Characterisation of an mRNA encoding a human ribosomal protein homologous to the yeast L44 ribosomal protein

183

Gene, 45 (1986) 183-191 Elsevier GENE

1699

Characterisation of an mRNA encoding a human ribosomal protein homologous to the yeast L44 ribosomal protein (Recombinant tamoxifen)

DNA; coordinate

regulation; multigene family; DNA; modulation by growth conditions;

Michael S. Davies, Avril Henney *, Walter H.J. Ward * and Roger K. Craig ** Cancer Research Campaign, Endocrine Tumour Molecular Biology Research Group, Courtauld Institute of Biochemistry. The Middlesex Hospital Medical School, Mortimer Street, London WlP 7PN (U.K.) Tel. 01-636-8333, Ext. 9343 (Received

February

(Revision

received

Znd, 1986) and accepted

June Sth, 1986)

SUMMARY

We describe the isolation and characterisation of a full-length cDNA sequence (pZH-21) of a human ribosomal protein (rp) mRNA isolated from a cDNA library constructed from the human ZR-75-l mammary tumour cell-line. The predicted protein is highly basic and shows 72% homology at the amino acid (aa) level with yeast rp L44. Comparative RNA blotting of ZR-75-i poly(A)’ RNA isolated from cells cultured in the presence of the anti-oestrogen tamoxifen demonstrates the presence of a number of mRNA species whose concentration is elevated co-ordinately 5-6-fold in the presence of 17goestradiol. Insulin in the presence of tamoxifen, also enhanced rp mRNA levels suggesting increased levels are a reflection of cell prol~eration as opposed to specific hormonal regulation. Genomic analysis demonstrates the presence of a family of related human sequences, and homology with rat and guinea pig rp genes, but not yeast DNA. The conservation of rp aa sequence, in the absence of detectable homology at the nucleotide (nt) level, points to an important common functions role of the L44 protein in ribosome structure and function in man and yeast.

INTRODUCTION

The biosynthesis of eukaryote ribosomes is a complex process involving the coordinate synthesis of four different molecules of RNA, and over 70 different species of protein, all of which appear to be * Present Imperial (U.K.)

addresses:

(W.H.J.W.)

Department

College of Science and Technology, Tel.Ol-589-5111,

Biology,

University

Ext. of

4678;

York,

(A.H.)

York

of Chemistry,

London YOl

SW7 2AZ

Department fDD

of

(U.K.)

Tel. 0904-5986 I, Ext. 5840. ** To whom addressed.

correspondence

present in equimolar amounts (Wool, 1979). Studies using cloned mamma&an rp cDNA probes have shown that these genes are dispersed through the genome on a number of different chromosomes (D’Eustachio et al., 1981), and that mammalian ribosomal genes are each present at 7 to 20 copies in the genome (Monk et al., 198 1). Many of these have subsequently proved to be pseudogenes (Dudov and

Abbreviations: otidc(s);

and reprint

requests

should

be

ribosomal

aa, amino

acid(s);

PA, polyacrylamide; protein(s);

SDS,

bp, base pair(s);

nt, nucle-

RBS, ribosome-binding

site; rp,

sodium

0.15 M NaCl, 0.015 M Na, . citrate,

0378-I 119/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

dodecyl pH 7.6.

sulphate;

SSC,

184

Perry, 1984; Wiedemann ent little is known late mammalian

and Perry, 1984). At pres-

of the mechanisms ribosomal

which modu-

gene expression,

though

evidence has accummulated to suggest that the synthesis of ribosomal RNAs and ribosomal proteins in proliferating

mammalian

lated (see Warner cDNAs described

cells is coordinately

regu-

et al., 1980). The nt sequences

for a number

et al., 1983; Nakanishi

and [ p3’P]ATP

were obtained

from

Dr. J.B. Taylor and guinea pig liver DNA the gift of Dr. J. Laird.

