions (200 ng of genomic DNA, 25 mM each

* This work was supported in part by National Institutes of HealthGrants HL38632-6 and CA72765-01 (to S. A. L.). The costs of publica-tion of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AF139894.

‡ Investigator of the Howard Hughes Medical Institute. To whomcorrespondence should be addressed: Depts. of Genetics and Medicine,Rm. 428 CRB, University of Pennsylvania School of Medicine, 415 CurieBlvd., Philadelphia, PA 19104. Fax: 215-898-1257; E-mail:Liebhaber@mail.med.upenn.edu.

1 The abbreviations used are: KH, K homology; UTR, untranslatedregion; haCP, human aCP; maCP, murine aCP; RT-PCR, reverse tran-scription-polymerase chain reaction; bp, base pair(s); FISH, fluores-cence in situ hybridization; ORF, open reading frame; EST, expressedsequence tag.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 35, Issue of August 27, pp. 24849–24857, 1999© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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primer, 1.5 mM MgCl2, 0.2 mM dNTPs, and 2.5 units of Taq polymerase(Perkin-Elmer)) were carried out using a thermocycler (Perkin-Elmer)programmed for 5 min of initial denaturation at 95 °C, followed by 30cycles for 1 min at 94 °C, 1 min 55 °C, and 3 min at 72 °C. The ampliconof predicted size (1081 bp) was cloned in the pUC19 vector (New Eng-land Biolabs, Inc.) and confirmed as the haCP-1 gene by sequencing.

RNA Isolation, RT-PCR, and 39-Rapid Amplification of cDNAEnds—Total cytoplasmic RNA was isolated from mouse erythroleuke-mia (MEL) cells, human epithelial cancer (HeLa) cells, and primarymouse tissues (RNeasy Mini kit, QIAGEN Inc.). RNA was treated with10 units of RNase-free DNase (Ambion Inc.) prior to final isolation. Toinitiate RT-PCR, 5 mg of total RNA was reverse-transcribed with Su-perscript II reverse transcriptase (Life Technologies, Inc.) using 2 pmolof a gene-specific primer. The subsequent PCRs were then performed asdescribed above. For selective amplification of aCP-1 mRNA, the RTstep was carried out with primer CP17, which was specific for the aCP-1mRNA (see Table I and Fig. 1). The subsequent PCR was carried outwith primers INV3 and INV4, which were universal for both aCP-1 andaCP-2. 39-Rapid amplification of cDNA ends was carried out between adT primer (59-CGG AAT TCC T18-39) and nested aCP-1-specific primersAM52 (first PCR) and AM50K (second PCR) (see Table I and Fig. 1).The annealing temperature in PCRs with the dT primer was decreasedto 42 °C; the number of cycles was increased to 35–40; and a finalelongation step (7 min at 72 °C) was added. The final amplificationproduct was cloned in the pUC19 vector and sequenced.

Relative Expression of aCP-1 and aCP-2 mRNAs by RT-PCR—5 mg oftotal cytoplasmic RNA from different mouse tissues was used for firststrand cDNA synthesis with 0.5 mg of dT18 primer using 10 units ofavian myeloblastosis reverse transcriptase (Promega). PCR was carriedout with the INV2 and MH14 aCP-specific primers (see Table I and Fig.2). Reverse primers were 59-end-labeled using [g-32P]ATP and T4

polynucleotide kinase (New England Biolabs, Inc.). Aliquots were re-moved every two cycles between cycles 16 and 30 and electrophoresedon a 2.5% MetaPhor-agarose gel (FMC Corp. BioProducts). The gel wasdried, and signals were quantified by PhosphorImager analysis (Molec-ular Dynamics, Inc.) and ImageQuantTM software. Logarithms of 32Pactivity incorporated into PCR products were plotted against the cor-responding PCR cycle, and intercepts on the abscissa corresponding tothe ratio of transcripts prior to PCR amplification were calculated (17).Common reaction mixtures were used for all reactions, and exposureswere done in parallel.

