MOLECULAR AND CELLULAR BIOLOGY, Apr. 1992, p. 1680-1686 0270-7306/92/041680-07$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 12, No. 4

Fine-Structure Map of the Human Ribosomal Protein Gene RPS14t JEAN-JACQUES DIAZt AND DONALD J. ROUFA* Center for Basic Cancer Research, Division of Biology, Kansas State University, Manhattan, Kansas 66506 Received 29 July 1991/Accepted 16 January 1992

We have used polymerase chain reaction-mediated chemical mutagenesis (J.-J. Diaz, D. D. Rhoads, and D. J. Roufa, BioTechniques 11:204-211, 1991) to analyze the genetic fine structure of a human ribosomal protein gene, RPS14. Eighty-three DNA clones containing 158 random single-base substitution mutations were isolated. Mutant RPS14 alleles were tested for biological activity by transfection into cultured Chinese hamster cells. The resulting data permitted us to construct a map of the S14-coding sequence that is comparable to available fine-structure genetic maps of many prokaryotic and lower eukaryotic gene loci. As predicted from the multiplicity of protein-protein and protein-RNA interactions required for ribosomal protein transport and assembly into functional ribosomal subunits, the distribution of null mutations indicated that S14 is composed of multiple, functionally distinct polypeptide domains. Two of the protein's internal domains, designated domains B and D, were essential for S14 biological activity. In contrast, mutations which altered or deleted S14's amino-terminal 20 amino acid residues (domain A) had no observable effect on the protein's assembly and function in mammalian ribosomes. Interestingly, S14 structural domains deduced by in vitro mutagenesis correlate well with the RPS14 gene's exon boundaries.

Genes encoding the 40S ribosomal subunit protein S14 (RPS14) have been isolated from human (21) and Chinese hamster (24) DNAs. Both mammalian S14 loci are composed of five exons whose protein-coding sequences are >91% identical. Indeed, the two genes encode exactly the same 151-amino-acid polypeptide (21, 22). In contrast, although human and hamster RPS14 introns are located at identical sites within the S14-coding sequence, they differ dramatically with respect to their lengths and base sequences (24). Nonetheless, human RPS14 is fully functional when transfected into cultured CHO cells (23). Whereas some information regarding transcriptional regulation of mammalian RPS14 is available (18, 19, 23), virtually nothing is known about the functional significance of amino acid sequence motifs within the S14 protein itself. Ribosomal proteins (r-proteins) have been described as multifunctional polypeptides, as they must (i) be efficiently transported to the cell's nucleolus, (ii) be accurately assembled into nascent 40S and 60S ribosomal subunits, and (iii) support the ribosome's participation in protein biosynthesis. Because each of these activities is believed to depend upon multiple protein-protein and protein-RNA interactions, a priori it was reasonable to expect that r-protein S14's primary structure might be composed of several distinct func-

technologies provide alternative molecular approaches to analyze protein-coding DNA sequences cloned from organisms not amenable to fine-structure analysis by recombinational genetics. In a previous report, we described tissue culture experiments designed to isolate nonfunctional (i.e., null) mutations in the human RPS14 locus (6). Although the tissue culture selection scheme used was successful, unanticipated somatic recombination and gene conversion events precluded the isolation of a comprehensive set of RPS14 missense mutations. In addition, selection in tissue culture did not permit us to recognize functional missense mutations in the S14-coding sequence. Rather, it yielded exclusively RPS14 null alleles that harbor missense and nonsense mutations. To overcome the experimental limitations imposed by genetic selection in tissue culture (6) and to mark the human RPS14 protein-coding sequence with a diverse array of missense mutations, we undertook the in vitro genetic analysis described in this report.

MATERIALS AND METHODS Enzymes, reagents, and cell lines. Commercial sources of tissue culture reagents as well as restriction endonucleases and other enzymes used in these experiments have been described elsewhere (2, 6, 7, 21, 23, 24). All enzymes were used according to instructions provided by the suppliers. Radioactive [a-32P]dCTP (800 Ci/mmol), [a-32P]UTP (400 Ci/mmol), and [35SJmethionine (1,153 Ci/mmol) were purchased from New England Nuclear Corp. Synthetic oligonucleotides were produced by The Midland Certified Reagent Co., Midland, Tex. DNA constructions were carried out in the plasmid vector pUC13 (29), using Escherichia coli TBi (Bethesda Research Laboratories, Inc.) as the bacterial host. For cell-free transcription, S14-coding sequences were introduced into the plasmid vector pGEM-1 and subcloned in E. coli C600 VCS, both of which were obtained from Stratagene, Inc. Emr-2-2 (13) is an emetine-resistant (emtb) mutant clone of CHO cells that harbors two recessive poirt

tional domains. Mutational genetics offers a powerful experimental approach for investigating functionally significant aspects of a protein's structure. However, with conventional techniques, the approach is most suitable for prokaryotic and lower eukaryotic organisms in which high-resolution genetic methods have been established. Fortunately, recombinant DNA *

Corresponding author. Electronic mail address:

MATr.KSU.KSU.EDU.