of

of rat and mouse rp have been

(see Meyuhas

[ c+32P]dCTP

Amersham International, Amersham (U.K.) or New England Nuclear Inc. Human lymphocyte DNA was the gift of Dr. S. Schifter, rat liver DNA the gift of

(b) Cell culture

and Perry, 1980; Nakamichi et al., 1985 and Tanaka

et al.,

1985). Here we describe the nt sequence of a full-length human rp cDNA sequence which we have isolated from a human mammary tumour cell-line ZR-75-1 cDNA library by differential screening using cDNA synthesised from poly(A) + RNA isolated from cells grown in the presence of 17/?-oestradiol, or the antioestrogen tamoxifen. The cloned sequence is complementary to a family of poly(A) + RNA species whose levels increase coordinately 5-6-fold during oestrogen-stimulated cell growth. The cDNA encodes a protein of deduced M, 12 452, which shows 72% homology with protein L44 from the large ribosomal subunit of yeast (Itoh and WittmannLiebold, 1978). Analysis of gene organisation by Southern blotting shows that the human rp mRNA we have characterised is a member of a family of closely related genes, which shows interspecies homology with the rat and guinea pig genomic DNA, but not with yeast genomic DNA.

MATERIALS AND METHODS

(a) Materials Tamoxifen was the gift of Imperial Chemical Industries plc, Pharmaceutical Division, Macclesfield, Cheshire (U.K.). Genescreen Plus membranes were obtained from New England Nuclear Inc., Boston, MA (U.S.A.) and foetal calf serum was purchased from Gibco Ltd., Paisley (U.K.). Insulin was obtained from the Sigma Chemical Company Ltd, Poole, Dorset (U.K.). The human mammary cell line ZR-75-1 (Engel et al., 1978) was the gift of Dr. J. Easty. All other enzymes, chemicals and solvents were obtained from sources described previously (Allison et al., 1981; Taylor et al., 1984).

ZR-75- 1 cells were maintained

in 50 % (v/v) mini-

mum Eagle’s medium, 50% (v/v) RPM1 1640, containing 15% (v/v) foetal calf serum and 0.5 pg insulin/ml. Endogenous steroid hormones were removed from serum using activated charcoal (stripped serum). For studies using 17/%oestradiol and anti-oestrogens, ZR-75-1 cells were incubated in the presence of stripped serum with a change of medium every 48 h for six days. The cells were then incubated for 48 h after the addition of either 17& oestradiol (lo- 8 M), tamoxifen (lo- 6 M), or in stripped serum alone. Medium was replaced after 24 h, all essentially as described by Westley and Rochefort (1980). (c) Isolation of RNA, cell-free protein synthesis and product analysis Total RNA was prepared by the SDS-proteinase K procedure of Hall et al. (1979), and poly(A) + RNA isolated using oligo(dT)-cellulose chromatography as described by Craig et al. (1976). Cell-free protein synthesis was carried out in a nuclease-treated reticulocyte lysate and two-dimensional PA gel electrophoretic

fluorography of analysis, and [ 3sS]methionine-labelled peptides were performed as described by Pascal1 et al. (198 1). (d) Construction and screening of a ZR-75-l cDNA library

cell-line

A cDNA library was constructed from poly(A) + RNA isolated from ZR-75-1 cells which had been incubated in the presence of 17/%oestradiol for 48 h after 6 days of hormone withdrawal. Briefly, size selected, doublestranded cDNA (400-2000 bp) was inserted into the &I site of pAT153 using homopolymeric dC-dG tails, and the resulting chimaeric plasmids used to transform Escherichiu coli RR1 (see Allison et al., 1981). Transformants

185

(1000) were picked in duplicate

onto nitrocellulose,

then screened

cDNA

with 32P-labelled

synthesised

To identify mRNA

from poly(A) + RNA isolated from cells grown in the

enhanced

presence of either 17goestradiol plasmid DNA preparations

of oestradiol,

or tamoxifen. (Ish-Horowitz

Rapid and

Burke, 1981) were made from colonies which hybridised the former most strongly, and DNA from each

and characterise

species

which

encode

relatively proteins

abundant present

at

levels as a result of growth in the presence a small cDNA library was constructed

(1800 clones) using total poly(A) + RNA isolated from ZR-75-1 cells grown in the presence of 17/Ioestradiol.

