Download Mapping of ribosomal protein S3 and internally nested snoRNA

SHORT COMMUNICATION
Mapping of Ribosomal Protein $3 and Internally Nested snoRNA
U15A Gene to Human Chromosome 11q13.3-q13.5
ROBERTO D. POLAKIEWICZ,*'t'1 DAVID J. MUNROE,t'~ S. N. JANI SALT,§ KAZIMIERZT. TYCOWSKI,¶
NORMA J. NOWAK,§ THOMAS B. SHOWS,§ DAVID E. HOUSMAN,t'$ AND DAVID C. PAGE*'t
*Howard Hughes Research Laboratories at Whitehead Institute, 9 Cambridge Center, Cambridge, Massachusetts 02142;
t Department of Biology and ~Center for Cancer Research, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139; §Department of Human Genetics, Roswell Park Cancer Institute, New York State Department
of Health, Buffalo, New York 14263; and ¶Howard Hughes Medical Institute, Department of Molecular Biophysics
and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06536-0812
Received July 6, 1994; revised September 26, 1994
T h e m a m m a l i a n r i b o s o m e is a m a s s i v e s t r u c t u r e
c o m p o s e d o f 4 R N A s p e c i e s a n d a b o u t 80 d i f f e r e n t
p r o t e i n s . O n e o f t h e s e r i b o s o m a l p r o t e i n s , $3, app e a r s to f u n c t i o n n o t o n l y in t r a n s l a t i o n b u t a l s o
as a n e n d o n u c l e a s e in r e p a i r o f U V - i n d u c e d D N A
d a m a g e . M o r e o v e r , t h e first i n t r o n o f h u m a n R P S 3
t r a n s c r i p t s is p r o c e s s e d to g e n e r a t e U15A, a s m a l l
n u c l e o l a r RNA. We l o c a l i z e d t h e n e s t e d R P S 3 / U 1 5 A
g e n e s to t h e i m m e d i a t e v i c i n i t y o f D l l S 3 5 6 a n d
D l l S 5 3 3 o n h u m a n c h r o m o s o m e 1 1 q 1 3 . 3 - q 1 3 . 5 usi n g a c o m b i n a t i o n o f s o m a t i c cell h y b r i d a n a l y s i s ,
f l u o r e s c e n c e i n s i t u h y b r i d i z a t i o n , a n d YAC/STS
c o n t e n t m a p p i n g . T h e s e f i n d i n g s a d d to t h e evidence that genes encoding ribosomal proteins are
s c a t t e r e d a b o u t t h e h u m a n g e n o m e . © 1995 Academic
Press, Inc.
In mammals, each ribosomal protein is typically
encoded by a single gene, from which a number of
silent, processed pseudogenes have been generated
(4). These pseudogenes complicate the mapping of
ribosomal protein genes to chromosomes, and this
explains, at least in part, why only 16 of the 80 or
more ribosomal protein genes have been chromosomally assigned (Refs. 1, 5 - 7 , 9, 10, 13, 16, 21).
The 16 genes that have been assigned map to 12
different chromosomes, suggesting that ribosomal
protein genes, unlike ribosomal RNA genes, are dispersed throughout the genome.
The RPS3 gene, not previously mapped, is of particular interest. First, the protein that it encodes
has two apparently distinct functions: (i) as a ribosomal protein, RPS3 contributes to the domain
where translation is initiated (2), and (ii) as an endonuclease, RPS3 apparently participates in repair of
UV damage (22; S. Linn, unpublished observations).
Second, U15A, a small nucleolar RNA ("snoRNA"),
is processed from the first intron of the RPS3 tran1 To whom correspondence should be addressed at the W h i t e h e a d
Institute. Telephone: (617) 258-8420. Fax: (617) 258-5578.
script (20). The function of snoRNA U15A is not well
understood, but it may act in ribosomal RNA processing (19). Third, the nested RPS3 and U15A
genes are overexpressed in colorectal carcinomas
(14). We have recently sequenced part of the RPS3/
U15A transcription unit (20). This facilitated chromosomal mapping by allowing us to design an intron-specific PCR assay that would not recognize
pseudogenes derived from processed RPS3 transcripts.
To assign RPS3/U15A to a h u m a n chromosome, we
used the polymerase chain reaction (PCR) to amplify
h u m a n - r o d e n t somatic cell hybrid DNAs with primers corresponding to the second intron of RPS3. This
PCR assay is specific to h u m a n genomic DNA; the
expected 263-bp product is observed with h u m a n but
not mouse or h a m s t e r DNA as template (Fig. 1A).