DROUFA@

t Contribution no. 92-56-J from the Kansas Agricultural Experiment Station. t Present address: Laboratoire de Biologie Moleculaire et Cellulaire, Faculte de Medecine Alexis Carrel, Lyon, France. 1680

FINE-STRUCTURE MAP OF RPS14

VOL. 12, 1992 d

c _0.

,

TABLE 1. Oligonucleotide primers used for PCR amplification and DNA sequence analysis of r-protein S14-coding sequences

,lu

b

B. 225bp

Primer

ai

P1

r i

L

P4

P3

Pi r

0E

I

-~

'_

_1 S

I I

Hpa I

slil

1681

-SOP 1

Xho

P2 P3 P4

Source

DNA sequence

5'-AGTCTGGAGACGACOGT-3' Exon I, sense strand 5'-GGTCATCAGCCTCGGACCTCA-3' Exon II, sense strand Exon III, antisense strand 5'-CAGCTCCTTGCACCTCTGGGC-3' pUC13, antisense strand 5'-CACAGGAAACAGCTATGACC-3'

IHd III

Tradion

FIG. 1. In vitro mutagenesis strategy for the human r-protein S14-coding sequence. A minimal expression gene encoding human RPS14 was modified from the plasmid clone pCS14-83 (19) and used as a duplex target DNA for PCR-mediated nitrous acid mutagenesis (7). S14 exons are indicated as stippled boxes labeled El (exon I) and EII-EV (fused exons II to V), noncoding flanking and intervening DNAs are represented by thin lines, and plasmid vector sequences are indicated by hatched boxes. As indicated by the arrow, the S14 protein-coding sequence initiates 3 bp downstream from the beginning of exon 11 (21). Four oligonucleotide primers, P1 to P4 (Table 1), were used to PCR amplify the mutagenized DNA as three overlapping fragments labeled A, B, and C. Restriction fragments a to d, which together comprise the entire S14-coding sequence, were cleaved from amplified DNAs and subcloned into a modified human S14 expression vector, pCS14-140. Reconstituted S14 expression genes therefore contained mutations in defined segments of the r-protein-coding sequence.

mutations affecting the 3' end of the RPS14 protein-coding sequence: Arg-149--*Cys and Arg-150---His (22). PCR-mediated chemical mutagenesis. A plasmid DNA clone encoding human r-protein S14 was mutagenized with nitrous acid as described previously (7). To maximize the yield of base substitution mutations evenly distributed throughout the S14-coding sequence, we constructed a convenient mutagenesis target DNA and minimal S14 expression vector (pCS14-140) from the human RPS14 DNA clone, pCS14-83 (19). The modified target DNA contained a 1,797-bp deletion (between SmaI and HpaI restriction sites) within RPS14 intron 1 and a 417-bp deletion (between XhoII and HindIII sites) in the downstream chromosomal DNA sequence (21). These deletions permitted us to use an exon I oligonucleotide (P1; Fig. 1) and the pUC13 antisense DNA primer (P4; Fig. 1) to amplify the 5' and 3' ends of the S14-coding sequence by means of the polymerase chain reaction (PCR). The target DNA used, therefore, contained 32 bp of upstream flanking sequence, human RPS14 exon I (54 bp), a short region of intron 1 (87 bp), fused exons II to V (501 bp), and 99 bp of downstream flanking DNA (Fig. 1). Construction of pCS14-140 involved destroying the NdeI and SspI cleavage sites within the vector sequence (originally derived from pUC13) by standard gap-filling and linker insertion methods. This treatment rendered the NdeI and SspI sites within pCS14-140 coding sequences unique (Fig. 1) and facilitated assembly of mutant S14 expression clones for biological testing. The S14 target DNA was mutagenized with 1 M nitrous acid at 22°C for 10 to 120 min. After treatment, the DNAs were PCR amplified as three overlapping nucleic acid fragments (A to C; Fig. 1), using four synthetic DNA primers (P1 to P4; Fig. 1 and Table 1), as described previously (7). S14-coding regions (labeled a to d in Fig. 1) were excised from the amplified DNAs by cleavage at flanking restriction sites. Region a was an 86-bp HpaI-Bsu36I DNA fragment derived from PCR product A, region b (157 bp) was a

