Gene, 118 (1992) 273-278 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

213

0378-l 119/92/$05.00

06580

The Drosophila (Recombinant

Stkphane

DNA;

melanogaster

gene cloning;

cDNA;

ribosomal protein L 17A-encoding

in situ hybridization;

Northern

blot; Diptera;

gene

Minute mutations)

Noselli and Alain Vincent

Centre de

Recherchede Biochimieet de Gtntrique Cellulaires du CNRS, 31062 Toulouse Cidex, France

Received

by H.M. Krisch:

29 January

1992; Revised/Accepted:

25 March/27

March

1992; Received

at publishers:

29 April 1992

SUMMARY

The structure and sequence of the gene encoding the Drosophila melanogaster homolog of the human and yeast largesubunit ribosomal protein L17A (rpL17A) is presented. The deduced amino acid (aa) sequence of 140 residues exhibits 870/, and 77% identity to that of the human (140 aa) and yeast (137 aa) rpL17As, respectively. The D. melanogaster rpLl7A gene is single copy and maps at 58F6-59A3, a chromosome region encompassing a previously characterized Minute locus, h4(2)1. Despite this extensive homology in their protein products, the D. melanogaster and yeast rpLl7A genes display different exon-intron structures, with the first D. melanogaster intron mapping within the 5’-untranslated mRNA leader. The rpLl7A gene gives rise to a single 600-nucleotide transcript present throughout development, and is located close to another similarly expressed gene. The 5’ end of the D. melanogaster rpLl7A mRNA contains a polypyrimidine tract displayed by several mammalian rp genes and involved in translational control of their expression.

INTRODUCTION

Ribosome biosynthesis is a fundamental process requiring the coordinate expression of different sets of genes, among which the 70 to 80 different rp genes (Wool, 1979). In D. melanogaster, an organism allowing a genetic analysis of this coordinate expression, the rp have been biochemically characterized in detail (Choi et al., 1984), but surprisingly only a few rp genes have been cloned. It is reasonably certain that at least some Minute mutations, a common class of haplo-insufficient mutations characterized by a delayed development, reduced viability and often small body size, are mutations in rp genes (reviewed by

Ashburner, 1989). This assumption has been verified in, at least, the case of gene rp49. P-element-mediated transformation of D. melanogaster with a DNA fragment containing the rp49 gene rescues the phenotypes associated with the Minute h4(3)99D mutation (Kongsuwan et al., 1985). We report here the structure and developmental expression of another D. melanogaster rp gene, identified by homology to the yeast and human rpLl7A genes, that maps at the same locus as a Minute mutation, M(2jl. Sequence comparison of the D. melanogaster and yeast rpLl7A genes shows that, despite extensive homology between their predicted products, the D. melanogaster and yeast genes differ in their intron-exon structures.

EXPERIMENTAL Correspondence to: Dr. A. Vincent, Centre de Recherche

de Biochimie

et

de GCn&ique Cellulaires du CNRS, 118 route de Narbonne, 31062 Toulouse CBdex (France) Tel. (33-61)33.59.56; Fax (33-61)33.58.86 Abbreviations: aa, amino acid(s); bp, base pair(s); plementary to RNA; D., Drosophila; kb, kilobase cleotide(s)

(number

tric point;

r, ribosomal;

or sequence);

SV40, simian virus 40;

cDNA, DNA comor 1000 bp; nt, nu-

ORF, open reading

rp, ribosomal

t.Fp,transcription

protein(s);

frame; PI; isoelec-

rp, gene encoding

start point(s).

AND DISCUSSION

rp;

(a) Isolation and identification of recombinant Drosophila melanogastev clones carrying sequence homology to the yeast (and human) rpLZ7A gene During the characterization of genomic targets for the D. melanogaster DNA-binding proteins Serendipity fi and 6 (Payre and Vincent, 1991; Noselli et al., 1992) we partially sequenced DNA fragments isolated from a mini-