Southern Blot Analysis—P1 DNAs were prepared using a high mo-lecular mass plasmid purification kit (MKb-100, Genome Systems,Inc.). Mouse genomic DNA (adult male BALB/c kidney tissue) andhuman genomic DNA (normal term placenta) were obtained fromCLONTECH. 100 ng of P1 DNA and 25 mg of genomic DNA weresubjected to overnight restriction cleavage with stated enzymes (NewEngland Biolabs, Inc.). The restriction fragments were separated by0.8% agarose gel electrophoresis at 20 V for 22 h, transferred to a nylonmembrane (Nytran Plus, Schleicher & Schull) under alkaline condi-tions, and immobilized by baking for 2 h at 80 °C. DNA templates forthe synthesis of aCP-1-specific RNA probes were obtained as PCRproducts of P1 clones 12172 (maCP-1) and 19548 (haCP-1) with primersT7-INV2 and MH14 (see Table I and Fig. 2). 32P-Labeled RNA probeswere synthesized using a MEGAshortscript kit (Ambion Inc.). 50 mCi of[32P]CTP (400 Ci/mmol) was added to 20 ml of transcription reactionmixture. RNA probes (specific activity of 10–20 Ci/mmol) were sepa-rated from unincorporated nucleotides on a Sephadex G-50 Quick Spincolumn (Roche Molecular Biochemicals). Prehybridization and hybrid-ization were performed in 0.5 M NaPO4 buffer (pH 7.2), 1.5 mM EDTA(pH 8.0), and 7% SDS. The probes were hybridized to the membranes at65 °C overnight.

FIG. 2. Alignment of aCP 3*-UTR sequences. The 39-UTRs of the following are presented: haCP-1 (GenBankTM accession number X78137)(13), maCP-1 (GenBankTM accession number AF139894) (this report), haCP-2 (GenBankTM accession number X78136) (13) extended using ESTclones (GenBankTM accession numbers W60916, AI183557, and N29959), maCP-2 (GenBankTM accession number X97982), rat (r) aCP-1 andaCP-2 (this report; see Appendix 2). Nucleotides that are identical among all mRNAs are indicated by hyphens, and all other sequences that differfrom haCP-1 (top line) are enumerated. The positions of the TAG termination codons are shown in boldface at the beginning of each sequence; theAATAAA poly(A) addition signal is underlined; and the poly(A) tails are shown. The positions of primers used in RT-PCRs are shown (see also TableI).

Fig. 1. maCP-1 gene ORF and alignment of mouse and human aCP-1 and aCP-2 cDNA coding regions. The haCP-1 (GenBankTM

accession number X78137) (13), maCP-1 (ORF from P1 clone 12173 (GenBankTM accession number AF139895) and cDNA coding region(GenBankTM accession number AF139894)), haCP-2 (GenBankTM accession X78136) (13), and maCP-2 (GenBankTM accession number L19661) (14)cDNAs and inferred protein sequences (single-letter code) are shown. Nucleotides and amino acids that are identical among all four genes areindicated by hyphens, and all other sequences are enumerated. The positions of primers used in the study for RT-PCRs are indicated by arrowsat appropriate positions on the cDNAs (see also Table I). The positions of the three KH domains are indicated by labeled and overlined.

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Antibodies and Western Blot Analyses—MEL cells were grown tosubconfluence, lysed in buffer (20 mM Tris (pH 7.4), 10 mM KCl, 1 mM

MgCl2, 1 mM dithiothreitol, 0.1% Triton X-100, 0.1% SDS, and anti-protease mixture (2 mg/ml leupeptin, 1 mg/ml pepstatin, 2 mg/ml apro-tinin, and 100 mg/ml phenylmethylsulfonyl fluoride)), sonicated, andthen heated at 95 °C for 5 min. After centrifugation, supernatant pro-tein concentrations were estimated using a Bio-Rad Bradford proteinassay kit. 50 mg of proteins was separated on each lane of a 12.5%SDS-polyacrylamide gel and electroblotted onto a nitrocellulose mem-brane (Protrane, Schleicher & Schull) in buffer containing 25 mM Tris,192 mM glycine, and 20% methanol. After overnight blocking in 13phosphate-buffered saline, 5% (w/v) dry milk, and 0.3% Tween, themembrane was incubated with rabbit antisera raised against aCP-1(1:6000 dilution) or aCP-2 (1:6000 dilution) (gifts of Dr. G. Gamarnik,University of California, San Francisco, CA) (8). The gels were devel-oped by incubation with horseradish peroxidase-conjugated donkeyanti-rabbit IgG (used at 1:7000 dilution; followed by ECL detection ofimmune complexes Amersham Pharmacia Biotech). Bacterially ex-pressed recombinant aCP-1 and aCP-2 with His6 tags were preparedand purified as detailed (18).