Bsu36I-NdeI fragment excised from PCR product B, and regions c (127 bp) and d (133 bp) were NdeI-SfiI and SfiI-SspI DNA fragments purified from PCR product C. Mutagenized DNA segments (a to d) were subcloned into pCS14-140 to generate four expression libraries, each of which harbored nitrous acid-induced point mutations within a different region of the human S14-coding sequence. Structural and functional analysis of mutant DNAs. Mutagenized human S14 DNA clones were screened for singlebase alterations by DNA sequence analysis (4, 7, 21, 22, 25) using primer oligonucleotides P1 to P4 (Table 1) and for their abilities to functionally complement a recessive emetine resistance RPS14 mutation carried in the Emr-2-2 line of CHO cells (6, 23). Accordingly, we transfected individual mutant S14 DNAs cloned in pCS14-140 into Emr-2-2 cells together with the selectable plasmid, pSV2Neo (26), and scored G418-resistant transformed colonies for their sensitivities to emetine. Under the test conditions used (see below), biologically active S14 DNAs rendered 80 to 85% of the transformants sensitive to emetine, whereas inactive DNAs (or DNAs that encode new emtb alleles) did not affect Emr-2-2 cells' drug resistance phenotype. Transfection assays were carried out by using the Polybrene-dimethyl sulfoxide procedure (15, 23, 24). Emr-2-2 cells (7 x 10 ) were transfected with a mixture of a cloned mutant S14 DNA (7 ,ug) and pSV2Neo (0.5 ,ug). Under these conditions, the molar ratio of pCS14-140 clones (4,220 bp) to pSV2Neo (5,825 bp) was approximately 20:1. Twenty-four hours later, cells were collected by trypsinization and transferred into six replicate 60-mm culture dishes. Three dishes were fed with medium containing G418 (1 mg/ml), and three were fed with G418-medium supplemented with emetineHCI (10-6 M). After 14 days, the culture dishes were fixed in 50% (vol/vol) methanol and stained with methylene blue. Biologically active mutant S14 DNAs yielded five- to sixfold more colonies in G418-medium than in medium containing G418 plus emetine. In contrast, inactive S14 DNAs yielded approximately the same number of colonies (±10%) in the two selective media (Fig. 2). Characterization of mutant S14 transcripts and polypeptides. To verify that transfected human S14 DNAs were expressed efficiently in CHO Emr-2-2 cells, mRNAs and r-proteins were purified from transformed cells after growth in G418 medium. Cytoplasmic S14 mRNAs (14) were assayed by S1 nuclease protection, using an antisense human RPS14 RNA probe labeled with [32P]UTP to a specific activity of =5 x 107 cpm/,ug (6, 23). Ribosomal proteins purified from transfected cells (2) were analyzed by twodimensional polyacrylamide gel electrophoresis (14, 23). First-dimension tube gels contained 4% (wt/vol) polyacrylamide, 0.2 M Tris-borate (pH 8.6), 8 M urea, and 10 mM EDTA and were electrophoresed for 2,000 V-h toward the cathode. Second-dimension slab gels included 12.5% (wt/ vol) polyacrylamide, 0.1 M BisTris-acetate, 6 M urea, and 1% (wt/vol) sodium dodecyl sulfate (pH 6.75) and were

1682 A

DIAZ AND ROUFA

s

o

n3,

MOL. CELL. BIOL. B

gs

MM fl2 C- T G-. A T-.C A-. G C-'G G-.T T-A A-.T A- C C-.A

.1 *~~~~~~~~1

Exon III

Exon 11

'ACC 1

I

25

50

I

75

Exon IV

T lI 100

Exon V

125

150

Nudeoddes nss

D

nv320

FIG. 2. Transfection assay for human RPS14 biological activity CHO Emr-2-2 cells were cotransfected with pSV2Neo and either a DNA encoding a functional RPS14 mutant allele (pMS14-19) (A and C) or an allele containing a missense mutation which inactivates S14 (pMS14-31) (B and D). Twenty-four hours later, the cells were replated into six replicate cultures. Three of the cultures were treated with G418 (1 mg/ml) (A and B), and three were treated with G418 plus 10' M emetine (C and D). The numbers of colonies which developed on each dish after 14 days in culture (n) are indicated.

electrophoresed toward the anode for 1,050 V-h. Following electrophoresis, gels were fixed in methanol-acetic acid (50%:7.5%) and stained with Coomassie brilliant blue.

FIG. 3. Distribution of base substitution mutations recovered in the human RPS14 protein-coding sequence. The horizontal axis represents the S14-coding sequence (453 nucleotides). Transition mutations are mapped above the axis with lines whose lengths indicate specific base substitutions (labeled at the left). Transversion mutations are mapped below the axis in a similar manner. S14 exon boundaries are indicated by brackets.

teins in cultured CHO cells. For example, the pattern of S14 proteins elaborated by Emr-2-2 cells transfected with mutant DNA pMS14-19 is illustrated in Fig. 4B. It clearly indicates two electrophoretically distinguishable S14 polypeptides: the endogenous Emr-2-2 S14 variant (S14a) and a novel r-protein (Sl4b) whose electrophoretic migration was consistent with the Arg-55---His mutation encoded by pMS14-19 DNA (Table 2). In contrast, transfection of several biologically inactive mutant DNAs did not result in variant S14 proteins that could be detected by electrophoresis, despite the fact that all of the mutated DNAs were transcribed efficiently in CHO cells (see above). pMS14-9 is an example in which the S14 Cys-31 residue was replaced by Trp (Table

A. W.T.

B.pMS14-19

C.pMS14-9

D.