Results of screening a panel of multichromosome hybrids indicated that the nested RPS3/U15A genes
reside on h u m a n chromosome 11 (Figs. 1A and 1B).
We screened a second panel of monochromosomal
hybrid DNAs to verify this result. As expected, the
only hybrid positive for RPS3/U15A was that which
retained h u m a n chromosome 11 (Fig. 1C).
To confirm and refine this localization, we assayed
cell hybrids retaining portions of h u m a n chromosome 11 and hybridized an RPS3/U15A-containing
YAC clone to h u m a n metaphase chromosomes in
situ. PCR analysis of subchromosomal hybrids (8,
17) (Fig. 2) allowed us to localize RPS3/U15A to
1 l q 1 3 - q 2 3 (Fig. 2). PCR screening of a chromosome
11-specific YAC library (15) enabled us to identify a
single RPS3/U15A-containing clone, YAC yRP9A2.
Fluorescence in situ hybridization analysis localized
yRP9A2 to l lq13 with negligible background elsewhere in the genome (Fig. 3A). Testing of yRP9A2
for the presence of other llq13 loci revealed that it
contains D l l S 5 3 3 and D l l S 3 5 6 (Fig. 3B), previously mapped to l l q 1 3 . 3 - q 1 3 . 4 and 11q13.4q13.5, respectively (18). These results are entirely
consistent with the somatic cell hybrid and in situ
hybridization studies (Fig. 3C). We conclude that
577
GENOMICS 25, 577--580 (1995)
0888-7543/95 $6.00
Copyright © 1995 by Academic Press, Inc.
All rights of reproduction in any form reserved.
A
=Ez=~
, - o = o ~ , ¢ = ~ © h . o o = ° g ° ~ © ~ ' . - = E , - , ,-- . -
-- 263
B
1
bp
g_
~.
4
~_
~.
Z
~.
.9_ 1~! _l .l.g .1~ .l~ 1.~. 1~ .1.7. 1.Q. 1~ g.Q g /
g2
X
Y
# of Concordant Hybrids
+/+
0
49
Total
9
1
8
9
2
5
7
5
6
11
4
6
10
5
5
10
4
5
9
6
5
11
0
11
11
1
5
6
6
5
I1 6
17 11
2
6
8
5
5
10
5
5
10
0
9
9
6
3
9
2
6
8
3
5
7
6
10 11
4
7
11
4
7
11
2
10
12
2
9
11
# of Discordant Hybrids
+1'-/+
Total
4
4
8
3
7
10
1
6
7
2
6
8
1
7
8
1
7
8
0
7
7
6
1
7
4
7
11
0
4
6
10
1
8
9
1
7
8
6
3
9
0
9
9
4
6
10
2
5
7
2
5
7
2
5
7
4
2
6
4
3
7
6
4
10
1
1
5
6
1
6
7
C
-
263bp
FIG, 1. Mapping of RPS3/U15A to human chromosome 11 by PCR analysis of human-rodent somatic cell hybrid DNAs. (A) Eighteen
human-rodent hybrids containing multiple human chromosomes (NIGMS panel 1). "Marker" lane contains ~bX174RF DNA digested with
HaeIII. Thirty PCR cycles (1 min at 94°C, 1 min at 61°C, 1 rain at 72°C) were carried out on 50 ng of genomic DNAs with primers (5'CATGGTCCCACCTATTCC-3' and 5'-GGGGGAAAAGTGACAATTCA-3 ') specific to the second intron of RPS3 (20). Reaction products were
analyzed by agarose gel electrophoresis in the presence of ethidium bromide. (B) Tabulation, by human chromosome, of number of concordant
and discordant hybrids from A. Symbols before slash denote presence (+) or absence ( - ) of 263-bp PCR product, and symbols after slash
denote presence (+) or absence ( - ) of chromosome. (C) Twenty-four hybrids containing single human chromosomes (NIGMS panel 2).
v-
--263
bp
FIG. 2. Mapping of RPS3/U15A to ll q 1 3 - q 2 3 by PCR analysis
of human-rodent somatic cell hybrid DNAs containing portions of
human chromosome 11. PCR was carried out as in Fig. I with genomic DNA from the following hybrids (8, 17) as template: J1 (retaining
an intact human chromosome 11), PBR-3 (retaining a human 11;12
translocation product carrying 11pter-q23.3), PBR-6 (retaining
llq23.3-qter; the reciprocal of PBR-3), TKR-2 (retaining an 11;2
translocation product carrying l l p t e r - q l 3 ) , TKR-33 (retaining
l l q l 3 - q t e r ; the reciprocal ofTKR-2), 15R1A (retaining l l p t e r - q l l ) ,
J l - l l (retaining l l p t e r - q l 2 ) , and EXR-5CSAZ (retaining an X ; l l
translocation product carrying l l q l 3 - q t e r ) .
the R P S 3 / U 1 5 A t r a n s c r i p t i o n unit is located in
l l q 1 3 . 3 - q 1 3 . 5 , near D l l S 5 3 3 and D11S356.
