- Email: [email protected]
Mechanism of interferon action Sequence of the human interferon-inducible RNA-dependent protein kinase (PKR) deduced from genomic clones
GENE AN
ELSEVIER
INTERNATIONAL dOURNAL GENES AND OENOME5
ON
Gene 178 (1996) 191-193
Short communication
Mechanism of interferon action Sequence of the human interferon-inducible RNA-dependent protein kinase (PKR) deduced from genomic clones 1 Kelli U Kuhen a, Xueyu Shen
b, Charles E. Samuel a,b,,
a Interdepartmental Graduate Program of Biochemistry and Molecular Biology, University of California, Santa Barbara, CA, USA b Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106, USA Received 19 March 1996; accepted 5 April 1996
Abstract
The pkr gene encoding the interferon-inducible, RNA-dependent protein kinase was isolated as lambda phage and P1 phage clones from human genomic DNA and characterized by restriction mapping, Southern blot analysis, and nucleotide sequencing. The genomic nucleotide sequence, when compared to that of previously determined cDNA sequences, revealed 17 exons encoding the 551-amino-acid PKR protein. We report herein the sequence of the human PKR protein kinase deduced from genomic clones.
Keywords: Translational control; Protein synthesis initiation factor elF-2c~; RNA-binding protein; Antiviral protein; Tumor suppressor; Nucleotide sequencing
The protein kinase P K R from human cells, also known as the P1/eIF-2~ protein kinase, P1, p68, DAI and dsI, is an interferon-inducible enzyme that is activated posttranslationally by RNA-dependent autophosphorylation (Samuel, 1993; Clemens, 1996). The best characterized substrate phosphorylated by P K R is the alpha subunit of eukaryotic protein synthesis initiation factor eIF-2 (Samuel, 1979, 1993; Clemens, 1996). Phosphorylation of eIF-2~ on serine 51 by P K R leads to an inhibition of translation in animals, because the eIF-2aP:GDP complex generated during the initiation stage of translation cannot beefficiently recycled by the guanine nucleotide exchange factor eIF-2B (Hershey, 1991). Thus, the P K R kinase plays an important role in the regulation of protein synthesis in interferon-treated and virus-infected cells (Samuel, 1991 ), and possibly also may play an important role in the control of cell growth * Corresponding author at the Department of Molecular, Cellular and Developmental Biology. Tel.: (+1-805) 893-3097; Fax: (+1-805) 893-4724; e-mail: [email protected] 1The sequences reported in this paper have been deposited in the GenBank data base (accession No. U50632 to U50648). Abbreviations: aa, amino acid(s); IFN, interferon; kb, kilobase(s); nt, nucleotide(s);PKR, RNA-dependentprotein kinase;pkr, geneencoding PKR; UTR, untranslated region(s). 0378-1119/96/$15.00© 1996 ElsevierScienceB.V. All rights reserved PH S0378-1119(96)00314-9
and differentiation in uninfected cells (Lengyel, 1993). Molecular cDNA clones of P K R from human Daudi cells (Meurs et al., 1990) and human amnion U cells (Thomis et al., 1992) have been reported. As an extension of our studies of the regulation and function of human PKR, we have isolated lambda and P1 phage genomic clones of P K R and determined the structure of the human pkr gene (Kuhen et al., 1996). The human pkr gene contains 17 exons and spans about 50-kb pairs. We now report the entire exon sequence of the human pkr gene determined from genomic clones. Human pkr genomic clones were isolated by screening two libraries, a human placenta genomic library in the 2 phage vector EMBL3 (Clontech) and a human foreskin fibroblast genomic library in the P1 phage vector pAD10SacBII (Shepherd et al., 1994), using fragments of the human P K R cDNA (Thomis et al., 1992) as probes. Isolated genomic clones were characterized by restriction mapping and Southern blot analyses, and exon containing fragments were subcloned into the pBluescript plasmid (Stratagene) for detailed restriction mapping and DNA sequencing by the dideoxy procedure using reverse or - 2 0 universal primers and custom-synthesized primers (Sambrook et al., 1989). The genomic exon nucleotide sequence of human pkr and the deduced amino acid
K t Kuhen et al./Gene 178 (1996) 191-193
192
V
G C G G C G G C GC,C G G C G C A G S " F T G C T C A T A C q ' V I ~ T G A C ~ T P G C ACCACAGGCACGACAAGCATAGAAAC
ATC CTAAACAATCTTCATCGAGGCATC
TCATAGACC~AG~TTCGCT<~GTA~ACTCGT~TGq~TGAACCAGCGG~'I~A C ~GGAC~CAAA'I~AGT~C
C TTC CTGGAT~GTA~T~TAA%~G
GGTCAC AGTGGCATTCAGCTC
HU
A ~ ~7-G T G A T C ~ A G C A G G ~ A T G G A G G A A C q ~ A A T A C M A G D L S A G F F M E
-271
i'l'l'l'tTfAAGCCq'IR2 iT~'i'I'IC~ I"~'~'iA C C A G T T T
- 181
ACC TCAAAAC T~AGCAG'I'~C T~
N T
S .