RESULTS In vitro mutagenesis of the human S14-coding sequence. The PCR-mediated chemical mutagenesis strategy described in Materials and Methods was designed to saturate the entire human S14-coding sequence with single-base substitution mutations. In this way, we isolated 83 DNA clones carrying 158 point mutations. As illustrated in Fig. 3, 141 of the mutations (89%) were transitions, and 17 (11%) were transversions. Mutant S14 DNAs were tested for biological activity by transfection into the emetine-resistant CHO cell line Emr-2-2. Forty-four of the mutant alleles (52%) encoded biologically active S14 proteins, as they restored Emr-2-2 cells to an emetine-sensitive phenotype. The remaining 39 DNAs (46%) were judged to encode RPS14 null alleles, because they did not complement drug resistance in transformed cells. Efficient transcription of all RPS14 null alleles was verified by S1 nuclease analysis of transfected cell messages (data not shown). As illustrated in Fig. 4, the Emr-2-2 S14 protein (S14a) can be resolved from wild-type S14 (S14c) by two-dimensional polyacrylamide gel electrophoresis (Fig. 4A) (13). On the basis of their altered protein-coding sequences, several mutant S14 DNAs were predicted to encode new electrophoretic variants of the S14 polypeptide. This provided a biochemical means to determine whether at least some of the mutant DNAs actually were expressed as functional r-pro-

(=
G C-134 T G-63 A G-92 A T-87 - C T-114 C T-158 C G-168 A T-185 C C-93- G T-160-- C A-182 T G-85- A T-122 - C G-164 A T-109 C 0 A-116 G C-191 - T A-182 G C-127 T G-85 -* A G-164 A T-123 C G-176 - T T-96- C A-152 -- G T-87-* C G-146 -- A G-71*A T-125 - C T-129 C T-158 C C-211 T G-197 A T-201 C C-ill T A-26 G G-27 A G-17 A G-39 A T-2 - C G-40 - A G-61 A T-98 C T-123 C A-24 C T--22> C T- -16 - C G-39 A T-21 C A-28> G C-8- A G-115 -* A C--27-* T C-58 - T G-259 A A-268 G C-320 T

N-26 -1-33 -* N-26 M-60

D V D T

C-31 S-70 K-50 T-45 V-21 C-31 G-29 N-38 I-53 -* V-56 V-62 C-31 C-54 K-61 -

S L E I

Functionb

Mutation

j

loNe

43

+

44

46 47 48 48a 50

T

ncd

K-10 -- E P-3 - H D-39 N

51 52

53 54 55 56 57

+ +

58

+ +

59 +

60

61

+ +

62

+ + + +

63

64 65 66

+

+

66a 67 68

+

+ + + +

69 72 73

nc

Q-20 - Trme E-87 - K I-90 - V T-107 -* I

C-218 G-225 G-243 G-258 G-331 A-310 G-229

T A A A A G A

G-251-A

Y

A W R M G-29 S F-41 S R-55 H F-37 L D-39 G A-64 V K-61 - R H-43 Y G-29 - S R-55 - H F-41 L G-59 V H-32 0 E-51 G G-29 G-49 D G-24 E V-42 A H-43 I-53 T P-71 - S R-66 Q D-67 F-37 K-9 R K-9 G-6 E Q-13 M-1 - nc V-14 nc V-21 M 1-33 -- T F-41 E-8 nc nc Q-13

Nucleic acid

74

ilL

G-312 A A-248 T C-289 - T C-270 T A-300 G C-314 - T G-232 A C-234 T C-235 T A-287 G C-289 T C-255 T G-293 T C-221 T G-350 A C-365 T C-341- T G-352 A AC-382f G-350 -A C-354 - A G-412 A C-365 T G-388 -A C-354 T C-360 T G-367 A G-358 A G-366 A G-367 A T-371 C G-383 A G-388 A T-464 - C C-365 --T G-388 A G-366 A G-391 - A G-358 A G-443 A C-354 - T C-355 T A-360 T C-408 - T C-448 T C-363 - T G-379 A G-362 A G-367 A T-435 C G-453 A A-24 -) G G-232 A C-276 - A A-327 - T G-259 A C-292 A

Mutation FuncC Clone tion tinNucleic acid Protein

Protein

A-73 - V M-75 - I V-81 K-86 G-111 R R-104 G A-77 T R-84 -K R-104 Q-83 -* L H-94 1-90 T-100 T-105 --+I A-78 - T A-78 Q-79 Trm

75 76 77

78 79 80 81

+

+

82 83 84

+ + +

86 88

nc nc

89

C-85 R-98 L A-74 V R-117 K S-122-- L S-114*F A-118 -1 FS' R-117-i K A-118 D-138 N S-122 - L E-130 K A-118 A-120 G-123 S A-120 T

90 +

92 93

G-259

A E-87-* K T L-119 F -1 FS AC-382 G-431 A G-432 A G-144 G-354 - T A-118 T-371 A M-124 K C-359 T A-120 V C-458 T nc C-468 T nc C-360 T A-120 C-447 T R-149 G-381 A G-127 G-429 - A K-143 G-430 -A G-144 R G-295 - T A-99 -+ S G-375 - A K-125 T-377 A I-126 -> N G-379 A G-127 R C-355