Chromosome Mapping—For fluorescence in situ hybridization(FISH), P1 plasmid DNA (clone 12173 for aCP-1 and clone 4716 foraCP-2) was labeled with digoxigenin-dUTP by nick translation. Labeledprobe was combined with sheared mouse DNA and hybridized to met-aphase chromosomes derived from mouse embryo fibroblasts in a solu-tion containing 50% formamide, 10% dextran sulfate, and 23 SSC.Specific hybridization signals were detected by incubating the hybrid-ized slides in fluoresceinated anti-digoxigenin antibodies, followed bycounterstaining with 4,6-diamidino-2-phenylindole. Chromosome iden-tity was confirmed with probes specific to the telomere (chromosome 6)or centromere (chromosome 15).

Interspecific backcross panels (Jackson Laboratory Mapping Re-source) contained 95 progeny ((C57BL/6J 3 Mus spretus)F1 3 C57BL/6J) and 94 progeny ((C57BL/6JEi 3 SPRET/Ei)F1 3 SPRET/Ei) (19).Using primers 59-TGC TGG AGG TGG GGG TGA T-39 and 59-TGA ACTACA ACA CAA CTG CTT T-39, corresponding to one of the maCP-2 geneintrons, we identified a PCR product length polymorphism (C57BL/6J,'220 bp; M. spretus, '190 bp) with which to follow segregation of Pcbp2alleles.

Estimation of Divergence Rates among aCP Gene Sequences—Theevolutionary distances between two homologous sequences were com-puted by “Method 1” of Ina (20), which includes a correction for multiplesubstitutions at single sites based on the two-parameter model ofKimura. Distances (K) were converted to rates (r) using the equationr 5 K/(2T), where T is the divergence time between the two species (21).

RESULTS

The maCP-1 Gene Lacks Introns—As part of our ongoingstudy of the aCP proteins, we initiated an analysis of the aCP-1and aCP-2 genes. The entire 11-kilobase aCP-2 gene has been

structurally defined and will be presented in detail elsewhere.2

This gene, which has been partially mapped by others (15),contains at least 12 introns. Two distinct mouse genomic P1clones containing the aCP-1 gene were identified (see “Exper-imental Procedures”). The full sequences of the maCP-1 geneon the two clones were determined and were identical to eachother. This maCP-1 gene sequence was surprising in that itrevealed an uninterrupted 1071-nucleotide open reading frame(ORF) (Fig. 1). The maCP-1 ORF was highly similar (94%sequence identity) to that of the previously reported haCP-1mRNA, and the inferred protein structure of maCP-1 wasidentical to haCP-1 with the exception of a single valine-for-alanine substitution at position 205 (Fig. 1). Thus, in markedcontrast to the multiexon aCP-2 gene, the maCP-1 gene lacksintrons.

The “Processed” maCP-1 Gene Is Expressed—To determinewhether the processed maCP-1 gene that we had identified wasa pseudogene or was expressed, we compared its sequence tothat of the maCP-1 mRNA. A 1.1-kDa fragment encompassingthe entire ORF of maCP-1 mRNA was generated by RT-PCRfrom poly(A) mRNA from MEL cells. The sequence of thisamplified region was identical to that of the maCP-1 gene (Fig.1). This sequence comparison was extended to include the39-untranslated region by 39-rapid amplification of cDNA endsamplification (see “Experimental Procedures”). The amplified

2 A. V. Makeyev, A. N. Chkheidze, and S. A. Liebhaber, manuscript inpreparation.

FIG. 3. Western blot detection of mouse aCP-1 and aCP-2. MELcell extract (lanes 1 and 4), recombinant aCP-1 (lanes 2 and 5), andrecombinant aCP-2 (lanes 3 and 6) were electrophoresed (SDS-poly-acrylamide gel), transferred to a nitrocellulose membrane, and probedwith rabbit antisera specific to either aCP-1 (lanes 1–3) or aCP-2 (lanes4–6). The specificities of the two antisera were documented by specificinteractions with the recombinant proteins as shown. RecombinantaCP-1 and aCP-2 were expressed from the pET28- and pQE-type vec-tors, respectively, and contained 34 (aCP-1) and 10 (aCP-2) additionalvector-derived amino acids. The positions of two molecular mass mark-ers are shown on the left.

