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Structural Organization of the Human Gene (PKR) Encoding an Interferon-Inducible RNA-Dependent Protein Kinase (PKR) and Differences from Its Mouse Homolog
SHORT COMMUNICATION Structural Organization of the Human Gene (PKR) Encoding an Interferon-Inducible RNA-Dependent Protein Kinase (PKR) and Differences from Its Mouse Homolog KELLI L. KUHEN,*,† XUEYU SHEN,* ELLIOT R. CARLISLE,* AMI L. RICHARDSON,* HEINZ-ULRICH G. WEIER,‡ HIDEO TANAKA,*,1 AND CHARLES E. SAMUEL*,†,2 *Department of Molecular, Cellular and Developmental Biology, and †Interdepartmental Graduate Program of Biochemistry and Molecular Biology, University of California, Santa Barbara, California 93106; and ‡Center for Molecular Cytogenetics, Life Sciences Division, University of California, Lawrence Berkeley National Laboratory, Berkeley, California 94720 Received March 7, 1996; accepted May 29, 1996
The gene encoding the interferon-inducible, RNAdependent protein kinase (PKR) was isolated as l phage and P1 phage clones from human genomic DNA libraries and characterized by Southern blot and nucleotide sequence analyses. Southern blot analyses were consistent with a single PKR gene, and genomic clones colocalized by fluorescence in situ hybridization to human chromosome 2p. Sequence analysis demonstrated that the human PKR gene consists of 17 exons and spans about 50 kb. The AUG translation initiation site for the 551-amino-acid PKR protein was located in exon 3; exon 17 was the largest exon and included the UAG translation termination site, AUUAAA polyadenylation signal, and putative C(A) 3* cleavage site. Two RNA-binding motifs, RI and RII , were present in exons 4 and 6, respectively, and the codon phasing of these exon junctions was conserved between them. The organization of the regulatory and catalytic subdomains of the PKR protein was remarkably preserved between the human and the mouse PKR genes; the amino acid junction positions for 13 of the 15 protein coding exons were exactly conserved. q 1996 Academic Press, Inc.
The RNA-dependent protein kinase (PKR)3 is an important regulator of gene expression in interferontreated and virus-infected animal cells (6, 25). PKR is a protein serine/threonine kinase that acquires enzySequence data from this article have been deposited with the GenBank/EMBL Data Libraries under Accession Nos. U50632 to U50648. 1 Present address: Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734, Japan. 2 To whom correspondence should be addressed. Telephone: (805) 893-3097. Fax: (805) 893-4724. 3 Abbreviations used: PKR, the RNA-dependent eIF-2a protein kinase inducible by interferon; IFN, interferon; FISH, fluorescence in situ hybridization; dsRNA, double-stranded RNA; bp, basepair; nt, nucleotide; UTR, untranslated region; PCR, polymerase chain reaction.
matic activity following autophosphorylation, a process mediated by RNA with double-stranded character (26). Protein synthesis initiation factor eIF-2 is the best characterized of the PKR substrates (6). Phosphorylation of eIF-2 on serine 51 of the a subunit (24) leads to an inhibition of translation (9, 26). Phosphorylation of transcription factor inhibitor I-kB, which leads to activation and nuclear translocation of the transcription factor NF-kB, also is catalyzed by PKR (15). PKR is a central component of the IFN-induced antiviral response (16, 25). PKR also is implicated in the control of cell proliferation (13). The amount of active PKR kinase present within cells is regulated at multiple levels: at the transcriptional level by interferon (IFN) treatment (18, 30, 31); at the translational level by an autoregulatory mechanism (1, 32); at the posttranslational level by the RNA-mediated autophosphorylation of PKR (3, 24, 33); and also at the posttranslational level by protein complex formation either with another PKR molecule (7, 19, 22) or with the p58 cellular inhibitor of PKR (12). The availability of cDNA clones encoding the human and mouse RNA-dependent protein kinase PKR (10, 18, 31) has permitted structure–function analyses of the domains of the kinase responsible for regulation and for catalysis (6, 26). The structure of the mouse PKR gene has also been established (30). Because of the possible role of PKR as a tumor suppressor (13) and because of the central role of PKR in the antiviral actions of IFN (25), it is important to establish the structure of the human PKR gene. We report herein the exon–intron organization of the human PKR gene and compare the structure of the human gene with that of the mouse gene encoding the PKR protein. A human genomic library in l phage vector EMBL3 SP6/T7 (Clontech) was screened by filter hybridization using fragments of the human PKR cDNA as probes (23, 31). Several overlapping l phage clones were isolated (Fig. 1D). These clones were characterized by restriction mapping and Southern blot analysis. Because GENOMICS
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FIG. 1. Physical map of the human PKR gene. (A) The structure of the gene is represented with regard to the organization of the exons and introns. Exons are indicated to scale by filled boxes, numbered 1–17; introns and the 5*- and 3*-flanking regions are indicated by the solid lines. The entire gene spans approximately 50 kb and contains 17 exons. Translation initiates in exon 3 and terminates in exon 17 as indicated. (B) The restriction map shows cleavage sites for endonucleases BamHI (B), XhoI (X), SacI (S), and HindIII (H). (C) The two P1 genomic clones (202, 963) span the entire length of the PKR gene and continue into flanking sequences. (D) Eleven of the overlapping l phage genomic clones (1, 3-4, 5B, 6A, 7-1, 8, 11, 18, 80, 97, 152) are shown to scale.