AC-407 AC-408 J T-467 - A C-398 --T C-414 - T C-355 T C-360

Function

to

+

+ + +

+

+ +

+

-2FS

nc T-133 I D-138 L-119 --F T A-120

+

S-122 G-123 - S M-124 R-128

T Q E-130>K nc S-122

L

E-130>K S-122 D-131

N

G-148

D

A-120>T A-118 L-119 A-120 P-136 R-150 R-121

G-127 R-121 G-123 G-145 L-151 E-8 A-78 -* A-92

+

F

C +

R H S +

T

G-109 E-87 -> K R-98

+ + +

A

a Cloned mutant genes (pMS14-1 to pMS14-93). b Mutant alleles' biological activities were assessed by transfection into emetine-resistant CHO EMr-2-2 cells. +, expression of the mutant gene rendered Emr-2-2 cells sensitive to emetine; -, it did not. Amino acids are specified by their standard single-letter abbreviations. Base substitutions which do not alter the affected codon's amino acid specification (i.e., silent mutations) are indicated without an arrow (e.g., V-21). d nc, the mutation affected a noncoding region of the DNA. e Trm, a premature polypeptide termination codon (TAG, TGA, or TAA). f A, single-base deletion. g FS, a frameshift in the protein-coding sequence.

1683

1684

DIAZ AND ROUFA

2). It was expected to encode an r-protein with electrophoretic properties similar to those of wild-type S14 (S14c; Fig. 4). However, ribosomes purified from cells transfected with pMS14-9 contained only the Emr-2-2 S14 polypeptide (Fig. 4C). Thus, substitution of Trp for Cys-31 appears to preclude the mutant S14 protein's assembly into functional 40S ribosomal subunits. Table 2 catalogs the structural and functional information determined for all 83 mutant S14 DNAs. Forty-one of the S14 alleles (49%) contained single-base substitution mutations, and 42 (51%) possessed between two and seven mutations each. Three DNAs in the latter group also carried one- or two-base deletions (pMS14-57, -77, and -89) which shifted the translational reading frame of downstream protein-coding sequences, and two alleles (pMS14-39 and -52) harbored premature polypeptide termination (nonsense) mutations. As expected, the five frameshift and nonsense alleles all were biologically inactive in the CHO cell transfection assay. Mutations summarized in Table 2 offer several insights into functional aspects of human r-protein S14's primary sequence. For example, the Cys-31 residue in S14 was mutated to Ser, Tyr, and Trp in mutant alleles pMS14-3, -7, and -9, respectively. In addition, naturally occurring insect, fungal, and plant S14 genes specify Ala at residue 31 (6). Although pMS14-3, -7, and -9 were biologically inactive in the rodent cell transfection assay (Table 2), the Drosophila melanogaster RPS14 gene containing Ala-31 previously was shown to function normally in CHO cells (15). Thus, our genetic data indicate that Cys and Ala both satisfy S14's functional requirements at position 31, whereas Ser, Tyr, and Trp do not. Finally, the distribution of amino acid substitutions within mutagenized RPS14 alleles suggested that the S14 polypeptide is organized into several functional domains (labeled A to E in Fig. 5). Four independent point mutations which mapped to the first 20 S14 amino acid residues (domain A) did not affect S14 function, demonstrating that the protein's amino terminus can be varied substantially without loss of biological activity. In contrast, two protein domains, B and D, appeared to be critical for normal r-protein activity, since most single amino acid replacements affecting these regions resulted in RPS14 null alleles. Domain B resides between Gly-24 and Gly-49; domain D resides between Thr-107 and Asp-131. Both of the missense mutations in domain B which are compatible with S14 function carry conservative amino acid replacements. On the other hand, four of the five mutations in domain D that do not appear to affect S14 activity are associated with nonconservative amino acid substitutions. Mutations in domain C (Ile-53 to Thr-105) for the most part yielded biologically active S14 alleles. Two of the four inactive S14 alleles that harbor single point mutations within domain C encoded nonconservative amino acid substitutions (pMS14-4 and -53), and one carried a premature polypeptide termination mutation (pMS14-52). Domain E (Thr-133 to Leu-151) contains only three point mutations (pMS14-65, -84, and -90) but is the polypeptide region affected by amino acid replacements which render CHO cell 40S ribosomal subunits resistant to emetine (6, 22). It is noteworthy that boundaries of the five functional domains defined by the distribution of null point mutations correspond closely with the S14 gene's protein-coding exons (exons II to V; Fig. 5). Domains A and B are contained within exon II, domain C is in exon III, domain D is in exon IV, and domain E is in exon V. The amino terminus of human S14 (domain A) is not

q,.~

MOL. CELL. BIOL.