FIG. 4. Detection of multiple alternatively spliced aCP-2mRNAs, but only a single aCP-1 mRNA. A, the region of the aCP-2gene located between the second and third KH domains is shown. Thesix consecutive exons in this region are arbitrarily labeled A–F (fullstructure of the aCP-2 gene will be detailed elsewhere; see Footnote 2).The alternatively spliced exons or exon segments are shown in solidblack. Exon splicing is indicated by the angled lines; exon F has twocompeting splice acceptor sites. The size of each of the alternativelyspliced exons is indicated, as are the positions of the two RT primers(INV7 and CP17) and the two PCR primers (INV3 and INV4) used inthe amplification reactions (horizontal arrows). The specificity of eachof the RT primers is indicated to the right of the respective arrows. B,shown are the results from the analysis of RT-PCR products. RNAsfrom mouse (MEL) and human (HeLa) cells were reverse-transcribedusing a primer common to aCP-1 and aCP-2 mRNAs (INV7) (lanes 2and 5; see Fig. 1) or specific to aCP-1 (CP17) (lanes 3 and 6; see Fig. 1)and then amplified between primers INV3 and INV4 (shown in A). Thefaint lower band in lane 3 was not reproducible. The control reactionslacking RT are shown in lanes 1 and 4, and the molecular size standards(lane M) are shown on the left.

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345-nucleotide 39-UTR terminated in a poly(A) tail located 14bases 39 to the second of two putative polyadenylation signals(AAUAAA). The sequence of this entire segment was an exactmatch to the corresponding region within the maCP-1 gene(Fig. 2). A compilation of 55 reported maCP-1 cDNA clones inthe GenBankTM EST data base failed to reveal any additionalor divergent sequences (see the list in Appendix 1).3 These datasupported the conclusion that maCP-1 mRNA was transcribedfrom the intronless aCP-1 gene.

Translation of the defined maCP-1 mRNA was confirmed bydetection of its protein product on Western blot analysis (Fig.3). Antisera specific for aCP-1 revealed a single band in MELcell extracts that was of slightly higher mobility than the pro-tein identified with the antisera specific for aCP-2 (Fig. 3, lanes1 and 4). These data, which were in full agreement with thepredicted molecular masses (37.5 and 38.6 kDa, respectively),demonstrated that processed aCP-1 mRNA was translationallyactive.

The Processed aCP-1 Gene Originated Prior to the Mamma-lian Radiation—The identical sequences of the intronlessmaCP-1 gene and the maCP-1 mRNA could be explained bytwo distinct models. The first model would posit that the pro-cessed aCP-1 gene was a pseudogene generated so recently thatthere had not been sufficient time for its sequence to divergefrom an originating retrotransposed maCP-1 mRNA. Thismodel would suggest that an expressed, intron-containingaCP-1 gene had been missed in the cloning survey. The secondmodel would posit that the intronless aCP-1 gene was in factfunctional and had brought with it or had acquired sufficientgene control elements to support substantial expression. Tocompare these two models, we estimated how long the pro-cessed aCP-1 gene had been in existence by searching for it inthe human genome. A segment of the haCP-1 gene encompass-ing the entire coding region was amplified from humangenomic DNA (see “Experimental Procedures”), and in parallel,a P1 clone containing the aCP-1 gene was isolated from agenomic library (see “Experimental Procedures”). The aCP-1gene sequences from both these sources were identical to eachother and were perfect matches to haCP-1 mRNA (GenBankTM

accession number X78137) (12). Thus, the intronless aCP-1gene was present in the human genome as well as the mousegenome. This finding established that the processed aCP-1gene had been generated over 80 million years ago, predatingthe mammalian radiation (21).

Human and Mouse Cells Contain Similar Sets of aCPmRNAs—Multiple splice variants of the maCP-2 transcript