G P P . . . .
F T F . . .
Q
V
I L
I
L .
N D
K N
AATAGAATTGCCCAGAAGAAAAGACTAACTGTAAATTATGAACAG N R I A Q K K R L T V N Y E
HU
AAT~AGAAGGA~ATCCATGGGGAATTACATAGGCCTTATC N S S E G L S M G N Y I
Hu b~
C
A E
K R
S P
V S
A A A . . . .
H E
G L
C AG G AA~A~C~T~GC E .
A
P
L .
g Q
A
V
E D
.
G R
F
H [
I
K
L E
A
Y .
AAGGA~C~
G
L
I
g C
K
C " K
C G C T A A A C q~fGC A T A T C T ~ L Q
R K
E .
M I
G
K N
Q
I L
L
S K
E .
E
G A
E K
G R S . . . .
~C~CAG T T A G T C C q r ~ T A T T A ~ A C K .
K T
AGATAq'FATCAGAAGAAAC Q K
E F P . . . .
A V
E M
V D
Y
S C
P H
S G
L .
I T
L .
-1
G
T S
K
~C~C
L .
.
T
G
T
S V
G T S
Q
T
E T S . . . .
V .
K .
S
L
.
D .
S
K
S P
.
HU
TCT~CTACTACGTG~AGTCCCAAAGCAACTCq~I~FAGTGACCAGcACAC~GCq~PCTGAAq~CATCATC~AAGGTGACT~AGCA S F A T T C E S Q S N S L V T S T L A S E S S S E G
MS
K
T
.
G
.
-
-
-
-
HU
D
T
S
E
I
N
S
N
S
MS HU MS
G N
HU
GAATTAAT~GCq~AGGTGGAT~GCCAAGT~AAAGCAA~CACA~AATTGACGGAAAGACTTACGTTATTAAACGTG~TAT E L I G S G G F G Q V F K A K H R I D G K T Y V
HU
N
K
A
E
R
E
HU MS
D Y D P . . . . .
E
T H
S
D .
90 30
60 59
C T C A G TGVAAATC T G A C T A C C T Gq%U C ~ ~ G ~ Y
.
L A A . . .
G E
Q .
.
Q .
K .
D .
HU MS
.
K
.
GCAGC C A~TTAGCTGT~AGATACq~TAAT N .
G N
R .
T
K Q
A
R .
- 91
ATAC C GTC AG AAGC AGGGAG TAG TACTTAAATATCAAG AAC T~ Y R Q K Q G V V L K Y Q E L
N
HU MS
E -
D .
C A~CTG AC TC A G G ~ C ~
C ~ C CA ~ A T A C ~ G ~ G ~
L
P S
AAGG~GCA~,CC
H .
~
E
bls
- 435
C CTGGCTA
~ ' ~ G ~ " P ~ ' I ~ I ~ A G / ~ " P ~ / ~ C A'I~ CACAC'P'~ CG'I~A~PA'P~'I~CG"I~A'I" I T ~ G A C - ~ 2
Hu
CACACTTGGTAGA
GAGGTC CATCC C AATAAAAATCAGGAGAC
G P
270 90 85
360 120
150 145 540 180 165 630 210 191
D
F S
A
S
S
M
T
N
N
Q
R
K
A
K
G A ~4~ A TVC ~ G C"A C CQ - - C A G A - - GTA~C C T T C CPT - AGC A T G - - AF~ C AASA~AGG T A T AEC T G TN G G AVC - - FG - ' T ~ G C - - T AG AG A ~- G- - -GA ~ k AET A AA
K TT~A~G
S
G
810219
R V
M S
D
F
K E
E D
!
270 245
I
K
R
V
K
Y
S K
L V
A S
P
R F D . . . .
.
E
.
.
.
.
.
. . . .
S
h
N
S
D
.
M V
.
.
E R
.
T N
.
.
.
Y
.
L
M
N
G
T
.
V L
D
K A
L
R
R
F
N
.
.
.
.
.
.
.
900 300
. 0
Y
N
G
C
W
D
G
F
330
D .
Y D P . . .
E
N
S M
K S
N D
S T
S .
R
S Y
K
T .
K R
360 322
HU
T G C C ITi" 1 C A ~ C C A A A T G G A A T T C T G ~ A T A A A G G G A C C'I~GAAC AATGGA~TGA~GAAGAGGC C L F I Q M E F C D K G T L E Q W I E K R R
G
GAGA/kAC T A G A C A ~ G I'~'I~I~ E K L D K V L
HU
A
MS
I
HU MS
T~AGTAGATACA2~CAAGTA~GATTGGAGACT~GGACT~TAAC F L V D T K Q V K I G D F G . . . . E R H I . . . . . . .