required for the protein's biological activity. pMS14-30 (Table 2) is a particularly interesting mutant allele. It includes three point mutations which dramatically affect the S14 aminoterminal sequence (Fig. 6). One of the mutations alters the translational initiator ATG to ACG and therefore should preclude the normal initiation of protein biosynthesis. A second mutation converts the Val-21 codon (GTG) to a methionine codeword (ATG) which, because it resides in a favorable nucleic acid context (11), was expected to restore the mutant mRNA's activity. The third mutation converts Val-14 (GTC) to Ile (ATC) upstream of the relocated initiator codon. If the pMS14-30 coding sequence were transcribed and translated into a truncated r-protein as proposed in Fig. 6, a variant S14 protein initiating at codon 21 might account for the DNA's biological activity (Table 2). To test this explanation for the pMS14-30 allele's biological activity in transfected CHO cells, we analyzed r-proteins purified from Emr-2-2 cells stably expressing pMS14-30 DNA by two-dimensional electrophoresis (Fig. 7). As shown in Fig. 7A, the pattern of Coomassie blue-stained r-proteins observed included an unusual polypeptide (AS14) significantly smaller and less basic than endogenous Emr-2-2 S14 (S14a). In vitro transcription and translation were used to determine whether AS14 is the protein encoded by pMS1430. pMS14-30 DNA was excised from pUC13 as a HpaIHindIII fragment (Fig. 1), subcloned between the SmaIHindIII sites of pGEM-1, and transcribed by using T7 RNA polymerase. The resulting mRNA was translated in a wheat germ cell extract containing [35S]methionine (3, 15) to produce a single [35S]polypeptide which comigrated in both electrophoretic dimensions with the AS14 protein (Fig. 7B). Therefore, we concluded that AS14 is the truncated polypeptide encoded by pMS14-30 and that it is assembled into biologically active, emetine-sensitive Chinese hamster cell ribosomes. Genedc Domains

-.

B

A

.............. ........ ...

.........

.

.. -.

,,,E-

D

C -

:i-...... ... 7..

iif

E

.. .. ....

E : :..dist :eplacementmut.ations a.c:::... . ' : . ' " : , ' .....r............ x throughout the human SB4 polypEptide.: ...... Th. .horizo

FIG 5 .A

.

l.. . . . . . . . . . . . . ] -'

.......

mut......di..ribute acd replacemet FIG.5. Mapof amino

sents the 151-amino-acid human S14 polypeptide sequence, with the amino terminus (N) at the left and the carboxyl terminus (C) at the right. Amino acid substitutions which abrogated the r-protein's biological activity are indicated above the horizontal axis; mutations which were compatible with S14 function are mapped below the axis. Only mutant alleles that carried single amino acid substitutions are illustrated. Each mutation is identified by its clone number (see Table 2). Amino acid substitutions were judged to be conservative (circles) or nonconservative (squares) according to the PAM-250 matrix (5). Polypeptide chain termination (nonsense) mutations are represented as triangles. S14 structural domains suggested by the distribution of missense mutations are shaded and labeled A to B. RPS14 protein-coding exons II to V are represented as rectangles below the map.

VOL. 12, 1992

A

FINE-STRUCTURE MAP OF RPS14

Promoter I

A.

ATGI ACG / ||GTG o ATG

I

11

-

1685

B.

V

,~~~~~~~~~l

[o Tran scription B

1

2

3

Translation 4

5

6

7

8

9

10

11

12

13

Met Ala Pro Arg Lys Gly Lys Glu Lys Lys Glu Glu Gln wild Type ATG GCA CCT CGA AAG GGG AAG GAA AAG AAG GAA GAA CAG

pIUL4 -30 AQG GCA CCT CGA AAG GGG AAG GAA AAG AAG GAA GAA CAG 14

15

16

17

18

19

20

21

22

23

24

25

26

Val Ile Ser Leu Gly Pro Gln Val Ala Glu Gly Glu Asn CTC GGA CCT CAG GTG GCT GAA GGA GAG AAT

Wild Type GTC ATC AGC I

I

pI14-30 ATC ATC AGC CTC GGA CCT CAG ATG GCT GAA OGA GAG AAT Hat Ala Glu Gly Glu Asn

...

...

FIG. 6. (A) Diagram showing that mutant allele pMS14-30 encodes a truncated r-protein which lacks the amino terminus of wild-type S14 protein. The mutant S14 sequence encoded by pMS14-30 is depicted. Rectangles represent the gene's exons (labeled in roman); lines represent the upstream flanking and intronic DNA sequences. The original initiator ATG codon, located 3 bp from the 5' end of exon II, was mutated to an ACG codeword. Simultaneously, the codon for Val-21 (GTG) was mutated to a replacement initiator codeword (ATG). As a result of both mutations, the pMS14-30 polypeptide was predicted to lack the first 20 amino acid residues contained in wild-type S14 (hatched box). (B) DNA and amino acid sequences involved in these mutations.