have been described. The most extensive alternative splicing ofaCP-2 exons occurs in the region encoding the segment be-tween the second and third KH domains (Fig. 4, exons B, C, andE) (15).4 In contrast, an intronless aCP-1 gene would be ex-pected to yield a single mRNA species. To confirm this predic-tion, total cytoplasmic mRNA was surveyed for aCP-2 andaCP-1 mRNAs. An initial RT-PCR analysis with primers uni-versal to all aCP mRNAs (Table I and Fig. 1) revealed acomplex set of mRNAs in MEL cells. The largest cDNA frag-ment (360 bp) corresponded to full-length maCP-2 cDNA, thenext to full-length maCP-1 cDNA (330 bp), as well as the aCP-2splice variant lacking the 39 6 3-bp alternatively spliced seg-ment, and the smallest fragment corresponded to maCP-2cDNA splice variants lacking the 93-bp segment (Fig. 4B, lane2). Each of these designations was confirmed by sequence anal-ysis (data not shown). In HeLa cells, the same set of amplifiedcDNAs was observed, as well as several additional smallerfragments of undetermined structure (Fig. 4B, lane 5). Thepresence of multiple aCP-2 mRNA spliced forms was furthersubstantiated by the EST data base, which revealed that 67.5%of 30 maCP-2 cDNAs and 70.4% of 71 haCP-2 cDNAs werealternatively spliced between the second and third KH do-mains. We next carried out an amplification with an RT primerspecific to aCP-1 mRNA. This analysis, which also encom-passed the second and third KH domains, detected a singleband (Fig. 4B, lanes 3 and 6). The sequence of this band (datanot shown) confirmed its assignment as maCP-1 mRNA. Inagreement with these data, EST data base analysis revealed asingle sequence for 55 mouse and 235 human aCP-1 cDNAclones. Thus, both mouse and human cells contained multiplespliced forms of aCP-2 mRNA and a single species of aCP-1mRNA.

The Tissue Distribution of aCP-1 mRNA Parallels That ofaCP-2—The relative expression of aCP-1 and aCP-2 mRNAswas compared in a variety of tissues. RT-PCR was carried outwith primers that were universal for human and mouse aCP-1and aCP-2 mRNAs and that were positioned so as to amplify aslightly larger fragment for aCP-2 than aCP-1 (Fig. 5, A andB). The bands representing each of the two mRNAs were quan-tified, and aCP-1/aCP-2 mRNA ratios were established for eachtissue (Fig. 5C). There were no major differences between therelative levels of aCP-1 and aCP-2 mRNAs in any of the tissuesstudied (Fig. 5C). Thus, expression of the processed aCP-1 geneparalleled that of the intron-containing aCP-2 gene in all tis-sues examined.

The Genes Encoding aCP-1 and aCP-2 Are Single Copy, Are

3 For further information, Appendices 1 and 2 may be obtained fromthe author upon request.

4 A. V. Makeyev, A. N. Chkheidze, and S. A. Liebhaber, unpublisheddata.

TABLE IOligonucleotides used for RT-PCR assays and for cloning

Sequences shown in boldface correspond to restriction sites added to facilitate cloning. The underlined sequence is the core T7 promoter. See Figs.1 and 2 for locations of each primer on cDNA.

Primer Orientation Sequence (59 3 39)

aCP1-specificAM50K 1 GGG GTA CCA CGC CAC CCA CGA CCTAM52 1 GCT CTC CCA GTC TCC ACA GGCP16 2 TTA AAA CCT GGA ATT ACC GACCP17 2 GGG GTA CCA CCC CAT SCC CTT CTC A

aCP-universal (or degenerate)INV2 1 ATG GGG WGC AGC TAG AAC AT7-INV2 1 TAA TAC GAC TCA CTA TAG GGA GAT GGG GWG CAG CTA GAA CAINV3 1 CGT GAC CAT YCC GTA CCINV4 2 TTT GGA ATG GTG AGT TCA TGINV6 1 CGG AAT TCC ATG GAY RCC GGT GTGINV7 2 GGG GTA CCT GTT CTA GCT GCW CCC CATMH14 2 GGG AGA AGC TTA TAA AAC TAA

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Located on Different Chromosomes, and Are in Linkage GroupsThat Are Conserved between Human and Mouse Genomes—Theformal possibility that a second intron-containing aCP-1 geneexisted in the genome was tested by Southern blot analysis.The aCP-1 and aCP-2 loci in the mouse and human genomeswere each specifically identified using discriminating probescorresponding to their unique 39-UTRs (see “Experimental Pro-cedures”). Each probe hybridized to a single band in four dif-ferent restriction digests of genomic DNA and to an identicallysized set of hybridizing bands generated from the correspond-ing mouse and human aCP-1 (P1) clones (Fig. 6 and data notshown). These data demonstrated that the aCP-1 and aCP-2genes were each present as unique loci in the mouse andhuman genomes.