Hu
T
h
R
.
Q
C T~
F
E
Q
I
Y
Y M S P g . . . . . .
T~ATGTATGTGACACq%GC L
D E
S F
S L
Y I H S . . . . .
Q K
O H
K
F
T~A~CATCAakA~-
K
F
K b L . . . .
E
S
T
K E
S
V A
T
K G
N
I
ATCTCTGAAAAAq~3ATGGA~GCGAACAAGGAGTAAGGGA S L K N D G K R T R S K A E .S R T
G
g G K E V . . . . . .
L .
D
I H R . . . .
L I
Y F
A .
D L . . .
L G . . .
K
b .
P
K
.
P .
F
E K
D
T
R
D
P
L
N E
R
D
G
I
T S E I . . . . .
I
L
S
R K
D
T .
I
b
HU
AAAAGCCCAGAGAAAAATGAACGACACACATGTTAGAGCCCTTCTGAA~GTATCCTGC2~CTGATATGCAGTTTTCCTTAAATTATCT K S P E K N E R H T C * N
S
K
K
N
A~I~CTAGGGAATATCAATAGATATTTAC
L .
F
T A
420 382 1350 450 413
E
D
K
K
1530 510
N
AA
162@474
V E
W .
K R
540 504
480 443
1710 551 515
*
Cq~AT~AATG~I~CCTTTAA
1170 390
A
~ y ~q ~ A C A G A C C T A C G G G A T G G C A T C A T C ~ A G A T A T A T ~ G A T A ~
MS
I
S G
I .
E
S
A
L
K
G A ~4~" k A A A "C T CT-q-~T A C A GF- - TATAAAC TEC - -SA A A G~ - - CK- - A AGI~- CA qT ~C G A C C T -G- C -E- C A TS C - -AAA.A~ A T A CKT ATAGG G A DC C T ~FA C T G.N q ~ G.~ G.- -.G . Q
T
I L
V
H%I MS
L
D
Q
G
Ms
L
C
T
.
b
T
V
K
V
S .
Hu
K
H
T
S .
H
240
H
L
K
K
F
V
E .
A
L
G
I
D S L . . . .
L
S
S
N
E
A
K Q
S
F
V
D
K
V
D
L
V
P D
.
L
MS
N
L
D
S
~-I-~T~'~A C T A i'i'~TIA C T A A T C T ~ C q ~ G C A G A A
l S 00
A ~ A G A A A G G ~ T t - ~ C ~ ~ i - ~ % C , C T T C A A A A A C A T T C T T A C A q ~ F T T A C T ~ q C C T ~ G C T C A T C T C T T T A T T C c~-~"~-~-~-~T~-~-~-~uA A A
1890
GACAGAGTCTCGCTCT~q~C
1980
C CAGGCq~GAGTGCAATGACACAGTCT~GCTC
CTGCCTCAGCCTCCTGAGTAGCTGGATTACAGGCATGTGCCACCCACCCAACTAA GT'I~GCCAGGCTGGTCq~
ACTGCAACq~TGC
CTCT~GGTTCAAGTGATTCTC
I-UI-I~GTG I'I'I-x-?AATA~GAC A G G G T ~ C A C
AAACTC C TGACCTCAAGTAATCCACCTGCCTCGGCCTCCCAAAGTGCTGGGAq~TACAGGGATGAGCCAC
GCCCAGC CTCATCTCTTTGTTCTAAAGATGGAAAAACCAC
CC C C A A A T T T T C
TTAq-TAAA~I-~CTACC G C l-lq-rA G G C C A ~ T A A G A T C G T T C T C T G C ~ATT, A ~ G T G A T G T A C A A C C a c t
tcggaaaacaat
CAT
2070
CGC
2160
t"l'c~ u A T A C T A T T A A T G A A T C A A T C A A T T C A T A T C T A T
2250
CTCACATAGCTTACAAGCCAGCTGGAGAAATATGGTAC
2340
t t @ g c a t tat ct a g t a a a g t t g a a t c c a t g t a t a c c c a c a t a
2430
Fig. 1, Nucleotide sequence of the human pkr exons determined from genomic clones and the deduced aa sequence. The A of the A U G translation initiation codon in exon 3 is denoted as n t + 1; the 5'-UTR of the mature m R N A begins with nt denoted as - 4 3 5 , upstream from the A at n t + 1. The open triangle symbols above the nt sequence indicate the positions of the exon junctions in the human gene, as determined by comparison of genomic sequence (GenBank accession Nos. U50632 to U50648) to the c D N A sequence (accession No. M85294). The exon junctions for the mouse gene (Tanaka and Samuel, 1995) are indicated by the filled circle symbols. The putative polyadenylation signal and C(a) cleavage site preceding the poly(A) tail of the c D N A (Thomis et al., 1992) are underlined.