DISCUSSION PCR-mediated nitrous acid mutagenesis (7) was used to mutate the human r-protein S14-coding sequence. Eightythree RPS14 alleles were isolated as recombinant DNA clones. We determined nucleotide sequence alterations carried by each mutated DNA as well as its encoded r-protein's biological activity after transfection into cultured rodent cells. In all, we recognized 158 base substitution mutations. Slightly more than half of the mutant alleles (52%) were biologically active, as they restored emetine-resistant CHO cells to the wild-type (i.e., drug-sensitive) phenotype. The remaining alleles (48%) encoded r-proteins which phenotypically were inactive in the CHO cell transfection assay. Nucleotide sequence data were used to identify and map the amino acid substitutions encoded by mutagenized DNAs. The mutations characterized resolved five distinct S14 polypeptide domains (labeled A to E in Fig. 5), two of which (domains B and D) appeared to be important for r-protein biological activity. Remarkably, the polypeptide structural domains corresponded quite closely with the boundaries of human RPS14 exons II to V. Others have proposed that modern eukaryotic exons may have derived from a limited number of ancestral exons whose recruitment into diverse loci provided an efficient mechanism for protein evolution (8-10, 16, 20). The correlation between human RPS14's mutationally defined functional domains and its exon structure is consistent with this hypothesis. Several yeast r-proteins contain nuclear localization signals (NLSs) close to their amino termini (1, 17, 28). The yeast r-protein NLSs are short, basic peptide motifs that display striking similarity to the simian virus 40 (SV40) T-antigen NLS, PKKKRKV (12). More recently, an NLS has been recognized within an internal domain of rat r-protein S2; although it is also a short basic peptide motif (RGGF), the S2 NLS does not resemble the SV40 T-antigen

FIG. 7. Assembly of the r-protein encoded by pMS14-30 into functional CHO cell ribosomes. The figure illustrates a two-dimensional polyacrylamide gel electrophoresis of r-proteins purified from CHO Emr-2-2 cells transfected with pMS14-30 DNA. (A) Coomassie blue-stained pattern of proteins observed in the S14 region of the gel; (B) diagram identifying each spot and an overlay of the autoradiogram used to detect the pMS14-30 polypeptide synthesized in vitro. The r-protein labeled AS14 below and to the right of the S20 protein (A) was not observed in ribosomes purified from control CHO Emr-2-2 cells (Fig. 4). However, this protein comigrates with the radioactive cell-free polypeptide product encoded by pMS14-30 DNA (B).

NLS (27). Mammalian S14 lacks peptide motifs with obvious similarity to either the SV40 T-antigen or human r-protein S2 NLS sequences, despite the fact the S14 protein is a very basic polypeptide (Arg + Lys = 18%) (23). In addition, because the amino-terminal 20 residues of human S14 (domain A) are functionally dispensable, domain A is unlikely to encode a necessary NLS. On the other hand, most of the missense mutations mapped to domains B (Gly-24 to Gly-49) and D (Thr-107 to Asp-131) abrogated S14's biological activity, indicating that these regions of the r-protein are critical for its normal function. Experiments to determine whether S14 domains B and/or D might encode an NLS now are in progress. In a previous attempt to isolate null alleles of -the mammalian RPS14 gene by tissue culture mutagenesis experiments, we surveyed large numbers of ethyl methanesulfonate-treated CHO cells carrying extra copies of a cloned emetine resistance RPS14 allele. The somatic genetic approach yielded only six point mutations that destroyed S14's biological activity (6). Four of the null alleles isolated in vivo encoded single amino acid replacements (Emr-36, Gly-

111--Arg; Emr-39, Gly-127-->Arg; Emr-75, Arg-150-+Gly; and Emr-80, Cys-85--*Phe), and two carried nonsense mutations (Emr-77, Gln-20 [CAA]--TAA; and Emr-84, Arg-98 [CGA]-*TGA). The low recovery of null alleles from tissue culture selection experiments indicated either that most of S14's amino acid residues might be mutated without loss of biological activity or that the majority of S14 missense mutations might confer a dominant lethal phenotype which could not be isolated in living cells. Data described in this report demonstrate that (i) 47 amino acid substitutions isolated in vitro abrogated S14's activity without conferring a dominant lethal phenotype on cultured rodent cells and (ii) at least 32 different amino acid substitutions were compatible with a sufficient level of S14 function to complement the Emr-2-2 emetine resistance mutation (Table 2). ACKNOWLEDGMENTS We express our appreciation to Dawn Slifer for expert help with tissue culture and to Carl G. Maki, Beth A. Montelone, and David A. Rintoul for help during preparation of the manuscript. J.J.D. is a Postdoctoral Cancer Research Fellow of The Wesley

1686

MOL. CELL. BIOL.

DIAZ AND ROUFA

Foundation of Wichita, Kans. Support for this study was provided by NIH grants GM38932 and GM23013. 16.

1.

2.

3. 4. 5.

6.

7. 8. 9. 10.

11.

12. 13. 14.

15.