Of these,

1000 were then

screened

for

was dot-blotted onto nitrocellulose, then rescreened using the hybridisation conditions described by

differences using 32P-labelled cDNA synthesised from total poly(A) + RNA isolated from cells grown

Thomas

in the presence

(1980).

these

RESULTS

AND

DISCUSSION

(a) Cloning of mRNA species present at enhanced levels in ZR-75-1 cells grown in the presence of 17jLoestradiol We have used the ZR-75 1 human mammary cellline to investigate changes in gene expression after incubation of cells in the presence of 17/I-oestradiol or the anti-oestrogen tamoxifen. Experiments using two-dimensional SDS-PA gel analysis of [ 35S]methionine-labelled ZR-75-1 cell proteins, demonstrated quantitative and in some instances qualitative changes in the pattern of 3sS-labelled proteins between cells incubated in the presence of steroid hormones and tamoxifen (A.H. and R.K.C., unpublished). To determine whether this reflected changes in mRNA accumulation, total poly(A) + RNA was isolated from cells incubated for 48 h in the presence of 17/$oestradiol(lO ’ M), tamoxifen (lo- 6 M), or neither, then translated in a rabbit reticulocyte lysate cell-free system, and the 35Slabelled newly synthesised proteins analysed by two-dimensional SDS-PA gel electrophoresis. The results (Fig. l,A,B,C) show that growth in the presence of 17/I-oestradiol resulted in quantitative and qualitative changes in the accumulation of mRNA species as judged by translational activity when compared with its absence or in the presence of tamoxifen as determined by the distribution of in vitro-synthesised 35S-radiolabelled proteins. One of these, 43.2 kDa, p1 5.7, which was induced in the presence of 17/3-oestradiol has the distinctive electrophoretic mobility of the actin gene family suggesting some changes, may reflect increased cell proliferation as opposed to a direct effect of oestrogen.

of 17goestradiol

119 appeared

to hybridise

or tamoxifen. preferentially

Of 32P-

labelled cDNA prepared from oestradiol-treated cells. Rapid plasmid DNA preparations were made on all of these, the DNA dotted in duplicate onto nitrocellulose, and the filters rescreened as described above. Nine recombinant cDNA plasmid preparations hybridised to the 32P-labelled cDNA from 17b-oestradiol-treated cells significantly more strongly than to a similar preparation from tamoxifen-treated cells. One of these (pZH-21) which contained a cDNA insert of about 600 bp was selected for further study. (b) Characterisation cloned into pZH21

and expression

of the mRNA

Nucleotide sequence analysis of the entire cDNA sequence cloned into pZH21 showed that it comprised 499 nt [excluding a poly(A) tail], and contained a single open reading frame with a 3’-noncoding end of 98 nt (Fig. 2). The 3’-noncoding region contained a polyadenylation recognition sequence (AATAAA) 19 nt from the site of polyadenylation,

whilst a potential

RBS (TCTCGTTT)

was located 40 nt upstream from the first ATG codon, which is therefore presumed to be the initiating ATG codon (Hagenbuchle et al., 1978). The open reading frame thus defined encoded a highly basic protein of 105 aa (M, 12452) excluding the initiating methionine residue. A computer search of protein data banks showed 72% homology between the predicted sequence, and that of the yeast large subunit rp L44 (M, 11849), a protein comprising 103 aa (Itoh and Wittmann-Liebold, 1978). Both the yeast and human rp are highly basic, the human protein containing 33 basic aa [21 lysine (20x), 12 arginine (11.4%)] evenly distributed throughout the molecule. The position of the four cysteine residues has been conserved between yeast and

186

1

PI 6-O

6.5

5.5

F

60K 4OK 35K Mol.

74K

HORMONE PI 6-O

6.5

5.5

!