214 Ofib

A

B I

aagcttttgcaatttcctgtgtgaacacgacatctgattggccagacctgccaacttgtt

61

cgaggtgggtgtaaaactccgatggtatgagtgcctgctaggattttagcaaagttcgac

121

ccaatccgtgctcattcttggttataattaggttattttttcgattttttgaggtatat~

181

totacgatagatcggcggtca~at~gt~ttt~~~TCCTTTTCGTTTTCGTTTCCGGCGRR

2t1

gtaagtagaataaaatctccactggttttttttaaatgctaataattgaaacatctcatt

301

ggctaactagCGARTRRTCTTGGRRTRflCCflGTCCGCGRGCRGCRRRRTGTCGRRGRGRG

1

II

361

gtaaaaatccgaacgaacgcgccgctaagcgtgcgaaatgtgacgggccggatgtggaga

421

gtgtcgaaattgtggtgtgcaaaactaataaccctctgtttttcggtctcatccagGRCG

S

K

6

R

G

R

481

TGGRGGTRCCGCGGGRGGCRRGTTCCGCRTCTCCCTCGGTTTGCCCGTGGGCGCCGTGRT GGTRGGKFRISLGLPUGRUH

7

GRRCTGTGCCGRCRRCRCCGGRGCCRRGRRCCTGTRCGTGRTCGCCGTCCRCGGRRTCCG

541

NCRONTGRKNLYUlRUnGlR

27

CGGTCGCCTTRRCCGTCTGCCCGCCGCTGGTGTCGGCGRCRTGTTCGTGGCCRCCGTGRR

601

GRLNRLPRRGUGOnFURTUK

t7 661

GRRGGGRRRGCCCGRGCTCRGGRRGRRGgtgagtgtggtgcccagtgctccggctgaatc

67 721

KGKPELRKK ___ ggtcatgtgctaaaagtgcccatgggtagcaacccgcccagtgaacacgtttttttcact

781

ttgtttttgtgggctttcttgaatgaaattcaacttgtttctgtttcaaagaagccaatt

841

tgaaattagttagctcttattacaatgggttgcttgaggttaataatcgtattttgtgct

901

acactatttgtcatttgttttgaacgggcagttttaatttctatcaattgcagagtattt

961

cattgtaaagtaatacgataatatagatagatttgtattagacgtggaaggtttctcctt

1021

acgcttccaataaatttcccccacatgttttgtgccggctaagatagcttatcgcaccaa

1081

actcacgtttttaatatttttgtttattgataagaatttaaaattgtagtgaagaggttt

1141

ataaaacggtttcactgcacttctcataattcccccacctataacaacgcacttctgcat

1201

aactaaccgatgctgcaggaggtttgcacactgtcgagcaactttgctaactactgtttt

1261

catttggttccagGTCRTGCCTGCCGTGGTTRTTCGGCRGCGCRRRCCGTTCRGGRGGRG U

76

fl

P

R

U

U

I

R

t)

R

_K__P__F__R__R_

R

GGRCGGGGTGTTTRTRTRCTTTGRGGRCRRTGCCGGGGTRRTRGTRRRCRRCRRGGGCGR

1321 92

OGUFIYFEONRGUIUNNKGE

I381

RRTGRRGGGCTCGGCCRTCRCTGGRCCGGTGGCCRRGGRRTGCGCCGRTCTGTGGCCCCG tlKGSRlTGPURKECROLWPR

l/Z 1441

TRTTGCRTCCRRTGCRRGCTCTRTRGCCTRRGGRGTTTCCTTTTC~CCCRCRRCG IRSNRSSIR

132

GRRRRCRGRTTGTTTTGRRTTCG

1501

Fig. 1. Molecular organization and scqucncc of the D. melurw~~~strr rpLl7A gene. (A) Top: Restriction map of the L). nwlunoguster fragment containing the rpLl7A gene. Bottom: Transcription map of the rpLI7A gene. The BanzHI-EcoRI (a), BanzHI-Hi~zdIII (c) fragments

(indicated

by horizontal

bars) were used as separate

probes

for Northern

blot analysis.

The positions

BunzHI-EcoRI genomic (b) and HindIII-EcoRI

of the introns

and the 3’ terminus

of the rpLl7A transcript were deduced from sequencing of the rpL17A cDNAs. Bent lines correspond to introns, open boxes to nontranslated regions, while the blackened boxes corrcspond to the ORF deduced from the nt sequence. (B) Genomic and cDNA nt sequences of the rpLl7A gene and the deduced rpL 17A aa sequence. The cDNA’s ters. The first A (a) of the genomic tative rsp is indicated

by a downward

The putative

nuclear

bipartite

nt sequence is in upper-cast Icttcrs. Intron and flanking sequences not present in the cDNAs are in lower-case letHind111 site is rcfcrrcd to as nt 1. The putativc TATA sequence and polyadenylation signal are underlined. The puarrow. The deduced

localization

aa sequence

signal is underlined

of the rpLl7A

with dashed

protein

lines. The GenBank

is indicated accession

under the nt sequence No. of the genomic

in italicized

sequence

numerals.

is M85295.