REFERENCES Bataille, N., T. Helser, and H. M. Fried. 1990. Cytoplasmic transport of ribosomal subunits microinjected into the Xenopus laevis oocyte nucleus: a generalized, facilitated process. J. Cell Biol. 111:1571-1582. Boersma, D., S. M. McGill, J. W. Mollenkamp, and D. J. Roufa. 1979. Emetine resistance in Chinese hamster cells. Analysis of ribosomal proteins prepared from mutant cells. J. Biol. Chem. 254:559-567. Brown, S. J., A. Jewell, C. G. Maki, and D. J. Roufa. 1990. A cDNA encoding human ribosomal protein S24. Gene 91:293296. Chen, I.-T., and D. J. Roufa. 1988. The transcriptionally active human ribosomal protein S17 gene. Gene 70:107-116. Dayhoff, M. 0. 1978. Atlas of protein sequence and structure. National Biomedical Research Foundation, Silver Spring, Md. Diaz, J.-J., D. D. Rhoads, and D. J. Roufa. 1990. Genetic analysis of a vital mammalian housekeeping locus using CHO cells that express a transfected mutant allele. Somat. Cell Mol. Genet. 16:517-528. Diaz, J.-J., D. D. Rhoads, and D. J. Roufa. 1991. PCR-mediated chemical (PMC) mutagenesis of cloned duplex DNAs. BioTechniques 11:204-211. Dorit, R. L., L. Schoenbach, and W. Gilbert. 1990. How big is the universe of exons. Science 250:1377-1382. Gilbert, W. 1978. Why genes in pieces. Nature (London) 271:501. Gilbert, W. 1979. Introns and exons: playgrounds of evolution, p. 1-10. In R. Axel, T. Maniatis, and C. F. Fox (ed.), Eucaryotic gene regulation: ICN-UCLA Symposium on Molecular and Cellular Biology. Academic Press, Inc., New York. Kozak, M. 1986. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc. Natl. Acad. Sci. USA 83:2850-2854. Lanford, R. E., and J. S. Butel. 1984. Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell 37:801-813. Madjar, J.-J., M. Frahm, S. M. McGill, and D. J. Roufa. 1983. Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells. Mol. Cell. Biol. 3:190-197. Madjar, J.-J., K. Nielsen-Smith, M. Frahm, and D. J. Roufa. 1982. Emetine resistance in Chinese hamster ovary cells is associated with an altered ribosomal protein S14 mRNA. Proc. Natl. Acad. Sci. USA 79:1003-1007. Maki, C. G., D. D. Rhoads, J.-J. Diaz, and D. J. Roufa. 1990. A

17.

18.

19. 20. 21. 22.

23. 24. 25. 26.

27.

28. 29.

Drosophila ribosomal protein functions in mammalian cells. Mol. Cell. Biol. 10:4524-4528. Marchionni, M. A., and W. Gilbert. 1986. The triosephosphate isomerase gene from maize: introns antedate and plant-animal divergence. Cell 46:133-141. Moreland, R. B., H. G. Nam, L. M. Hereford, and H. M. Fried. 1985. Identification of a nuclear localization signal of a yeast ribosomal protein. Proc. Natl. Acad. Sci. USA 82:6561-6565. Nakamichi, N., D. D. Rhoads, and D. J. Roufa. 1983. The Chinese hamster cell emetine resistance gene: analysis of cDNA and genomic sequences encoding ribosomal protein S14. J. Biol. Chem. 258:13236-13242. Overman, P. F., D. D. Rhoads, and D. J. Roufa. Multiple regulatory elements ensure accurate expression of a human ribosomal protein gene. J. Biol. Chem., in press. Perler, F., A. Estratiadis, P. Lomedico, W. Gilbert, R. D. Kolodner, and J. Dodgson. 1980. The evolution of genes: the chicken preproinsulin gene. Cell 20:555-566. Rhoads, D. D., A. Dixit, and D. J. Roufa. 1986. Primary structure of human ribosomal protein S14 and the gene that encodes it. Mol. Cell. Biol. 6:2774-2783. Rhoads, D. D., and D. J. Roufa. 1985. Emetine resistance in Chinese hamster cells: structures of wild-type and mutant ribosomal protein S14 mRNAs. Mol. Cell. Biol. 5:1655-1659. Rhoads, D. D., and D. J. Roufa. 1987. A cloned human ribosomal protein gene functions in rodent cells. Mol. Cell. Biol. 7:3767-3774. Rhoads, D. D., and D. J. Roufa. 1991. Molecular evolution of the mammalian ribosomal protein gene, RPS14. Mol. Biol. Evol. 8:503-514. Sanger, F., and A. R. Coulson. 1975. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J. Mol. Biol. 94:441-448. Southern, P. J., and P. Berg. 1982. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1:327-341. Suzuki, K., J. Olvera, and I. G. Wool. 1991. Primary structure of rat ribosomal protein S2. A ribosomal protein with arginineglycine tandem repeats and RGGF motifs that are associated with nucleolar localization and binding to ribonucleic acids. J. Biol. Chem. 266:20007-20010. Underwood, M. R., and H. M. Fried. 1990. Characterization of nuclear localizing sequences derived from yeast ribosomal protein L29. EMBO J. 9:91-99. Vieira, J., and J. Messing. 1982. The pUC plasmids, a p.13mp7derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259-268.