the typical insert size of the l phage genomic clones was about 15 kb, and only one l clone that included the exon 12 region was obtained, a P1 phage genomic library in the pAD10SacBII vector (28) was screened by PCR. Two overlapping P1 clones were isolated (202 and 963), each with inserts of about 85 kb covering the entire human PKR gene. A composite map of the human PKR gene was determined (Fig. 1B). The precise exon–intron organization of the PKR gene was established by sequencing the plasmid subclones and by comparison of the genomic sequences to the previously determined cDNA sequences (18, 31). The human PKR gene contains 17 exons and spans about 50 kb (Fig. 1). Figure 2 summarizes the exon–intron organization of the human PKR gene. Exons range from 18 to 840 bp. Introns range from 78 bp to about 9.5 kb. All splice sites conform to the GT-AG rule (20). Exons 1 and 2, together with the first 16 nt of exon 3, specify the 5*untranslated region. The AUG translation initiation site for the 551-amino-acid human PKR protein is included in exon 3. The RNA binding subdomain motifs RI and RII (17) are located in exons 4 and 6, respectively. The K296R mutation, which substitutes arginine for lysine at amino acid position 296 of the C-terminal kinase catalytic subdomain II and thus destroys kinase activity (11, 32), is present in exon 11. The 3*-terminal exon 17, the largest exon, includes the UAG translation termination site and the 3*-UTR. The structural organization of the human PKR gene determined from overlapping l phage clones was consistent with that of two independently isolated P1 phage clones that spanned all 17 exons (Fig. 1). The structure determined from the l and P1 clones was
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confirmed by restriction mapping and Southern blot comparison with digests of genomic DNA isolated from human amnion U cells (data not shown). The pattern of bands was consistent with the presence of a single copy of the PKR gene in the haploid genome of the human. Metaphase spreads from normal human lymphocytes hybridized with l- and P1-derived probes (34, 35) showed signals on the short arm of chromosome 2 (data not shown), in agreement with the previously determined assignment of the human PKR gene to a single locus at 2p21–p22 (2, 29). The available genomic sequence, including genomic sequence 5* of the first PKR exon, was analyzed for the presence of Alu family DNA repeat elements (27). Alu elements were identified 5* of exon 1 and within the first intron of the PKR gene (data not shown). Another Alu-like element had previously been identified in the 3*-UTR of PKR at nt 1865 to 2076 (18, 31), which corresponds to sequences within the last exon. Insertion of Alu-type DNA repeats during primate evolution has been reported to lead to increased instability, thus contributing to genetic variability and causing inheritable disorders in humans (27). The presence of Alu repeats in the human PKR gene, on the other hand, seems to have no adverse effect on its expression. The nucleotide sequence previously determined for the human PKR cDNA included a 5*-UTR of 186 nt (18, 31). 5*-end cDNA clones were isolated by the 5*RACE procedure (5) from a human placenta cDNA library using the Marathon-Ready cDNA system (Clontech). The 5*-RACE cDNA clones included the previously reported 186-nt sequence along with an addi-
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FIG. 2. Comparison of the exon organization of the human and mouse PKR genes. The structures of the human (top) and mouse (bottom) PKR genes are compared with regard to the organization and sizes of the exons and introns. Introns are denoted by inverted triangles, with intron sizes specified in kilobases above the triangles; filled triangles indicate conserved intron positions between the human and the mouse PKR genes. Exons are indicated to scale, with exon sizes specified in nucleotides (nt) below the schematic. Open regions in the schematics depict the 5*- and 3*-untranslated regions; the internal shaded region corresponds to the open reading frame.