WITHDRAWN PI 6-O

6.5

40K 35K

5-5

E

40K 35K

Mol. wt.

Mol. wt.

14K + OESTRADIOL Fig.

1. In vitro translation

+TAMOXIFEN

of poly(A) + RNA isolated from ZR75-1 cells cultured

SDS-PA

gel analysis

of 32S-labelled

poly(A)’

RNA isolated

from ZR-75-1

(C) plus tamoxifen

(IO-“M).

proteins

cells cultured

Isoelectric

calculated

on the basis of the relative

anhydrase

(30 kDa)

reticulocyte

protein;

and lysozyme arrow

indicates

synthesised

focussing mobility

(14.3 kDa) the putative

in a reticulocyte

in the presence

cell-free

of (A) ch~coal-stripped

in the first dimension of 14C-methylated

under different hormonal lysate

in the final dimension.

serum

Arrowheads

system

Two-dimensional directed

serum alone; (8) plus oestradiol

used a pH 5-7 ampholite,

bovine

conditions.

synthesising

albumin denote

and M,s of resolved

(69 kDa),

ovalbumin

the position

proteins

(46 kDa),

of the major

by total (IO- ‘M); were

carbonic

endogenous

actin (see B).

human rp, whilst an additional cysteine residue is present towards the C-terminal end of the human rp. Maximum conservation of sequence was at the N-terminal end. ~ydroph~icity studies (Fig. 3) reveal conserved hydrophilic domains distributed along each protein. These do not reflect duplication of a single primordial sequence, as judged by dot-

matrix analysis of the human nt sequence. Such conservation of rp sequence points to common functional role(s) of the L44 protein in ribosome structure and function in yeast and man. Studies on the expression of the human rp RNA transcripts by Northern analysis of poly(A) + RNA isolated from human tissues and cell-lines of mam-

187

3 yeast

210.

VV

n

u

V”

-

-l-

-2

I

I

20

40

60

80

100

120

20

40

60

80

100

120

I

3I

=-

.."

1

1

4

-31 sequence Fig. 3. Comparative

average

L44 and the homologous Hopp Fig. 2. Restriction a human

rp cDNA

sequence

analysis

(Maxam

map, nt sequence cloned

and Gilbert,

and across

all restriction

the

published

Wittmann-Liebold,

sequences

Lincoln’s

of the

yeast

generated

residues

Inn Fields, L44

with the human

aa are boxed, and potential

were

London

has been aligned with rp

patterns

of yeast rp

rp, calculated

according

to

(1981), using the programme

described

by

Larsen

and Messing

regions

and negative

(1983). Positive values indicate

average

is taken over 7 aa.

values

hydrophobic

regions.

hydrophilic The running

(Sanger et al.,

using facilities provided

aa sequence

hydrophilicity human

cleavage

on both strands

1978), ‘best fit’ being obtained

of the four yeast cysteine Conserved

was performed

Unit,

human

aasequence

Nucleotide

using the chemical

The nt and the aa sequence

Computing

(U.K.). The deduced

pZH-21.

of

sites used for cloning into M13, or for

with other known

by the ICRF

aa sequence

1977) and dideoxynucleotide

Sequencing

3’ or 5’ end-labelling. compared

in the plasmid

was performed

1980) procedures.

and deduced

and Woods

position

(Itoh

and

by alignment counterparts.

RBS and polyadenylation

sites underlined.