Three o t h e r r i b o s o m a l p r o t e i n genes have been
m a p p e d to h u m a n c h r o m o s o m e 11, b u t no two genes
a p p e a r to be in close proximity: R P S 1 7 m a p s to
l l p l 3 - p t e r (6). R P S 2 5 m a p s to 11q23.3 (9). RPS30,
while m a p p i n g to l l q 1 3 (10), the same b a n d as
RPS3, is not p r e s e n t on YAC y R P 9 A 2 (not shown).
Thus, t h e rule t h a t r i b o s o m a l p r o t e i n genes are
s c a t t e r e d a b o u t the h u m a n genome (3) c o n t i n u e s to
hold.
B a n d 1 l q 1 3 is a f r e q u e n t site of s t r u c t u r a l abnormality, amplification, or loss of h e t e r o z y g o s i t y in
c e r t a i n h u m a n cancers, including m u l t i p l e endocrine n e o p l a s i a t y p e 1, b r e a s t and s q u a m o u s cell
carcinomas, a n d B-cell n e o p l a s m s (11). We do not
k n o w w h e t h e r R P S 3 or U 1 5 A p l a y s a n y role in the
d e v e l o p m e n t of such n e o p l a s m s , b u t we note t h a t
the genes are o v e r e x p r e s s e d in colorectal carcinom a s (14) and t h a t the 3;21 t r a n s l o c a t i o n s o b s e r v e d
579
SHORT COMMUNICATION
C
A
J
B
,•zP
Ii
~I~RPS3/U15A
-500~
- 263 ~o
-183~
FIG. 3. (A) Mapping of RPS3/U15A to 11q13 by fluorescence in situ hybridization (FISH) ofYAC yRP9A2 to human metaphase chromosomes. FISH was performed basically as described (8, 15), using the digoxygenin-labeled"IRE-bubble" PCR product (12) from YAC yRP9A2.
Arrows indicate fluorescent hybridization signals on DAPI-banded chromosomes. (B) Presence of three STSs (DllS533, DllS356, and RPS3/
U15A) in YAC yRP9A2 demonstrated by PCR. Primers for DllS533 and DllS356, as described by Smith et al. (18). (C) Idiogram of
chromosome 11 summarizing localization of RPS3/U15A by subchromosomal hybrid analysis, FISH, and YAC/STS content mapping.
in myelodysplasia disrupt another ribosomal protein gene, RPL22 (13). Given RPS3's role in DNA
repair (22; S. Linn, unpublished observations), it
would be of great interest to know whether any heritable disorders of DNA repair map to l l q 1 3 .
6.
7.
ACKNOWLEDGMENTS
We gratefully acknowledge Joan Steitz's role in helping to initiate the mapping of RPS3/U15A, Roger Eddy's excellent technical
assistance, and Renee Reijo's and Naoya Kenmochi's comments
on the manuscript. This work was supported by the Howard
Hughes Medical Institute and the National Institutes of Health
(CA087775 to D.J.M., HG00333 to T.B.S., and HG0029 to D.E.H.).
R.D.P. was the recipient of a Human Frontier Science Program
postdoctoral fellowship.
8.
9.
REFERENCES
10.
1. Antoine, M., and Fried, M. (1992). The organization of the intron-containing human $6 ribosomal protein (rpS6) gene and
determination of its location at chromosome 9p21. Hum. Mol.
Genet. 1: 565-570.
2. Bommer, U. A., Lutsch, G., Stahl, J., and Bielka, H. (1991).
Eukaryotic initiation factors elF-2 and elF-3: Interactions,
structure and localization in ribosomal initiation complexes.
Biochimie 73: 1007-1009.
3. D'Eustachio, P., Meyuhas, O., Ruddle, F., and Perry, R. P.
(1981). Chromosomal distribution of ribosomal protein genes in
the mouse. Cell 24: 307-312.
4. Davies, B., Feo, S., Heard, E., and Fried, M. (1989). A strategy
to detect and isolate an intron-containinggene in the presence
of multiple processed pseudogenes. Proc. Natl. Acad. Sci. USA
86: 6691-6695.
5. Davies, B., and Fried, M. (1993). The structure of the human
intron-containing$8 ribosomal protein gene and determination
11.
12.
13.
14.
of its chromosomal location at 1p32-p34.1. Genomics 15: 6875.