tional 249 nt of 5*-sequence that corresponded exactly to sequence found in the 5*-genomic lambda clones 1 and 8. Thus, the 5*-UTR of the human PKR gene is 435 nt (Fig. 2). The significance of the finding that the human PKR mRNA has a larger 5*-UTR and one additional exon compared to the mouse PKR mRNA, which possesses a 159-nt 5*-UTR (30), is unclear. The 3*-UTR, 720 nt, is completely within exon 17, the largest exon of the PKR gene at 840 nt (Fig. 2). Based on the cDNA sequence obtained for oligo(dT)primed cDNA clones of human PKR (18, 31) and our genomic sequence data, it is likely that the 3* cleavage site and polyadenylation of the PKR nascent transcript occur after the C(A) at nt 2373 following the AUUAAA polyadenylation signal positioned at nt 2343–2348 (4). No additional candidate polyadenylation signals were found in the genomic sequence within 300 nt downstream of the proposed AUUAAA, although an additional candidate AUUAAA polyadenylation signal was identified upstream at nt 2253 to 2258, followed 19 nt later by a C(A) at nt position 2277. However, sequence analysis of cDNA clones does not support the possibility that this second alternative polyadenylation signal is utilized. Comparison of the human PKR gene structure with that previously determined for the mouse PKR gene (30) revealed that the exon–intron organization was highly conserved between human and mouse. As shown by the schematic diagrams in Fig. 2, the human and mouse PKR genes contained 17 and 16 exons, respec-
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tively. The human PKR gene possesses one additional exon in the 5*-UTR region. The human PKR gene, which spans about 50 kb (Fig. 1), is significantly larger than the 28-kb mouse gene (30). The principal difference in size between the two genes is due to the larger size of some of the introns of the human gene compared to those of the mouse gene, as illustrated by introns I, IV, X, XI, and XIV, which were 2.5 to 7.3 kb larger in the human gene (Fig. 2). TABLE 1 Homology Comparison of Protein Coding Exons of Human and Mouse PKR Genes Size (nt)
Identity
Size (aa)
Identity
Similarity
No.
Hu
No.
Ms
(nt, %)
Hu
Ms
(aa, %)
(aa, %)
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
135 121 149 127 77 94 35 63 123 159 181 129 102 54 840
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
132 121 137 121 80 49 35 51 123 120 181 132 102 57 750
41.9 35.6 33.6 40.7 40.3 38.8 37.1 33.3 41.7 35.0 33.3 39.3 33.3 38.9 53.2
40 40 50 42 26 31 12 21 41 53 60 43 34 18 40
39 40 46 40 27 16 12 17 41 40 60 44 34 19 40
61.5 69.2 47.8 57.5 42.9 20.0 50.0 35.3 78.0 60.0 71.7 74.4 61.8 64.7 57.5
76.9 87.2 65.2 67.5 64.3 46.7 58.3 52.9 80.5 75.0 81.7 83.7 70.6 70.6 70.0
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FIG. 3. Codon phases and sizes of R motif exons. The codon phasing at the exon–intron junctions is shown for the human and mouse PKR genes. The two RNA-binding R motifs are found in exons 4 (RI) and 6 (RII) in the human gene and in exons 3 (RI) and 5 (RII) in the mouse gene. The sizes of the exons are shown in amino acids (aa). Human, Hs, Homo sapiens; mouse, Mm, Mus musculus. Identical amino acid residues and similar residues are shown as white letters on black and gray backgrounds, respectively.