K.L. Kuhenet al./Gene178 (1996) 191-193 sequence are shown in Fig. 1. The h u m a n pkr gene contains 17 exons. Exons 1 (18 nt) and 2 (401 nt) and part of exon 3 (16 nt) constitute the 5-UTR of the m a j o r P K R transcript. E x o n 3 includes the A U G translation initiation site as established by ribosome protection analysis with the c D N A - d e r i v e d transcripts (Thomis et al., 1992). The translation termination c o d o n for synthesis of the deduced 551-aa P K R protein is located in exon 17, the largest exon (840 nt) that also includes the complete 720 nt 3'-UTR that terminates at nt 2373, the site of poly(A) addition. C o m p a r i s o n of the exon organization of the h u m a n pkr gene to that previously established for the mouse pkr gene ( T a n a k a and Samuel, 1995) shows that 13 of the 15 amino-acid-coding junctions are exactly conserved (Fig. 1). W h e n the nt sequence of the h u m a n pkr exons determined from genomic clones (Fig. 1) was compared to the reported c D N A sequences (Meurs et al., 1990; Thomis et al., 1992), the following differences were found: in exon 2 of the 5'-UTR, nt - 17 is a G as reported by T h o m i s et al. (1992) rather than a C as found by Meurs et al. (1990); in exon 3 of the 5'-UTR, nt - 1 6 is a G as reported by T h o m i s et al. (1992) rather than a C as found by Meurs et al. (1990); in exon 17 of the 3'UTR, n t + 1878 is a T in agreement with Meurs et al. (1990) rather than a C as found by T h o m i s et al. (1992); and also in exon 17 of the 3'-UTR, a deletion of two A residues following nt 2348 was found which gives the subsequent sequence of A 12 instead of A 14, in agreement with Meurs et al. (1990) rather than T h o m i s et al. (1992). Finally, the exon 1 sequence - 4 3 5 to - 4 1 8 and the 5' portion of the exon 2 sequence corresponding to nt - 4 1 7 to - 1 8 7 represents new 5'-UTR sequence that was not included in the incomplete c D N A clone sequence previously described (Meurs et al., 1990; T h o m i s et al., 1992). This new 5'-UTR sequence was obtained by the Y - R A C E procedure using c D N A prepared from h u m a n placenta R N A , and contained one difference from the genomic D N A sequence: nt - 3 3 1 was a C in the genomic sequence and a G in the c D N A sequence. The complete c D N A of h u m a n P K R is, therefore, 2808 bp in length excluding the 3' poly(A) ÷ tail.
Acknowledgement This w o r k was supported in part by Research G r a n t AI-20611 from the N a t i o n a l Institutes of Health.
193
References Clemens, M.J. (1996) Protein kinases that phosphorylate eIF2 and eIF-2B, and their role in eukaryotic cell translational control. In." Translational Control. Cold Spring Harbor Press, Cold Spring Harbor, NY, pp. 139-172. Hershey, J.W.B. (1991) Translational control in mammalian cells. Annu. Rev. Biochem. 60, 715-755. Kuhen, K.L., Shen, X., Carlisle, E.R., Richardson, A.L., Weier, H-U.G., Tanaka, H. and Samuel, C.E. (1996) Structural organization of the human gene encoding an interferon-inducible RNA-dependent protein kinase (PKR) and differences from its mouse homolog. Genomics 36, 197-201. Lengyel, P. (1993) Tumor suppressor genes. News about the interferon connection. Proc. Natl. Acad. Sci. USA 90, 5893 5895. 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. Sambrook, J., Fritsch, E.F. and Maniatis, G. (1989) Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Samuel, C.E. (1979) Phosphorylation of protein synthesis initiation factor eIF-2 in interferon-treated human cells by a ribosomeassociated protein kinase possessing site-specificity similar to hemin-regulated rabbit reticulocyte kinase. Proc. Natl. Acad. Sci. USA 76, 600-604. Samuel, C.E. ( 1991) Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology 183, 1-11. Samuel, C.E. (1993) The eIF-2a protein kinases, regulators of translation in eukaryotes from yeasts to humans. J. Biol. Chem. 268, 7603-7606. Shepherd, N.S., Pfrongner, B.D., Coulby, J.N., Ackerman, S.L., Vaidyanthan, G., Sauer, R.H., Balkenhol, T.C. and Sternberg, N. (1994) Preparation and screening of an arrayed human genomic library generated with the PI cloning system. Proc. Natl. Acad. Sci. USA 91, 2629-2633. Tanaka, H. and Samuel, C.E. (1995) Sequence of the murine interferon-inducible RNA-dependent protein kinase (PKR) deduced from genomic clones. Gene 153, 283-284. Thomis, D.C., Doohan, J.P. and Samuel, C.E. (1992) Mechanism of interferon action, eDNA structure, expression and regulation of the interferon-induced, RNA-dependent P1/eIF-2c~ protein kinase from human cells. Virology 188, 33-46.