The chromosome map positions of the maCP-1 and maCP-2genes were determined by in situ hybridization and recombi-

nation mapping. The mouse aCP-2 gene (Pcbp2) was localizedby FISH to cytoband 15F1 (Fig. 7C). In full agreement with thisassignment, an interspecific backcross analysis (see “Experi-mental Procedures”) demonstrated the Pcbp2 locus as nonre-combinant with the marker D15Mit16, placing Pcbp2 58–61centimorgans distal to the centromere of mouse chromosome15. This map position of the maCP-2 locus shared an extensiveregion of synteny with the previously reported map position ofthe human aCP-2 gene (PCBP2) (human chromosome12q13.12-q13.13; Oxford Grid) (22). The maCP-1 gene waslocalized by FISH analysis to cytoband 6D1 (Fig. 7, A and B).This region was syntenic with human chromosome 2p12-p13(Oxford Grid), which is the map position of the haCP-1 gene(PCBP1) (22). These data demonstrated synteny of correspond-ing aCP-1 and aCP-2 loci in mouse and human genomes andconfirmed the orthologous nature of their relationships.

DISCUSSION

The present report demonstrates that the highly conservedRNA-binding protein aCP-1 is encoded by a processed gene.This conclusion derived from the following three lines of evi-dence. 1) The sequence of the aCP-1 gene was colinear with andidentical to its mRNA (Figs. 1 and 2). 2) This processed aCP-1gene was the only aCP-1 locus in the mouse and human ge-nomes (Fig. 6). 3) In contrast to the multiple alternativelyspliced aCP-2 mRNAs, only a single aCP-1 mRNA could beidentified (Fig. 4 and Appendix 1). This processed aCP-1 geneencodes an abundant and functionally active protein (Fig. 3) (8,9).

Based on the data presented, the most likely origin of theprocessed aCP-1 gene was via retrotransposition of a fullyprocessed aCP-2 mRNA. For this gene to be vertically trans-mitted, such an event would have had to occur in the germ line.The observation that the aCP-2 mRNA is widely expressed(Fig. 5) and is present in testis, unfertilized mouse egg, and intwo-cell stage mouse embryo (see EST data base data in Ap-pendix 1) supports this model. Characteristic features of retro-posons such as a remnant poly(A) tail and/or direct repeatsflanking the region of homology between the processed gene

FIG. 5. Parallel expression of aCP-1 and aCP-2 mRNAs in dif-ferent mouse tissues. A, a representative quantitative RT-PCR ofaCP-1 and aCP-2 mRNAs (mouse heart mRNA) is shown. Primer MH14was used for RT, and cDNA fragments were amplified with primersINV2 and MH14 (Table I and Figs. 1 and 2). The amplified aCP-1 andaCP-2 cDNA fragments were of the predicted sizes (219 and 234 bp,respectively). Aliquots of the reaction were sampled at the indicatedcycle number. B, shown are the amplification kinetics of the RT-PCRamplification shown in A. The relative quantities of aCP-1 and aCP-2,quantified by PhosphorImager analysis, are shown for each cycle num-ber indicated. The equivalent exponential amplification phases ofaCP-1 and aCP-2 cDNAs validated the accuracy of the measured aCP-1/aCP-2 mRNA ratio (1:1.6 in the heart). C, shown are the relativelevels of aCP-1 and aCP-2 mRNAs in different mouse tissues. All valuesare taken from RT-PCRs at exponential amplification phase. rel. units,relative units.

FIG. 6. Detection of single aCP-1 and aCP-2 loci in mouse andhuman genomes by Southern blot analysis. Mouse or humangenomic DNA and DNA from P1 clones containing the maCP-1 andhaCP-1 P1 genes (clones 12172 and 19548, respectively) were digestedwith HindIII (H) or SacI (S), resolved on an agarose gel, and hybridizedwith a 32P-labeled probe specific to aCP-1 (see “Experimental Proce-dures”). The positions of the DNA molecular size markers are shown onthe right.

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and originating gene (23, 24) tend to be present in retrotrans-posed genes dating less than 40 million years and are uni-formly absent in those whose origins preceded avian and mam-malian divergence ('300 million years ago). The absence of aconvincing remnant poly(A) tail and direct repeats associatedwith the aCP-1 gene suggests a remote origin.

We have estimated the date of origin for the aCP-1 genebased on the extent of its sequence divergence (Tables II–IV).The accuracy of this approach was increased by supplementingthe mouse and human sequence data with the rat aCP genesequences identified from EST data bases (Appendix 2). Theexpected numbers of substitutions per 100 sites have verysimilar values for each orthologous pairing (Table III), and thisdivergence rate was much less than that observed in theparalogous pairings (aCP-1 versus aCP-2 in mouse, rat, andhuman) (Table IV). The orthologous relationships of the aCP-1and aCP-2 genes in these three species, along with the local-ization of the mouse and human aCP-1 genes within corre-sponding regions of known synteny (Fig. 7), supported theconclusion that the retrotransposition generating the aCP-1gene predated mammalian radiation.