The organization of the regulatory and catalytic subdomains of the PKR protein was remarkably preserved between the human and the mouse PKR genes. The amino acid junction positions for 13 of the 15 protein coding exons were exactly conserved. Each of the respective 15 protein coding exons were comparably sized between the human and the mouse PKR genes, with the exception of exons 8, 10, and 12, which were larger in the human than the respective cognate pair exons 7, 9, and 11 in the smaller mouse gene (Table 1). These three smaller mouse exons, 7, 9, and 11, correspond to the regions possessing the deletions previously noted in the mouse PKR cDNA relative to the human PKR cDNA (31). While the sequence homology between respective human and mouse exons was typically 35 to 40% identity at the nucleotide level, at the deduced amino acid level the identity was typically about 60% in each exon (Table 1). Curiously, the highest identity at the nucleotide level was found between the 78-nt human intron XVI and the 78-nt mouse intron XV sequences, which were 72% identical. Comparison of the positions of introns within the codons of the human and mouse PKR genes revealed a nonrandom distribution. Intron phase 0 before the first base of the codon occurred in 7 of 14 introns (50%) in the protein coding region of the human gene; 0 of 14 were intron phase 1 after the first base, and 7 of 14 (50%) were intron phase 2 after the second base of the junction codon. By comparison, when a large set of animal genes were recently analyzed, phase 0 introns represented nearly half (48%) of the total and phase two only 22% (14). The two RNA binding motifs, designated RI and RII (17, 26), were located in exons 4 and 6, respectively. Interestingly, the codon phasing was conserved between the exons that specify the RI and RII binding motifs, in both the human and the mouse PKR genes (Fig. 3). This conserved phasing would readily accommodate exon skipping involving one of the R motif exons, while still retaining the open reading frame of the mRNA encoding the downstream catalytic subdomains of the PKR protein. This is of potential biologic significance, because recombinant PKR mutants have been generated that fail to bind dsRNA, but that still
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can be activated by heparin (8, 21). Although it is conceivable that PKR variants may exist naturally that are activated by effectors other than dsRNA, PCR analysis of human placenta and kidney cDNA libraries did not reveal the presence of PKR splice site variants lacking one or both R motif exons. ACKNOWLEDGMENTS This work was supported in part by Research Grant AI-20611 from the National Institute of Allergy and Infectious Diseases, U.S.Public Health Service (C.E.S.) and by a grant from the Director, Office of Energy Research, Office of Health and Environmental Research, Department of Energy, under Contract DE-AC-03-76SF00098 (H.-U.G.W.). The assistance of Dr. Yong Liu in the preparation of Fig. 3 is gratefully acknowledged.
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SHORT COMMUNICATION 8. George, C. X., Thomis, D. C., McCormack, S. J., Svahn, C. M., and Samuel, C. E. (1996). Characterization of the heparin-mediated activation of PKR, the interferon-inducible RNA-dependent protein kinase. Virology 221: 180–188. 9. Hershey, J. W. B. (1991). Translational control in mammalian cells. Annu. Rev. Biochem. 60: 715–755. 10. Icely, P. L., Gros, P., Bergeron, J. J. M., Devalult, A., Afar, D. E. H., and Bell, J. C. (1991). TIK, a novel serine/threonine kinase, is recognized by antibodies directed against phosphotyrosine. J. Biol. Chem. 266: 16073–16077. 11. Katze, M. G., Wambach, M., Wong, M.-L., Garfinkel, M., Meurs, E., Chong, K., Williams, B. R. G., Hovanessian, A. G., and Barber, G. N. (1991). Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated 68,000-Mr protein kinase in a cell-free system. Mol. Cell. Biol. 11: 5497–5505. 12. Lee, T. G., Tang, N., Thompson, S., Miller, J., and Katze, M. G. (1994). The 58,000-dalton cellular inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (PKR) is a member of the tetratricopeptide repeat family of proteins. Mol. Cell. Biol. 14: 2331–2342. 13. Lengyel, P. (1993). Tumor-suppressor genes: News about the interferon connection. Proc. Natl. Acad. Sci. USA 90: 5893– 5895. 14. Long, M., Rosenberg, C., and Gilbert, W. (1995). Intron phase correlations and the evolution of the intron/exon structure of genes. Proc. Natl. Acad. Sci. USA 92: 12495–12499. 15. Maran, A., Maitra, R. K., Kumar, A., Dong, B., Xiao, W., Li, G, Williams, B. R. G., Torrence, P. F., and Silverman, R. H. (1994). Blockage of NF-kB signaling by selective ablation of an mRNA target by 2-5A antisense chimeras. Science 265: 789–792. 16. Mathews, M. B. (1993). Viral evasion of cellular defense mechanisms: Regulation of the protein kinase DAI by RNA effectors. Semin. Virol. 4: 247–257. 17. McCormack, S. J.,Thomis, D. C., and Samuel, C. E. (1992). Mechanism of interferon action: Identification of a RNA binding domain within the N-terminal region of the human RNA-dependent P1.eIF-2a protein kinase. Virology 188: 47–56. 18. Meurs, E., Chong, K., Galabru, J., Thomas, N. S. B., Kerr, I. M., Williams, B. R. G., and Hovanessian, A. G. (1990). Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell 62: 232–236. 19. Ortega, L. G., McCotter, M. D., Henry, G. L., McCormack, S. J., and Samuel, C. E. (1996). Mechanism of interferon action: Biochemical and genetic evidence for the intermolecular association of the RNA-dependent protein kinase PKR from human cells. Virology 215: 31–39. 20. Padgett, R. A., Grabowski, P. J., Konarska, M. M., Seiler, S., and Sharp. P. A. (1986). Splicing of messenger RNA precursors. Annu. Rev. Biochem. 55: 1119–1150. 21. Patel, R. C., Stanton, P., and Sen, G. C. (1994). Role of the amino-terminal residues of the interferon-induced protein kinase and its activation by double-stranded RNA and heparin. J. Biol. Chem. 269: 18593–18598.