ELSEVIER
INTERNATIONAL dOURNAL GENES AND OENOME5
ON
Gene 178 (1996) 191-193
Short communication
Mechanism of interferon action Sequence of the human interferon-inducible RNA-dependent protein kinase (PKR) deduced from genomic clones 1 Kelli U Kuhen a, Xueyu Shen
b, Charles E. Samuel a,b,,
a Interdepartmental Graduate Program of Biochemistry and Molecular Biology, University of California, Santa Barbara, CA, USA b Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106, USA Received 19 March 1996; accepted 5 April 1996
Abstract
The pkr gene encoding the interferon-inducible, RNA-dependent protein kinase was isolated as lambda phage and P1 phage clones from human genomic DNA and characterized by restriction mapping, Southern blot analysis, and nucleotide sequencing. The genomic nucleotide sequence, when compared to that of previously determined cDNA sequences, revealed 17 exons encoding the 551-amino-acid PKR protein. We report herein the sequence of the human PKR protein kinase deduced from genomic clones.
Keywords: Translational control; Protein synthesis initiation factor elF-2c~; RNA-binding protein; Antiviral protein; Tumor suppressor; Nucleotide sequencing
The protein kinase P K R from human cells, also known as the P1/eIF-2~ protein kinase, P1, p68, DAI and dsI, is an interferon-inducible enzyme that is activated posttranslationally by RNA-dependent autophosphorylation (Samuel, 1993; Clemens, 1996). The best characterized substrate phosphorylated by P K R is the alpha subunit of eukaryotic protein synthesis initiation factor eIF-2 (Samuel, 1979, 1993; Clemens, 1996). Phosphorylation of eIF-2~ on serine 51 by P K R leads to an inhibition of translation in animals, because the eIF-2aP:GDP complex generated during the initiation stage of translation cannot beefficiently recycled by the guanine nucleotide exchange factor eIF-2B (Hershey, 1991). Thus, the P K R kinase plays an important role in the regulation of protein synthesis in interferon-treated and virus-infected cells (Samuel, 1991 ), and possibly also may play an important role in the control of cell growth * Corresponding author at the Department of Molecular, Cellular and Developmental Biology. Tel.: (+1-805) 893-3097; Fax: (+1-805) 893-4724; e-mail: [email protected] 1The sequences reported in this paper have been deposited in the GenBank data base (accession No. U50632 to U50648). Abbreviations: aa, amino acid(s); IFN, interferon; kb, kilobase(s); nt, nucleotide(s);PKR, RNA-dependentprotein kinase;pkr, geneencoding PKR; UTR, untranslated region(s). 0378-1119/96/$15.00© 1996 ElsevierScienceB.V. All rights reserved PH S0378-1119(96)00314-9
and differentiation in uninfected cells (Lengyel, 1993). Molecular cDNA clones of P K R from human Daudi cells (Meurs et al., 1990) and human amnion U cells (Thomis et al., 1992) have been reported. As an extension of our studies of the regulation and function of human PKR, we have isolated lambda and P1 phage genomic clones of P K R and determined the structure of the human pkr gene (Kuhen et al., 1996). The human pkr gene contains 17 exons and spans about 50-kb pairs. We now report the entire exon sequence of the human pkr gene determined from genomic clones. Human pkr genomic clones were isolated by screening two libraries, a human placenta genomic library in the 2 phage vector EMBL3 (Clontech) and a human foreskin fibroblast genomic library in the P1 phage vector pAD10SacBII (Shepherd et al., 1994), using fragments of the human P K R cDNA (Thomis et al., 1992) as probes. Isolated genomic clones were characterized by restriction mapping and Southern blot analyses, and exon containing fragments were subcloned into the pBluescript plasmid (Stratagene) for detailed restriction mapping and DNA sequencing by the dideoxy procedure using reverse or - 2 0 universal primers and custom-synthesized primers (Sambrook et al., 1989). The genomic exon nucleotide sequence of human pkr and the deduced amino acid
K t Kuhen et al./Gene 178 (1996) 191-193
192
V
G C G G C G G C GC,C G G C G C A G S " F T G C T C A T A C q ' V I ~ T G A C ~ T P G C ACCACAGGCACGACAAGCATAGAAAC
ATC CTAAACAATCTTCATCGAGGCATC
TCATAGACC~AG~TTCGCT<~GTA~ACTCGT~TGq~TGAACCAGCGG~'I~A C ~GGAC~CAAA'I~AGT~C
C TTC CTGGAT~GTA~T~TAA%~G
GGTCAC AGTGGCATTCAGCTC
HU
A ~ ~7-G T G A T C ~ A G C A G G ~ A T G G A G G A A C q ~ A A T A C M A G D L S A G F F M E
-271
i'l'l'l'tTfAAGCCq'IR2 iT~'i'I'IC~ I"~'~'iA C C A G T T T
- 181
ACC TCAAAAC T~AGCAG'I'~C T~
N T
S .