mary origin, showed that the cloned cDNA sequence was complementary to a number of poly(A) + RNA species. Examination of the relative amount and size of rp mRNA in ZR-75-1 cells treated for 48 h after hormone withdrawal for 6 days (see MATERIALS AND section d) by either 17/?-oestradiol METHODS, (IOO’M) or tamoxifen (10e6M) in the presence or absence of insulin, revealed the presence of a complex RNA population containing a number of com-

ponents ranging from 460 to 630 nt in size (Fig. 4A). Treatment with 17goestradiol in the absence of insulin resulted in a 5-6-fold increase in all rp mRNA species when compared with the equivalent tamoxifen-treated cells. The difference was less obvious when insulin was present throughout the experiment, since in the presence of insulin the basal ribosomal mRNA levels were significantly higher in the tamoxifen-treated cell preparation. The heterogeneous nature of the poly(A) + RNA species identitied by Northern blotting was not a reflection of size heterogeneity of the poly(A) tail, since removal of the poly(A) tail by treatment with RNaseH in the presence of excess oligo(dT) (Donis-Keller, 1979) resulted in an overall reduction in size of 60-80 nt (not shown). Reanalysis of the same filter using a mouse actin cDNA probe, revealed a single 1600 nt band, whose relative levels fluctuated under the different hormonal regimes in a similar manner to the ribosomal protein mRNA species (not shown). The

188

B

A . III

ii

I

iv

ii

I

iii

-517 -396 -298-221_

Fig. 4. Effect of hormones homologous presence

to pZH-21

of tamoxifen

and distribution was determined

hormonal

regimes were separated

onto Biodyne A nylon membrane, DNA fragments

as described

described

in ZR-75-1

* M). Samples

(ii) human

(B) The distribution

tibroblasts;

(iii) human

in the presence

of human rp mRNA species

or absence

of insulin,

(5 pg) of total poly(A) + RNA isolated from ZR-75 gel electrophoresis

to nick-translated

by Taylor et al. (1984). Size determinations

(iv) + tamoxifen. mammary

cells cultured

by 1.1 y0 (w/v) agarose

and the filter was hybridised

(1630, 517, 396, 298 and 221 nt) electrophoresed

(iii) + 1‘l/&oestradiol; tumour;

L44 rp mRNA species. (A) The relative concentration

by RNA blotting

(10 - 6 M) or 17)%oestradiol(lO

under the appropriate autoradiographed

of human

**

32P-labelled

under denaturing

pZH-21

conditions,

DNA (10’ cmp/pg),

were based on the relative mobility ofHinfl-digested

in parallel.

(i) + insulin + 17)?-oestradiol,

and in the

1 cells cultured blotted

washed

and

pATl53

(ii) + insulin + tamoxifen

of total poly(A) + RNA (5 pg each) isolated from (i) benign human mammary

placenta,

and (iv) lactating

guinea

pig mammary

gland

was determined

as

in panel A.

same rp mRNA species were also present in poly(A) + RNA isolated from human placenta, human mammary tumour tissue and cultured human mammary tibroblasts (Fig. 4B), whilst examination of polysomal poly(A)+ RNA from the lactating guinea pig mammary gland also showed the presence of homologous guinea pig mRNA species of similar size (Fig. 4B). It is apparent from these studies that the human rp cloned into pZH-2 1 is a member of a small family of related mRNAs, which are expressed in the human tissues, cell lines and tumours so far examined. The accumulation of the human rp mRNAs appears to be regulated in a coordinate fashion as judged by their expression in ZR-75-1 cells grown in the presence or absence of 17goestradiol. It is unlikely that the increased levels of rp mRNA reflect a direct regulation of rp gene expression by oestrogen, in view of the similar variations observed in actin mRNA accumulation, and the effects of insulin on basal rp mRNA levels. Thus the increase in rp mRNA levels, though coordinately regulated, probably reflects in

this instance a generalised pleiotropic effect, a secondary response presumably due to the mitogenic effects of oestrogen and insulin on the ZR-75-1 cell line. Similar fluctuations were also observed during studies on the MCF-7 human mammary tumour cell line in the presence of 17/$oestradiol or the antioestrogen

tamoxifen

(A.H.,

M.S.D.

and

R.K.C.,

unpublished). A small coordinated increase in rp mRNA levels has also been reported during rat liver regeneration (Faliks and Meyuhas, 1982). (c) Analysis of human ribosomal genes Analysis of the structure of the human ribosomal gene(s) by Southern blotting of human lymphocyte DNA revealed a complex banding pattern, suggesting the presence in the genome of a number of related genes (Fig. 5A). Digestion with EcoRI gave rise to four strongly hybridising bands of 20.5, 11.2, 6.7, and 5.2 kb and a number of discrete bands which hybridised the pZH-21 cDNA probe less strongly. Digestions with the enzyme PstI, which has a single