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22. Patel, R. C., Stanton, P., McMillan, N. M. J., Williams, B. R. G., and Sen, G. C. (1995). The interferon-inducible double-stranded RNA-activated protein kinase self-associates in vitro and in vivo. Proc. Natl. Acad. Sci. USA 92: 8283–8287. 23. Sambrook, J., Fritsch, E. F., and Maniatis, G. (1989). Molecular Cloning, a Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory. 24. Samuel, C. E. (1979). Phosphorylation of protein synthesis initiation factor eIF-2 in interferon-treated human cells by a ribosome-associated protein kinase possessing site-specificity similar to hemin-regulated rabbit reticulocyte kinase. Proc. Natl. Acad. Sci. USA 76: 600–604. 25. Samuel, C. E. (1991). Antiviral actions of interferon. Interferonregulated cellular proteins and their surprisingly selective antiviral activities. Virology 183: 1–11. 26. Samuel, C. E. (1993). The eIF-2a protein kinases, regulators of translation in eukaryotes from yeasts to humans. J. Biol. Chem. 268: 7603–7606. 27. Schmid, C., and Maraia, R. (1992). Transcriptional regulation and transpositional selection of active SINE sequences. Curr. Opin. Genet. Dev. 2: 874–882. 28. Shepherd, N. S., Pfrogner, B. D., Coulby, J. N., Ackerman, S. L., Vaidyanathan, G., Sauer, R. H., Balkenhol, T. C., and Sternberg, N. (1994). Preparation and screening of an arrayed human genomic library generated with the P1 cloning system. Proc. Natl. Acad. Sci. USA 91: 2629–2633. 29. Squire, J., Meurs, E. G., Chong, K. L., McMillan, N. A. J., Hovanessian, A. G., and Williams, B. R. G. (1993). Localization of the human interferon-induced, ds-RNA activated p68 kinase gene (PRKR) to chromosome 2p21–p22. Genomics 16: 768–770. 30. Tanaka, H., and Samuel, C. E. (1994). Mechanism of interferon action: Structure of the mouse PKR gene encoding the interferon-inducible RNA-dependent protein kinase. Proc. Natl. Acad. Sci. USA 91: 7995–7999. 31. Thomis, D. C., Doohan, J. P., and Samuel, C. E. (1992). Mechanism of interferon action: cDNA structure, expression and regulation of the interferon-induced, RNA-dependent Pl/eIF-2a protein kinase from human cells. Virology 188: 33–46. 32. Thomis, D. C., and Samuel, C. E. (1992). Mechanism of interferon action: Autoregulation of RNA-dependent P1/eIF-2a protein kinase (PKR) expression in transfected mammalian cells. Proc. Natl. Acad. Sci. USA 89: 10837–10841. 33. Thomis, D. C., and Samuel, C. E. (1993). Mechanism of interferon action: Evidence for intermolecular autophosphorylation and autoactivation of the interferon-induced, RNA-dependent protein kinase PKR. J. Virol. 67: 7695–7700. 34. Weier, H. U. G., Rhein, A. P., Shadravan, F., Collins, C., Polikoff, D. (1995). Rapid physical mapping of the human trk protooncogene (NTRK1) gene to human chromosome 1q21–22 by P1 clone selection, fluorescence in situ hybridization (FISH) and computer-assisted microscopy. Genomics 26: 390–393. 35. Weier, H-U. G., George, C. X., Greulich, K. M., and Samuel, C. E. (1995). The interferon-inducible, double-stranded RNAspecific adenosine deaminase gene (DSRAD) maps to human chromosome 1q21.1–21.2. Genomics 30: 372–375.
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The gene encoding the interferon-inducible, RNAdependent protein kinase (PKR) was isolated as l phage and P1 phage clones from human genomic DNA libraries and characterized by Southern blot and nucleotide sequence analyses. Southern blot analyses were consistent with a single PKR gene, and genomic clones colocalized by fluorescence in situ hybridization to human chromosome 2p. Sequence analysis demonstrated that the human PKR gene consists of 17 exons and spans about 50 kb. The AUG translation initiation site for the 551-amino-acid PKR protein was located in exon 3; exon 17 was the largest exon and included the UAG translation termination site, AUUAAA polyadenylation signal, and putative C(A) 3* cleavage site. Two RNA-binding motifs, RI and RII , were present in exons 4 and 6, respectively, and the codon phasing of these exon junctions was conserved between them. The organization of the regulatory and catalytic subdomains of the PKR protein was remarkably preserved between the human and the mouse PKR genes; the amino acid junction positions for 13 of the 15 protein coding exons were exactly conserved. q 1996 Academic Press, Inc.