G P P . . . .
F T F . . .
Q
V
I L
I
L .
N D
K N
AATAGAATTGCCCAGAAGAAAAGACTAACTGTAAATTATGAACAG N R I A Q K K R L T V N Y E
HU
AAT~AGAAGGA~ATCCATGGGGAATTACATAGGCCTTATC N S S E G L S M G N Y I
Hu b~
C
A E
K R
S P
V S
A A A . . . .
H E
G L
C AG G AA~A~C~T~GC E .
A
P
L .
g Q
A
V
E D
.
G R
F
H [
I
K
L E
A
Y .
AAGGA~C~
G
L
I
g C
K
C " K
C G C T A A A C q~fGC A T A T C T ~ L Q
R K
E .
M I
G
K N
Q
I L
L
S K
E .
E
G A
E K
G R S . . . .
~C~CAG T T A G T C C q r ~ T A T T A ~ A C K .
K T
AGATAq'FATCAGAAGAAAC Q K
E F P . . . .
A V
E M
V D
Y
S C
P H
S G
L .
I T
L .
-1
G
T S
K
~C~C
L .
.
T
G
T
S V
G T S
Q
T
E T S . . . .
V .
K .
S
L
.
D .
S
K
S P
.
HU
TCT~CTACTACGTG~AGTCCCAAAGCAACTCq~I~FAGTGACCAGcACAC~GCq~PCTGAAq~CATCATC~AAGGTGACT~AGCA S F A T T C E S Q S N S L V T S T L A S E S S S E G
MS
K
T
.
G
.
-
-
-
-
HU
D
T
S
E
I
N
S
N
S
MS HU MS
G N
HU
GAATTAAT~GCq~AGGTGGAT~GCCAAGT~AAAGCAA~CACA~AATTGACGGAAAGACTTACGTTATTAAACGTG~TAT E L I G S G G F G Q V F K A K H R I D G K T Y V
HU
N
K
A
E
R
E
HU MS
D Y D P . . . . .
E
T H
S
D .
90 30
60 59
C T C A G TGVAAATC T G A C T A C C T Gq%U C ~ ~ G ~ Y
.
L A A . . .
G E
Q .
.
Q .
K .
D .
HU MS
.
K
.
GCAGC C A~TTAGCTGT~AGATACq~TAAT N .
G N
R .
T
K Q
A
R .
- 91
ATAC C GTC AG AAGC AGGGAG TAG TACTTAAATATCAAG AAC T~ Y R Q K Q G V V L K Y Q E L
N
HU MS
E -
D .
C A~CTG AC TC A G G ~ C ~
C ~ C CA ~ A T A C ~ G ~ G ~
L
P S
AAGG~GCA~,CC
H .
~
E
bls
- 435
C CTGGCTA
~ ' ~ G ~ " P ~ ' I ~ I ~ A G / ~ " P ~ / ~ C A'I~ CACAC'P'~ CG'I~A~PA'P~'I~CG"I~A'I" I T ~ G A C - ~ 2
Hu
CACACTTGGTAGA
GAGGTC CATCC C AATAAAAATCAGGAGAC
G P
270 90 85
360 120
150 145 540 180 165 630 210 191
D
F S
A
S
S
M
T
N
N
Q
R
K
A
K
G A ~4~ A TVC ~ G C"A C CQ - - C A G A - - GTA~C C T T C CPT - AGC A T G - - AF~ C AASA~AGG T A T AEC T G TN G G AVC - - FG - ' T ~ G C - - T AG AG A ~- G- - -GA ~ k AET A AA
K TT~A~G
S
G
810219
R V
M S
D
F
K E
E D
!
270 245
I
K
R
V
K
Y
S K
L V
A S
P
R F D . . . .
.
E
.
.
.
.
.
. . . .
S
h
N
S
D
.
M V
.
.
E R
.
T N
.
.
.
Y
.
L
M
N
G
T
.
V L
D
K A
L
R
R
F
N
.
.
.
.
.
.
.
900 300
. 0
Y
N
G
C
W
D
G
F
330
D .
Y D P . . .
E
N
S M
K S
N D
S T
S .
R
S Y
K
T .
K R
360 322
HU
T G C C ITi" 1 C A ~ C C A A A T G G A A T T C T G ~ A T A A A G G G A C C'I~GAAC AATGGA~TGA~GAAGAGGC C L F I Q M E F C D K G T L E Q W I E K R R
G
GAGA/kAC T A G A C A ~ G I'~'I~I~ E K L D K V L
HU
A
MS
I
HU MS
T~AGTAGATACA2~CAAGTA~GATTGGAGACT~GGACT~TAAC F L V D T K Q V K I G D F G . . . . E R H I . . . . . . .