189

0

A ii

I

Fig. 5. Genomic Human

analysis

lymphocyte

of the human

DNA samples

on the basis of size by electrophoresis &I-cut

gel-purified

a2P-labelled

step was omitted. The membrane

L44 rp gene and homology

(10 ng) were digested cDNA

was washed

with other species. (A) Genomic

analysis

of the human

with PstI (lane i), PstI + EcoRI (lane ii), and EcoRI

on a 0.8 y0 (w/v) agarose

pZH-21

iii

ii

I

iii

gel. This was blotted onto Genescreen

(5 x IO’ cpm/ng)

as described

by Edbrooke

L44 rp gene.

(lane iii) and separated

Plus membrane,

and probed

with

et al. (1985) except that the depurination

twice in 2 x SSC, 1% (w/v) SDS for 5 min at room temperature,

twice for 15 min at 65°C

then once in 0.1 x SSC at 65°C for 15 min, prior to autoradiography. in kb on the left margin. (lane iii) were digested wash was omitted.

(B) Human

lymphocyte

with EcoRI, electrophoresed,

The sizes of HindIII-digested

The sizes ofHindIII-digested phage I DNA fragments are indicated DNA (10 ng) (1ane i), guinea pig liver DNA (10 ng) (lane ii) and rat liver DNA (10 ng) blotted,

hybridised

and washed

phage i DNA fragments

site within the pZH-21 cDNA sequence, and double digests with PstI + EcoRI, produce a comparable complex pattern of bands, some of which hybridise the pZH-2 1 cDNA probe more strongly than others. Hybridisation to EcoRI-digested rat and guinea pig genomic DNA preparations (Fig. 5B) also revealed a complex pattern of bands, indicating the presence of a closely related rp gene family in these species. No hybridisation was observed to EcoRI-digested

as described

are indicated

above, except that the final 0.1 x SSC

in kb on the right margin.

yeast genomic DNA, under comparable hybridisation conditions. Studies on the structure of the human rp genes by Southern blotting indicate, in common with analogous sequences in rat and mouse, that genes encoding individual human ribosomal proteins comprise a family of related genes (see D’Eustachio et al., 1981; Monk et al., 1981), some of which may ultimately prove to be pseudogenes (see Peled-Yalif

190

et al., 1984; Dudov and Perry, 1984; Wiedemann and Perry, 1984). Recently the structures of a human rp L32 pseudogene and L32 cDNA sequence, which are closely related to characterised mouse rp gene sequences have also been described (Young and Trowsdale, 1985). A preliminary analysis using mouse-human and rat-human hybrid cells (M.S.D., S. Povey and R.K.C., unpublished) provides evidence that as in the mouse, the human L44 rp gene family is located on more than one chromosome (see D’Eustachio et al., 1981). Homology between mouse, human and hamster rp genes has been reported previously (D’Eustachio et al., 1981; Monk et al., 1981). We extend these observations, demonstrating conservation of rp gene sequences between human, rat and guinea pig, and moreover, expression of homologous human and guinea pig rp mRNA species. Strong homology between the aa sequence of rat rp S 17 and yeast rp 51 has recently been reported (Nakanishi et al., 1985). This, and our own demonstration of strong homology between a human rp and yeast rp L44 indicate conservation of primary aa sequence of at least some rp has occurred between evolutionarily remote species, in spite of the apparent absence of homology at the nt level between human and yeast genes, and reports of varying interphyla cross-hybridisation between mouse, Xenopus and Drosophila genomic DNA (Meyuhas, 1985). The availability of cloned human rp mRNAs will now permit a rigorous examination of the factors which modulate rp gene expression in human cell lines and tissues.

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