The molecular clock, based on the divergence rate of the

aCP-1 39-UTR sequences subsequent to human/rodent andmouse/rat divergence (80 and 15 million years ago, respec-tively) (16), predicted that the processed aCP-1 gene was gen-erated as much as 400–500 million years ago (Fig. 8). Theabsolute rate of sequence divergence at the aCP-1 and aCP-2loci has been remarkably slow. A statistical analysis of 2820orthologous rodent and human sequences provided the averagevalue and the distribution range of divergence for both coding

TABLE IICoding sequence comparison of mouse and human aCP orthologs

Property aCP-1 aCP-2

Identity (protein), % 99.7 100Identity (cDNA), % 94.2 97.6Aligned length, bp 1065 1095Synonymous distance, Ks (S.E.),

substitutions/100 sites19.7 (2.7) 7.5 (1.5)

Synonymous rate (31029) (S.E.),substitution/site/year

1.23 (0.17) 0.47 (0.09)

Nonsynonymous distance, Ka (S.E.),substitutions/100 sites

0.3 (0.3) 0

Nonsynonymous rate (31029)(S.E.), substitution/site/year

0.02 (0.02) 0

FIG. 7. Chromosomal localization ofthe murine aCP-1 and aCP-2 genes. Aand D, representative FISH analyses formaCP-1 and maCP-2 loci, respectively.Red arrows point to gene-specific signals,and white arrows point to chromosome-specific telomeric (A) and centromeric (D)signals (the centromeric signal at chromo-some 15 is not well visualized in the re-production). The chromosomal localiza-tion of the mouse aCP-2 gene (Pcbp2) wasdetermined by FISH using P1 clone 4716as a probe. A single predominant signalwas obtained on chromosome 15 at a po-sition that corresponds to cytoband 15F1.FISH analysis using the encompassing P1clone (clone 12173) as a probe localizedthe maCP-1 locus (Pcbp1) to mouse chro-mosome 6 at a position that correspondsto cytoband 6D1. B and C, idiograms ofmouse chromosomes 6 and 15, respec-tively, indicating the positions of themaCP-1 (Pcbp1) and maCP-2 (Pcbp2) loci.

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sequences and UTRs (25). Very high coding sequence identity(97.6%) placed the aCP-2 gene at an extreme position, reveal-ing that it is one of the most highly conserved sequences in thedistribution of aligned mouse/human nucleotide identities. Thecoding sequence identity of the aCP-1 gene (94.2%) was alsohighly significantly above the reported average value (85.2%)(25). In fact, aCP-1 and aCP-2 were both among the 10 mosthighly conservative proteins in this extensive comparisongroup. Unexpectedly, a high level of conservation was alsomaintained in the 39-UTRs of human, mouse, and rat aCPmRNAs (Table III). Although it is generally assumed that39-UTRs of eukaryotic mRNAs are under less selective pressurethan coding regions, the rate of sequence divergence within39-UTRs of the aCP mRNAs was an order of magnitude lowerthan the average rate of nucleotide substitutions in an exten-sive survey of orthologous 39-UTRs (25). This conservation ofthe 39-UTR suggests the existence of a unexplained functionalconstraint on its structure.

Coexpression of functional retroposons and their intron-con-taining parental genes has been identified and characterized inseveral studies. Among the first of these sets to be discoveredwere autosomal processed genes that had originated from con-

stitutively expressed genes located on the X chromosome (phos-phoglycerate kinase 2 (Pgk-2) (26, 27), the E1a subunit ofpyruvate dehydrogenase (Pdha-2) (28, 29), the zinc finger pro-tein (Zfa) (30), and glucose-6-phosphate dehydrogenase(G6pd-2) (31)). Evolutionary fixation of this set of retrotrans-posons was suggested as a mechanism to escape X inactivation(26, 28). However, this observation could not be generalized asexamples of X-linked retrotransposons originating from auto-somal precursors were also observed (32). Finally, situationshave been reported in which functional processed genes andtheir originating intron-containing genes demonstrate a simi-lar broad range of expression (the mouse S-adenosylmethi-onine decarboxylase gene (Amd2) (33) and the human eukary-otic initiation factor 4E2 gene (34)). Thus, it is difficult tosupport general rules correlating expression patterns of origi-nating genes and retrotransposed gene copies.