Hu
T
h
R
.
Q
C T~
F
E
Q
I
Y
Y M S P g . . . . . .
T~ATGTATGTGACACq%GC L
D E
S F
S L
Y I H S . . . . .
Q K
O H
K
F
T~A~CATCAakA~-
K
F
K b L . . . .
E
S
T
K E
S
V A
T
K G
N
I
ATCTCTGAAAAAq~3ATGGA~GCGAACAAGGAGTAAGGGA S L K N D G K R T R S K A E .S R T
G
g G K E V . . . . . .
L .
D
I H R . . . .
L I
Y F
A .
D L . . .
L G . . .
K
b .
P
K
.
P .
F
E K
D
T
R
D
P
L
N E
R
D
G
I
T S E I . . . . .
I
L
S
R K
D
T .
I
b
HU
AAAAGCCCAGAGAAAAATGAACGACACACATGTTAGAGCCCTTCTGAA~GTATCCTGC2~CTGATATGCAGTTTTCCTTAAATTATCT K S P E K N E R H T C * N
S
K
K
N
A~I~CTAGGGAATATCAATAGATATTTAC
L .
F
T A
420 382 1350 450 413
E
D
K
K
1530 510
N
AA
162@474
V E
W .
K R
540 504
480 443
1710 551 515
*
Cq~AT~AATG~I~CCTTTAA
1170 390
A
~ y ~q ~ A C A G A C C T A C G G G A T G G C A T C A T C ~ A G A T A T A T ~ G A T A ~
MS
I
S G
I .
E
S
A
L
K
G A ~4~" k A A A "C T CT-q-~T A C A GF- - TATAAAC TEC - -SA A A G~ - - CK- - A AGI~- CA qT ~C G A C C T -G- C -E- C A TS C - -AAA.A~ A T A CKT ATAGG G A DC C T ~FA C T G.N q ~ G.~ G.- -.G . Q
T
I L
V
H%I MS
L
D
Q
G
Ms
L
C
T
.
b
T
V
K
V
S .
Hu
K
H
T
S .
H
240
H
L
K
K
F
V
E .
A
L
G
I
D S L . . . .
L
S
S
N
E
A
K Q
S
F
V
D
K
V
D
L
V
P D
.
L
MS
N
L
D
S
~-I-~T~'~A C T A i'i'~TIA C T A A T C T ~ C q ~ G C A G A A
l S 00
A ~ A G A A A G G ~ T t - ~ C ~ ~ i - ~ % C , C T T C A A A A A C A T T C T T A C A q ~ F T T A C T ~ q C C T ~ G C T C A T C T C T T T A T T C c~-~"~-~-~-~T~-~-~-~uA A A
1890
GACAGAGTCTCGCTCT~q~C
1980
C CAGGCq~GAGTGCAATGACACAGTCT~GCTC
CTGCCTCAGCCTCCTGAGTAGCTGGATTACAGGCATGTGCCACCCACCCAACTAA GT'I~GCCAGGCTGGTCq~
ACTGCAACq~TGC
CTCT~GGTTCAAGTGATTCTC
I-UI-I~GTG I'I'I-x-?AATA~GAC A G G G T ~ C A C
AAACTC C TGACCTCAAGTAATCCACCTGCCTCGGCCTCCCAAAGTGCTGGGAq~TACAGGGATGAGCCAC
GCCCAGC CTCATCTCTTTGTTCTAAAGATGGAAAAACCAC
CC C C A A A T T T T C
TTAq-TAAA~I-~CTACC G C l-lq-rA G G C C A ~ T A A G A T C G T T C T C T G C ~ATT, A ~ G T G A T G T A C A A C C a c t
tcggaaaacaat
CAT
2070
CGC
2160
t"l'c~ u A T A C T A T T A A T G A A T C A A T C A A T T C A T A T C T A T
2250
CTCACATAGCTTACAAGCCAGCTGGAGAAATATGGTAC
2340
t t @ g c a t tat ct a g t a a a g t t g a a t c c a t g t a t a c c c a c a t a
2430
Fig. 1, Nucleotide sequence of the human pkr exons determined from genomic clones and the deduced aa sequence. The A of the A U G translation initiation codon in exon 3 is denoted as n t + 1; the 5'-UTR of the mature m R N A begins with nt denoted as - 4 3 5 , upstream from the A at n t + 1. The open triangle symbols above the nt sequence indicate the positions of the exon junctions in the human gene, as determined by comparison of genomic sequence (GenBank accession Nos. U50632 to U50648) to the c D N A sequence (accession No. M85294). The exon junctions for the mouse gene (Tanaka and Samuel, 1995) are indicated by the filled circle symbols. The putative polyadenylation signal and C(a) cleavage site preceding the poly(A) tail of the c D N A (Thomis et al., 1992) are underlined.