275 library made of size-fractionated fragments

EcoRI-BamHI

consensus

genomic

stream

and inserted into the pKS + plasmid (Stratagene,

AATAAA

signal

site. Moreover,

32 bp upthe dis-

tance separating the stop codon and the polyadenylation signal (14 bp) is conserved between the human and D. melanogaster cDNAs. The tsp has not been determined in this study but it can be noted that a proposed consensus sequence for the eukaryotic transcription start PyA(Py)S (where A is the tsp at nt + 1) is found 6 bp upstream from the 5’ end of the cDNA, and 28 bp downstream from a possible TATA box (Fig. 1B; Breathnach and Chambon,

La Jolla, CA). One fragment (named EB6), which turned out to be a false positive, showed striking homology to the 3’ end of the yeast and human rpLl7A genes (Leer et al., 1984; Berchtold and Berger, 1991). The 2.5-kb EB6 fragment was used to isolate DNA from an Oregon R cDNA library made from poly(A)+ RNA of 8-12-h-old D. melanogaster embryos (Brown and Kafatos, 1988). Two independent cDNA clones were isolated, slightly differing in their 5’ and 3’ ends. A physical map of the genomic and cDNA clones carrying homology to the yeast rpLl7A gene is shown in Fig. 1A and the corresponding nt sequences are given in Fig. 1B. The cDNA sequence had a length of 537 nt, containing 63 nt of 5’-noncoding, 420 nt of proteincoding and 55 nt of 3’-noncoding sequences. The ATG codon at nt position 348 is preceded by a sequence motif (CAAA) identical with the consensus sequence of the D. melanoguster 5’ flanking site of the ATG start codon (Cavener, 1987). The ORF which starts at this ATG encodes a putative protein product of 140 aa with the same size and 87% sequence identity to the human rpL17A protein (see section d). The 3’-noncoding sequence contains a

1981). Because of the sequence homology between clone EB6 and the yeast and human rpLI 7A genes, we designate this D. melunogaster

gene as the rpLl7A

gene.

(b) The Drosophila melanogaster rpLI7A gene maps to 58F6-59A3, a region of the second chromosome that contains a Minute locus All D. melunoguster rp genes for which probes are available have been shown to be single-copy genes except for the S14 gene that is present in two adjacent, almost identical copies (Brown et al., 1988). Southern blot analysis under high stringency conditions of genomic DNA cut with dif-

A 1

polyadenylation

from the polyadenylation

B

2

3

kb

Fig. 2. Hybridization (Fig. 1A). Genomic

to gcnomic

11.4

-

6.2

-

4.4 3.7

-

2.6

-

1.9 1.7

-

1.1 0.9

-

0.6

-

DNA.

(Panel A) Southern

DNA (5 ng) was digested

with BamHI

blot analysts

under stringent

(lane l), EcoRI

conditions

(lane 2) or BarnHI

of D. melanogasrer

+ EcoRI

Oregon

R DNA using probe c

(lane 3). A single band of hybridization

is de-

tected in each lane. (Plate B) In situ hybridization to squashes of third instar larvae salivary gland polytene chromosomes (Oregon R stock). The genomic BarnHI-EcoRI (probe a, Fig. IA) was digoxigenin-labelled by random priming using the Genius kit (Boehringer-Mannheim). Hybridization to chromosome squashes

and detection

were done according

59A3 on the right arm of the second

chromosome.

to De Frutos

(1990). A single hybridization

signal (see arrowhead)

was observed

at position

58F6-

276 ferent restriction endonucleases reveals a single band in each lane, indicating that the rpL17A gene is present at a single copy in the D. melanogaster genome (Fig. 2A). In situ hybridization of the EcoRI-BamHI EB6 fragment to salivary gland polytene chromosomes of the D. melan-