The RNA-dependent protein kinase (PKR)3 is an important regulator of gene expression in interferontreated and virus-infected animal cells (6, 25). PKR is a protein serine/threonine kinase that acquires enzySequence data from this article have been deposited with the GenBank/EMBL Data Libraries under Accession Nos. U50632 to U50648. 1 Present address: Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734, Japan. 2 To whom correspondence should be addressed. Telephone: (805) 893-3097. Fax: (805) 893-4724. 3 Abbreviations used: PKR, the RNA-dependent eIF-2a protein kinase inducible by interferon; IFN, interferon; FISH, fluorescence in situ hybridization; dsRNA, double-stranded RNA; bp, basepair; nt, nucleotide; UTR, untranslated region; PCR, polymerase chain reaction.
matic activity following autophosphorylation, a process mediated by RNA with double-stranded character (26). Protein synthesis initiation factor eIF-2 is the best characterized of the PKR substrates (6). Phosphorylation of eIF-2 on serine 51 of the a subunit (24) leads to an inhibition of translation (9, 26). Phosphorylation of transcription factor inhibitor I-kB, which leads to activation and nuclear translocation of the transcription factor NF-kB, also is catalyzed by PKR (15). PKR is a central component of the IFN-induced antiviral response (16, 25). PKR also is implicated in the control of cell proliferation (13). The amount of active PKR kinase present within cells is regulated at multiple levels: at the transcriptional level by interferon (IFN) treatment (18, 30, 31); at the translational level by an autoregulatory mechanism (1, 32); at the posttranslational level by the RNA-mediated autophosphorylation of PKR (3, 24, 33); and also at the posttranslational level by protein complex formation either with another PKR molecule (7, 19, 22) or with the p58 cellular inhibitor of PKR (12). The availability of cDNA clones encoding the human and mouse RNA-dependent protein kinase PKR (10, 18, 31) has permitted structure–function analyses of the domains of the kinase responsible for regulation and for catalysis (6, 26). The structure of the mouse PKR gene has also been established (30). Because of the possible role of PKR as a tumor suppressor (13) and because of the central role of PKR in the antiviral actions of IFN (25), it is important to establish the structure of the human PKR gene. We report herein the exon–intron organization of the human PKR gene and compare the structure of the human gene with that of the mouse gene encoding the PKR protein. A human genomic library in l phage vector EMBL3 SP6/T7 (Clontech) was screened by filter hybridization using fragments of the human PKR cDNA as probes (23, 31). Several overlapping l phage clones were isolated (Fig. 1D). These clones were characterized by restriction mapping and Southern blot analysis. Because GENOMICS
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36, 197–201 (1996) ARTICLE NO. 0446
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FIG. 1. Physical map of the human PKR gene. (A) The structure of the gene is represented with regard to the organization of the exons and introns. Exons are indicated to scale by filled boxes, numbered 1–17; introns and the 5*- and 3*-flanking regions are indicated by the solid lines. The entire gene spans approximately 50 kb and contains 17 exons. Translation initiates in exon 3 and terminates in exon 17 as indicated. (B) The restriction map shows cleavage sites for endonucleases BamHI (B), XhoI (X), SacI (S), and HindIII (H). (C) The two P1 genomic clones (202, 963) span the entire length of the PKR gene and continue into flanking sequences. (D) Eleven of the overlapping l phage genomic clones (1, 3-4, 5B, 6A, 7-1, 8, 11, 18, 80, 97, 152) are shown to scale.