K.L. Kuhenet al./Gene178 (1996) 191-193 sequence are shown in Fig. 1. The h u m a n pkr gene contains 17 exons. Exons 1 (18 nt) and 2 (401 nt) and part of exon 3 (16 nt) constitute the 5-UTR of the m a j o r P K R transcript. E x o n 3 includes the A U G translation initiation site as established by ribosome protection analysis with the c D N A - d e r i v e d transcripts (Thomis et al., 1992). The translation termination c o d o n for synthesis of the deduced 551-aa P K R protein is located in exon 17, the largest exon (840 nt) that also includes the complete 720 nt 3'-UTR that terminates at nt 2373, the site of poly(A) addition. C o m p a r i s o n of the exon organization of the h u m a n pkr gene to that previously established for the mouse pkr gene ( T a n a k a and Samuel, 1995) shows that 13 of the 15 amino-acid-coding junctions are exactly conserved (Fig. 1). W h e n the nt sequence of the h u m a n pkr exons determined from genomic clones (Fig. 1) was compared to the reported c D N A sequences (Meurs et al., 1990; Thomis et al., 1992), the following differences were found: in exon 2 of the 5'-UTR, nt - 17 is a G as reported by T h o m i s et al. (1992) rather than a C as found by Meurs et al. (1990); in exon 3 of the 5'-UTR, nt - 1 6 is a G as reported by T h o m i s et al. (1992) rather than a C as found by Meurs et al. (1990); in exon 17 of the 3'UTR, n t + 1878 is a T in agreement with Meurs et al. (1990) rather than a C as found by T h o m i s et al. (1992); and also in exon 17 of the 3'-UTR, a deletion of two A residues following nt 2348 was found which gives the subsequent sequence of A 12 instead of A 14, in agreement with Meurs et al. (1990) rather than T h o m i s et al. (1992). Finally, the exon 1 sequence - 4 3 5 to - 4 1 8 and the 5' portion of the exon 2 sequence corresponding to nt - 4 1 7 to - 1 8 7 represents new 5'-UTR sequence that was not included in the incomplete c D N A clone sequence previously described (Meurs et al., 1990; T h o m i s et al., 1992). This new 5'-UTR sequence was obtained by the Y - R A C E procedure using c D N A prepared from h u m a n placenta R N A , and contained one difference from the genomic D N A sequence: nt - 3 3 1 was a C in the genomic sequence and a G in the c D N A sequence. The complete c D N A of h u m a n P K R is, therefore, 2808 bp in length excluding the 3' poly(A) ÷ tail.
Acknowledgement This w o r k was supported in part by Research G r a n t AI-20611 from the N a t i o n a l Institutes of Health.
193
References Clemens, M.J. (1996) Protein kinases that phosphorylate eIF2 and eIF-2B, and their role in eukaryotic cell translational control. In." Translational Control. Cold Spring Harbor Press, Cold Spring Harbor, NY, pp. 139-172. Hershey, J.W.B. (1991) Translational control in mammalian cells. Annu. Rev. Biochem. 60, 715-755. Kuhen, K.L., Shen, X., Carlisle, E.R., Richardson, A.L., Weier, H-U.G., Tanaka, H. and Samuel, C.E. (1996) Structural organization of the human gene encoding an interferon-inducible RNA-dependent protein kinase (PKR) and differences from its mouse homolog. Genomics 36, 197-201. Lengyel, P. (1993) Tumor suppressor genes. News about the interferon connection. Proc. Natl. Acad. Sci. USA 90, 5893 5895. 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. Sambrook, J., Fritsch, E.F. and Maniatis, G. (1989) Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Samuel, C.E. (1979) Phosphorylation of protein synthesis initiation factor eIF-2 in interferon-treated human cells by a ribosomeassociated protein kinase possessing site-specificity similar to hemin-regulated rabbit reticulocyte kinase. Proc. Natl. Acad. Sci. USA 76, 600-604. Samuel, C.E. ( 1991) Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology 183, 1-11. Samuel, C.E. (1993) The eIF-2a protein kinases, regulators of translation in eukaryotes from yeasts to humans. J. Biol. Chem. 268, 7603-7606. Shepherd, N.S., Pfrongner, B.D., Coulby, J.N., Ackerman, S.L., Vaidyanthan, G., Sauer, R.H., Balkenhol, T.C. and Sternberg, N. (1994) Preparation and screening of an arrayed human genomic library generated with the PI cloning system. Proc. Natl. Acad. Sci. USA 91, 2629-2633. Tanaka, H. and Samuel, C.E. (1995) Sequence of the murine interferon-inducible RNA-dependent protein kinase (PKR) deduced from genomic clones. Gene 153, 283-284. Thomis, D.C., Doohan, J.P. and Samuel, C.E. (1992) Mechanism of interferon action, eDNA structure, expression and regulation of the interferon-induced, RNA-dependent P1/eIF-2c~ protein kinase from human cells. Virology 188, 33-46.