A E

L

P

F

M

kb

ogaster Oregon R stock indicated that the rpLl7A gene is located at position 58F6-59A3 on the right arm of the second chromosome (Fig. 2B). A strong Minute mutation, A4(2)Z,genetically defined as an haplo-insufficient mutation characterized by a delay in puparium formation, viability 80-90”, of wild type and low fertility, has been previously localized at 58F (Lindsley and Grell, 1968), but so far, no gene has been cloned from this region. From molecular cloning studies, it is reasonably certain that at least some other Minute mutations are in rp (for review see Kay and Jacobs-Lorena, 1987; Ashburner, 1989), as originally suggested by Vaslet et al. (1980), and shown in the case of the rp4Y gene (Kongsuwan et al., 1985). (c) The rpLl7A gene encodes a single 600-nt mRNA expressed throughout the fly life cycle The 2.5-kb EB6 genomic fragment hybridizes to two polyadenylated RNAs species. They are 4 kb and 0.6 kb long, respectively, and are present throughout the whole fly life cycle (Fig. 3A). The most abundant transcript is the 0.6-kb RNA that accumulates to higher levels in embryos and adult females, and is detected at lower levels during the other developmental stages. Northern blot hybridization to poly(A)’ RNA from 0-lo-h-old embryos was repeated, using as separate probes the BamHI-Hind111 and HindIIIEcoRI fragments derived from the EB6 insert (Fig. 3B). The BamHI-Hind111 fragment ‘lights up’ the 4-kb RNA species while the HirzdIII-EcoRI probe, which includes the entire rpLl7A sequence, hybridizes to the 0.6-kb transcript. This result identifies the 0.6-kb RNA expressed throughout development as the rpLl7A gene transcript, consistent with a similar pattern of expression shown by other D. meianogaster rp genes. (d) Comparison of the Drosophila melanogastev rpL17A aa sequence to its yeast and human homologs Comparison between the eubacterial and archaebacterial homologs of the yeast rpL17A protein (protein L14) and the yeast and human rp has recently been reported (Berchtold and Bergcr, 1991). In summary, it appears that the eubacterial and chloroplast L14 proteins have a smaller size (120-130 aa) than the archaebacterial (132 aa), yeast ( 137 aa) or human proteins (140 aa), and display from 29 “/, to 78:; identity with the human protein. The D. melanogaster rpL17A protein is more closely sequence related to its human (87”/, identity/94a/0 similarity) than its yeast homolog (77 “/, identity/8 1% similarity; Fig. 4A). Like its human and yeast homologues, the D. melanogaster rpL17A

-

4-

0.6

Cg. 3. Northern hybridization scribed

(Vincent

probe a (Fig.

blot nnalys~s. Staging

in high-stringency

of embryoa.

conditions

et al., 1984). (Panel

A) The blot was hybridircd

and dcwith

1A). Poly(A)’ RNA (2 pg per lane) was isolated from 0-10-h

embryos (E), third-ins&

larvae (L), pupae(P),

males (M). Two RNA species were detected (Panel B) Probes to 0-10-h

RNA isolatmn

were done as previously

adult females (F) and adult at each developmental

b (1) and E (2) wcrc separately

embryonic

stage.

used for hybridization

poly(A)‘RNAs.

protein is very basic (calculated p1 of 11.47), a characteristic correlating with its nuclear import. While no motif closely related to the SV40 T antigen or a recently identified D. melanogaster nuclear localization signals (Noselli and Vincent, 1991; for review see Silver, 1991) is found in the rpL17A protein, a consensus bipartite motif is present between aa positions 74 and 90 (Fig. 1B; Dingwall and Laskey, 1991). This motif, responsible for nuclear targeting of nucleoplasmin (Robbins et al., 1991), is made of two short stretches of basic aa separated by a IO-aa spacer, and is found in nearly half of known nuclear proteins. In spite of their remarkably similar protein products the intron-exon organization of the D. melanogaster and yeast rpLl7A genes is quite different (the structure of the human gene is at present not known). There is a single intron in the yeast gene which interrupts the coding region after aa position 15 (position 18 in the D. melanogaster protein). This intron has no counterpart in D. melanogaster. However, there are three introns in the D. melanogaster gene that are not found in the yeast gene. The second and third of