the typical insert size of the l phage genomic clones was about 15 kb, and only one l clone that included the exon 12 region was obtained, a P1 phage genomic library in the pAD10SacBII vector (28) was screened by PCR. Two overlapping P1 clones were isolated (202 and 963), each with inserts of about 85 kb covering the entire human PKR gene. A composite map of the human PKR gene was determined (Fig. 1B). The precise exon–intron organization of the PKR gene was established by sequencing the plasmid subclones and by comparison of the genomic sequences to the previously determined cDNA sequences (18, 31). The human PKR gene contains 17 exons and spans about 50 kb (Fig. 1). Figure 2 summarizes the exon–intron organization of the human PKR gene. Exons range from 18 to 840 bp. Introns range from 78 bp to about 9.5 kb. All splice sites conform to the GT-AG rule (20). Exons 1 and 2, together with the first 16 nt of exon 3, specify the 5*untranslated region. The AUG translation initiation site for the 551-amino-acid human PKR protein is included in exon 3. The RNA binding subdomain motifs RI and RII (17) are located in exons 4 and 6, respectively. The K296R mutation, which substitutes arginine for lysine at amino acid position 296 of the C-terminal kinase catalytic subdomain II and thus destroys kinase activity (11, 32), is present in exon 11. The 3*-terminal exon 17, the largest exon, includes the UAG translation termination site and the 3*-UTR. The structural organization of the human PKR gene determined from overlapping l phage clones was consistent with that of two independently isolated P1 phage clones that spanned all 17 exons (Fig. 1). The structure determined from the l and P1 clones was
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confirmed by restriction mapping and Southern blot comparison with digests of genomic DNA isolated from human amnion U cells (data not shown). The pattern of bands was consistent with the presence of a single copy of the PKR gene in the haploid genome of the human. Metaphase spreads from normal human lymphocytes hybridized with l- and P1-derived probes (34, 35) showed signals on the short arm of chromosome 2 (data not shown), in agreement with the previously determined assignment of the human PKR gene to a single locus at 2p21–p22 (2, 29). The available genomic sequence, including genomic sequence 5* of the first PKR exon, was analyzed for the presence of Alu family DNA repeat elements (27). Alu elements were identified 5* of exon 1 and within the first intron of the PKR gene (data not shown). Another Alu-like element had previously been identified in the 3*-UTR of PKR at nt 1865 to 2076 (18, 31), which corresponds to sequences within the last exon. Insertion of Alu-type DNA repeats during primate evolution has been reported to lead to increased instability, thus contributing to genetic variability and causing inheritable disorders in humans (27). The presence of Alu repeats in the human PKR gene, on the other hand, seems to have no adverse effect on its expression. The nucleotide sequence previously determined for the human PKR cDNA included a 5*-UTR of 186 nt (18, 31). 5*-end cDNA clones were isolated by the 5*RACE procedure (5) from a human placenta cDNA library using the Marathon-Ready cDNA system (Clontech). The 5*-RACE cDNA clones included the previously reported 186-nt sequence along with an addi-
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FIG. 2. Comparison of the exon organization of the human and mouse PKR genes. The structures of the human (top) and mouse (bottom) PKR genes are compared with regard to the organization and sizes of the exons and introns. Introns are denoted by inverted triangles, with intron sizes specified in kilobases above the triangles; filled triangles indicate conserved intron positions between the human and the mouse PKR genes. Exons are indicated to scale, with exon sizes specified in nucleotides (nt) below the schematic. Open regions in the schematics depict the 5*- and 3*-untranslated regions; the internal shaded region corresponds to the open reading frame.
tional 249 nt of 5*-sequence that corresponded exactly to sequence found in the 5*-genomic lambda clones 1 and 8. Thus, the 5*-UTR of the human PKR gene is 435 nt (Fig. 2). The significance of the finding that the human PKR mRNA has a larger 5*-UTR and one additional exon compared to the mouse PKR mRNA, which possesses a 159-nt 5*-UTR (30), is unclear. The 3*-UTR, 720 nt, is completely within exon 17, the largest exon of the PKR gene at 840 nt (Fig. 2). Based on the cDNA sequence obtained for oligo(dT)primed cDNA clones of human PKR (18, 31) and our genomic sequence data, it is likely that the 3* cleavage site and polyadenylation of the PKR nascent transcript occur after the C(A) at nt 2373 following the AUUAAA polyadenylation signal positioned at nt 2343–2348 (4). No additional candidate polyadenylation signals were found in the genomic sequence within 300 nt downstream of the proposed AUUAAA, although an additional candidate AUUAAA polyadenylation signal was identified upstream at nt 2253 to 2258, followed 19 nt later by a C(A) at nt position 2277. However, sequence analysis of cDNA clones does not support the possibility that this second alternative polyadenylation signal is utilized. Comparison of the human PKR gene structure with that previously determined for the mouse PKR gene (30) revealed that the exon–intron organization was highly conserved between human and mouse. As shown by the schematic diagrams in Fig. 2, the human and mouse PKR genes contained 17 and 16 exons, respec-
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tively. The human PKR gene possesses one additional exon in the 5*-UTR region. The human PKR gene, which spans about 50 kb (Fig. 1), is significantly larger than the 28-kb mouse gene (30). The principal difference in size between the two genes is due to the larger size of some of the introns of the human gene compared to those of the mouse gene, as illustrated by introns I, IV, X, XI, and XIV, which were 2.5 to 7.3 kb larger in the human gene (Fig. 2). TABLE 1 Homology Comparison of Protein Coding Exons of Human and Mouse PKR Genes Size (nt)
Identity
Size (aa)
Identity
Similarity
No.