Feo, S., Davies, B., and Fried, M. (1992). The mapping of seven
intron-containingribosomal protein genes shows they are unlinked in the human genome. Genomics 13: 201-207.
Fisher, E. M., Beer, R. P., Brown, L. G., Ridley, A., McNeil,
J. A., Lawrence, J. B., Willard, H. F., Bieber, F. R., and Page,
D. C. (1990). Homologous ribosomal protein genes on the human
X and Y chromosomes: Escape from X inactivation and possible
implications for Turner syndrome. Cell 63: 1205-1218.
Golden, W. L., von Kap-Herr, C., Kurth, B., Wright, R. M.,
Flickinger, C. J., Eddy, R., Shows, T. B., and Herr, J. C. (1993).
Refinement of the localization of the gene for human intraacrosomal protein SP-10 (ACRV1) to the junction of bands q23q24 of chromosome 11 by in situ hybridization. Genomics 18:
446-449.
Imai, T., Sudo, K., and Miwa, T. (1994). Assignment of the
human ribosomal protein $25 gene (RPS25) to chromosome
1lq23.3 by sequence analysis of the marker D11S456. Genomics
20: 142-143.
Kas, K., Schoenmakers, E., Van de Ven, W., Weber, G., NordenskjSld, M,, Michiels, L., Merregaert, J., and Larsson, C. (1993).
Assignment of the human FAU gene to a subregion of chromosome llq13. Genomics 17: 387-392.
Lammie, G. A., and Peters, G. (1991). Chromosome 11q13 in
human cancer. Cancer Cells 3: 413-420.
Munroe, D. J., Haas, M., Bric, E., Whitton, T., Aburatani, H.,
Hunter, K., Ward, D., and Housman, D. E. (1994). IRE-bubble
PCR: A rapid and efficient method for amplification of human
DNA sequences from complex background. Genomics 19: 506514.
Nucifora, G., Begy, C. R., Erickson, P., Drabkin, H. A., and
Rowley, J. D. (1993). The 3;21 translocation in myelodysplasia
results in a fusion transcript between AML1 gene and the gene
for EAP, a highly conserved protein associated with the Epstein-Barr virus small RNA EBER 1. Proc. Natl. Acad. Sci.
USA 90: 7784-7788.
Pogue-Geile, K., Geiser, J. R., Shu, M., Miller, C., Wool, I. G.,
580
SHORT COMMUNICATION
Meisler, A. I., and Pipas, J. M. (1991). Ribosomal protein genes
are overexpressed in colorectal cancer: Isolation of a cDNA encoding the human $3 ribosomal protein. Mol. Cell. Biol. 11:
3842-3849.
15. Qin, S., Zhang, J., Isaacs, C. M., Hagafuchi, S., Jani Sait,
S. N., Abel, K. J., Higgins, M. J., Nowak, N. J., and Shows,
T. B. (1993). A chromosome 11 YAC library. Genomics 16: 580585.
16. Rhoads, D. D., Dixit, A., and Roufa, D. J. (1986). Primary structure of human ribosomal protein $14 and the gene that encodes
it. Mol. Cell. Biol. 6: 2774-2783.
17.
Sanford, J., Kim, B.-P., Deaven, L. L., Jones, C., Higgins,
M. J., Nowak, N. J., and Shows, T. B. (1993). A human chromosome 11 NotI end clone library. Genomics 15: 653-658.
18.
Smith, M. W., Clark, S. P., Hutchinson, J. S., Wei, Y. H., Churu-
19.
20.
21.
22.
kian, A. C., Daniels, L. B., Diggle, K. L., Gen, M. W., Romo,
A. J., Lin, Y., Selleri, L., McElligott, D. L., and Evans, G. A.
(1993). A sequence-tagged site map of human chromosome 11.
Genomics 17: 699-725.
Sollner-Webb, B. (1993). Novel intron-encoded small nucleolar
RNAs. Cell 75: 403-405.
Tycowski, K. T., Shu, M.-D., and Steitz, J. A. (1993). A small
nucleolar RNA is processed from an intron of the human gene
encoding ribosomal protein $3. Genes Dev. 7: 1176-1190.
Webb, G. C., Baker, R. T., Coggan, M., and Board, P. G. (1994).
Localization of the human UBA52 ubiquitin fusion gene to chromosome band 19p13.1-p12. Genomics 19: 567-569.
Wilson, D. M., III, Deutsch, W. A., and Kelley, M. R. (1993).
Cloning of the Drosophila ribosomal protein $3: Another multifunctional ribosomal protein with AP endonuclease DNA repair
activity. Nucleic Acids Res. 21: 2516.