Since retroposons appear to integrate randomly (23), themost surprising and improbable event in the generation of afunctional processed gene is the acquisition of functional pro-moter elements. Theoretically, this can result from mutation ofsequences adjacent to the insertion site or insertion within oradjacent to a preexisting transcription unit (35). In the latercase, the origin of transcription should be 59 to the direct repeatflanking the retrotransposition site (glutamine synthetase)(36). As the fortuitous acquisition of promoters by either ofthese two mechanisms must be exceedingly rare, other mech-anisms may also be involved. One possibility (Pgk-2 gene) (37)is that transcription could be initiated 59 to the normal startsite via an aberrant initiation event (38, 39) or from a bona fideminor or alternative promoter. In either case, the resultanttranscript could then incorporate proximal (major) promoterelements within the retrotransposed unit (40). Consistent withthis model, EST-derived sequence data suggest that the aCP-2gene has a minor promoter 59 to the predominant housekeepingpromoter (GenBankTM accession AA317361). This mechanism,which can be further explored, would explain the parallel ex-pression profiles of the retrotransposed aCP-1 locus and theoriginating aCP-2 locus (Fig. 5).

The stringent conservation of aCP-1 gene structure and cod-ing potential, predating the mammalian radiation, suggeststhat this processed gene has taken on a nonredundant andessential role. The most prominent structural differences be-tween the aCP-1 and aCP-2 proteins is that the former con-tains a 31-amino acid auxiliary domain located between RNA-binding KH domains II and III. This region is excluded frommost aCP-2 isoforms due to extensive alternative splicing ofthe aCP-2 transcript in this region. Since the aCP-1 geneoriginated as a copy of the fully spliced aCP-2 mRNA, theaCP-1 retrotransposition served to fix the high level expressionof an aCP containing this auxiliary domain (Fig. 4A). The factthat aCP-1 binds to the 39-UTR stability motif of a2-globinmRNA in vitro (18) and inhibits translation of erythroid 15-lipoxygenase and Papillomavirus type 16 L2 protein mRNAs intransfected cells establishes its functional capacity. Whetherspecificity of such actions, or additional actions, of this aCP

TABLE III39-UTR sequence comparison of mouse, human, and rat aCP orthologs

PropertyMouse vs. human Rat vs. human Mouse vs. rat

aCP-1 aCP-2 aCP-1 aCP-2 aCP-1 aCP-2

Identity, % 96.4 96.4 97.9 97.2 98.8 98.6Aligned length, bp 335 356 332 362 340 360Mutation distance, K (S.E.),

substitutions/100 sites3.7 (1.1) 3.7 (1.0) 2.1 (0.8) 2.8 (0.9) 1.2 (0.6) 1.4 (0.6)

Mutation rate (31029) (S.E.),substitutions/site/year

0.23 (0.07) 0.23 (0.06) 0.13 (0.05) 0.17 (0.06) 0.07 (0.04) 0.09 (0.04)

TABLE IV39-UTR sequence comparison of mouse, human, and rat aCP paralogs

Property HumanaCP-1/aCP-2

MouseaCP-1/aCP-2

RataCP-1/aCP-2

Identity, % 87.6 84.4 85.4Aligned length, bp 330 326 328Mutation distance, K (S.E.),

substitution/100 sites13.6 (2.2) 17.5 (2.5) 16.4 (2.5)

FIG. 8. Evolutionary tree of the aCP-1 and aCP-2 genes. Thetime scale in millions of years (Myr) is shown on the left. The positionsof the mouse, rat, and human aCP-1 and aCP-2 genes are indicated.The putative generation of the aCP-1 gene by retrotransposition of theaCP-2 cDNA is indicated by the curved arrow to distinguish this eventfrom a typical gene duplication event.

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isoform can be related to the region between the two last KHdomains remains to be demonstrated.

Acknowledgments—We thank Martin Holcik for contributions toPcbp2 chromosomal mapping. The manuscript was assembled with theexpert secretarial assistance of Jessie Harper.

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Aleksandr V. Makeyev, Alexander N. Chkheidze and Stephen A. LiebhaberIntron-containing Paralog

mRNA Stabilization, Are Coexpressed from an Intronless Gene and Its CP-2, Implicated inαCP-1 and αA Set of Highly Conserved RNA-binding Proteins,

doi: 10.1074/jbc.274.35.248491999, 274:24849-24857.J. Biol. Chem. 

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