Hu
No.
Ms
(nt, %)
Hu
Ms
(aa, %)
(aa, %)
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
135 121 149 127 77 94 35 63 123 159 181 129 102 54 840
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
132 121 137 121 80 49 35 51 123 120 181 132 102 57 750
41.9 35.6 33.6 40.7 40.3 38.8 37.1 33.3 41.7 35.0 33.3 39.3 33.3 38.9 53.2
40 40 50 42 26 31 12 21 41 53 60 43 34 18 40
39 40 46 40 27 16 12 17 41 40 60 44 34 19 40
61.5 69.2 47.8 57.5 42.9 20.0 50.0 35.3 78.0 60.0 71.7 74.4 61.8 64.7 57.5
76.9 87.2 65.2 67.5 64.3 46.7 58.3 52.9 80.5 75.0 81.7 83.7 70.6 70.6 70.0
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FIG. 3. Codon phases and sizes of R motif exons. The codon phasing at the exon–intron junctions is shown for the human and mouse PKR genes. The two RNA-binding R motifs are found in exons 4 (RI) and 6 (RII) in the human gene and in exons 3 (RI) and 5 (RII) in the mouse gene. The sizes of the exons are shown in amino acids (aa). Human, Hs, Homo sapiens; mouse, Mm, Mus musculus. Identical amino acid residues and similar residues are shown as white letters on black and gray backgrounds, respectively.
The organization of the regulatory and catalytic subdomains of the PKR protein was remarkably preserved between the human and the mouse PKR genes. The amino acid junction positions for 13 of the 15 protein coding exons were exactly conserved. Each of the respective 15 protein coding exons were comparably sized between the human and the mouse PKR genes, with the exception of exons 8, 10, and 12, which were larger in the human than the respective cognate pair exons 7, 9, and 11 in the smaller mouse gene (Table 1). These three smaller mouse exons, 7, 9, and 11, correspond to the regions possessing the deletions previously noted in the mouse PKR cDNA relative to the human PKR cDNA (31). While the sequence homology between respective human and mouse exons was typically 35 to 40% identity at the nucleotide level, at the deduced amino acid level the identity was typically about 60% in each exon (Table 1). Curiously, the highest identity at the nucleotide level was found between the 78-nt human intron XVI and the 78-nt mouse intron XV sequences, which were 72% identical. Comparison of the positions of introns within the codons of the human and mouse PKR genes revealed a nonrandom distribution. Intron phase 0 before the first base of the codon occurred in 7 of 14 introns (50%) in the protein coding region of the human gene; 0 of 14 were intron phase 1 after the first base, and 7 of 14 (50%) were intron phase 2 after the second base of the junction codon. By comparison, when a large set of animal genes were recently analyzed, phase 0 introns represented nearly half (48%) of the total and phase two only 22% (14). The two RNA binding motifs, designated RI and RII (17, 26), were located in exons 4 and 6, respectively. Interestingly, the codon phasing was conserved between the exons that specify the RI and RII binding motifs, in both the human and the mouse PKR genes (Fig. 3). This conserved phasing would readily accommodate exon skipping involving one of the R motif exons, while still retaining the open reading frame of the mRNA encoding the downstream catalytic subdomains of the PKR protein. This is of potential biologic significance, because recombinant PKR mutants have been generated that fail to bind dsRNA, but that still
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can be activated by heparin (8, 21). Although it is conceivable that PKR variants may exist naturally that are activated by effectors other than dsRNA, PCR analysis of human placenta and kidney cDNA libraries did not reveal the presence of PKR splice site variants lacking one or both R motif exons. ACKNOWLEDGMENTS This work was supported in part by Research Grant AI-20611 from the National Institute of Allergy and Infectious Diseases, U.S.Public Health Service (C.E.S.) and by a grant from the Director, Office of Energy Research, Office of Health and Environmental Research, Department of Energy, under Contract DE-AC-03-76SF00098 (H.-U.G.W.). The assistance of Dr. Yong Liu in the preparation of Fig. 3 is gratefully acknowledged.
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