277 Whether

A 1

Hs

20

10

30

40

MSKRGRGGS&UCFRISLGLPVGAVINCADNTGAKNLYIISVKGIKGRLN

IIIIIIII!!I

IIIIIIIIIIIII!IIIIIIIIIIII!I!I

a similar translational

control

is involved

in reg-

ulation of the D. melanogaster rpLl7A gene will be the topic

M

of future studies. IIIIIII

Dm MSKRGRGGTAGGKFRISLGLPVGAvMNCADNTGAKNLyvIAvMGIRGRLN II I! I IIIIIIIIIIII!IIIIII II!III!III I SC MS---GNGAQGTKFRISLGLPVGAIMNCADNSGARNLYIIAVKGSGSRLN

I II ACKNOWLEDGEMENTS

PAWIRQRKSYRRKDGVFLYFED Ii.5 RLPAAGVGDl.FJMATVICKGKPE~ IllIllIIII !IIIIIIIIIIIIII IIIIIIIII !ll!llll!llII Dm RLPAFdZVGDMFVATVKKGKPELRXKVMP AVVIRRKPFRRRDGVFIYFED IIIII ‘III ~lllllllllllllllll!l!lf I ‘IIIIIII’IIII LRKKVMPAIVVRQAKSbGVFhFED SC RLPAASiGDMVh'MCKGKPE

t-/s NAGVIVNNK&hlKGSAITG&.KECADLW&IASNRGSI~ IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Dm NAGVIVNNXGEMKGSAITGPVAKKCADLWPRIASNAsSIA IIIII I IIIIIIIIIIIII IIIIlIIlI!IlI! SC NAGVIMiPKGEMRGSAITGPVGKECADLWPRvAsNSG&

We wish to thank N. Brown for a gift of Drosophila embryonic cDNA library. We are grateful to members of the laboratory for discussions and to H.M. Bourbon for critical reading of the manuscript. We wish to thank Joelle Maurel for editorial assistance. This work was supported by CNRS and Ligue Nationale Contre le Cancer. S.N. was supported by a fellowship from the Association pour la Recherche contre le Cancer.

II

B rpLl7AATTTCCCTCCTTTTCGTTTTC

IIII

531

I

I

IIIIIIIIIIIl

ATTTTCTTTCTTTTCGTTTCC

Fig. 4. Sequence

comparisons.

REFERENCES

(A) Comparison

of the D. melanogusrer

rpLl7A aa sequence with its yeast and human homologs. Orientation of each polypeptide is from N (left) to C terminus (right). Identical aa are marked

by solid vertical lines; similar aa are connected

The following exchanges D=E;

N=Q;

R=K;

were considered I=L=M=V;

as conservative F=Y=W.

Homo supietls, D. melunogrrster and Saccharonzyces ment of 5’untranslated

nt sequences

by dashed

lines.

ones: A = S = T;

Hs, Dm, SC refer to cerevisiae. (B) Align-

of the D. melunogaster rpLl7A

S.?l genes. For the rpLl7A gene this sequence begins at the putative (see section a and the arrow in Fig. 1B). Identical nt are marked

and tsp by

vertical lines

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these introns interrupt the protein coding region at aa position 5 and after aa position 75, respectively. Both yeast and D. melunoguster introns are located in a rpL17A protein segment showing 100% identity between yeast, human and D. melanoguster. Interestingly, the first D. melunogaster intron maps within the 5’-untranslated mRNA leader. A similar structure has been reported for the yeast rp29 and D. melunogaster rpS31 genes (Mitra and Warner, 1984; Itoh et al., 1989). This gene organization suggests the possibility of c&acting regulatory elements located downstream from the tsp of some D. melanoguster or yeast rp genes, as reported for several mouse rp genes (Hariharan et al., 1991). Among the few D. melcmogaster rp genes whose structure is known, several have introns (from one to three) while another is intronless. Therefore, a general role of pre-mRNA processing in the coordinate expression of rp genes seems excluded. Alignment of the leader sequences of the D. melanogaster rpLl7A and S31 genes shows that both contain an identical 5’ polypyrimidine tract (Fig. 4B). A similar polypyrimidinerich structure is found 5’ of transcripts from several Xenopus and mammalian rp genes and has been shown to be involved in translational control of the corresponding mRNAs (Mariottini and Amaldi, 1990; Levy et al., 1991).

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