[CANCER RESEARCH 41, 2215-2220,
0008-5472/81
/0041-OOOOS02.00
June 1981]
Comparison of RNase T, Fingerprints of U1, U2, and U3 Small Nuclear
RNA's of HeLa Cells, Human Normal Fibroblasts, and Novikoff
Hepatoma Cells1
Katsuhiko Nohga, Ramachandra Reddy, and Harris Busch
Department of Pharmacology,
Baylor College of Medicine, Houston, Texas 77030
ABSTRACT
MATERIALS
To determine whether there are differences between the U1,
U2, and U3 small nuclear RNA's of human cancer cells (HeLa
Log-phase cultures of HeLa cells and IMR-90 cells were
grown in Eagle's minimal essential medium supplemented with
5% fetal calf serum and 5% calf serum and in McCoy's medium
cells) and human normal fibroblasts (IMR-90 cells), and be
tween these uridine-rich small nuclear RNA's of human and
AND METHODS
Novikoff hepatoma cells, the cells were first incubated in Ea
gle's medium with [32P]P, to label these RNA's uniformly.
5A supplemented with 10% fetal calf serum and 10% calf
serum, respectively.
Preparation of 32P-labeled 4 to 8S RNA's from HeLa Cells,
No differences were found between the RNase T, fingerprints
of the purified U1, U2, and U3 RNA's of HeLa cells and IMR90 cells. The RNase T! fingerprints of U1 RNA's from human,
IMR-90 Cells, and Novikoff Hepatoma Cells. The harvested
Novikoff hepatoma cells were incubated in Eagle's medium
with 32P¡
as described previously (12). For HeLa cells and IMR-
tissues were very similar to that of the U1 RNA of Novikoff
hepatoma cells. The RNase T, fingerprints of U2 and U3 RNA's
90 cells, the procedure used was similar to that described for
Novikoff hepatoma cells, except that the Eagle's medium was
from human tissues had many similarities to those
hepatoma cells, but a few differences were found,
point mutation of the U-U-Gp in the rat U2 RNA to
-»A) in human U2 RNA. Unlike the three U3 RNA's
supplemented with 20%, rather than 10%, fetal calf serum.
The precipitation of citric acid nuclei and isolation of RNA with
sodium dodecyl sulfate-phénol were carried out as described
previously (22). The 4 to 8S RNA fraction was separated by
sucrose gradient centrifugation (22).
Purification of U1, U2, and U3 RNA's. Nuclear 4 to 8S RNA
of Novikoff
such as a
A-U-Gp (U
of Novikoff
hepatoma cells, U3 RNA from human tissues appears to be
only one species.
These results indicate that U1, U2, and U3 RNA's of human
cancer cells are essentially the same as those of human normal
cells. In addition, the uridine-rich small nuclear RNA's appear
obtained by sucrose gradient centrifugation was subjected to
electrophoresis
on 10% polyacrylamide gels (40 x 20 cm)
containing 7 M urea (5). To separate the U1, U2, and U3 RNA's
from other snRNA species, electrophoresis was carried out
until the 4S RNA migrated to the bottom of the gel. The RNA's
to be conserved through evolution.
INTRODUCTION
The snRNA's,2 which have been purified and sequenced in
this laboratory (21), have been reported recently to be impli
cated in processing of precursor RNA's (10, 13, 23). Lerner ef
were visualized by méthylèneblue staining and/or autoradiography. The U1, U2, and U3 RNA bands were excised, and RNA
was extracted as described by Winter and Brownlee (25). The
purity of these RNA's was sufficient to carry out the RNase T,
fingerprinting. If further purification was required, the RNA's
of human cancer cells and human normal fibroblasts using
RNase T, fingerprinting. In this study, U1, U2, and U3 RNA's
were rerun on 12% acrylamide-7 M urea gels.
Chemicals. RNase T,, RNase U2, and pancreatic RNase
were obtained from Calbiochem, La Jolla, Calif. RNase T2 was
obtained from Sankyo, Tokyo. Nuclease PI was purchased
from Sigma Chemical Co., St. Louis, Mo. The PEI-cellulose
sheets were obtained from Brinkman Instruments, Westbury,
N. Y.
Fingerprinting of RNA Fragments. Complete digestion of
RNA with RNase T, and separation of the resulting oligonucleotides on cellulose acetate were carried out as described by
Brownlee ef a/. (2). The second dimension was carried out by
an improved homochromatography
on PEI-cellulose sheets
using the C-15 homomixture (3).
of Novikoff hepatoma cells were also compared with those from
human cells.
RESULTS
al. (10) suggested that a U1 ribonucleoprotein
particle is in
volved in splicing hnRNA to mature RNA. The possibility has
been suggested that U2, U4, U5, and U6 snRNA's may be also
involved in processing of hnRNA, since they are associated
with hnRNA (6, 7, 9, 14) and are precipitated by the same
antibodies (11 ). U3 RNA is localized to the nucleolus and was
shown to be hydrogen bonded to precursor RNA's (15, 28).
Accordingly, U3 RNA might be involved in cleaving precursor
rRNA's as suggested previously (18).
The present study was carried out in order to determine
whether there are differences between U1, U2, and U3 RNA's
' These studies were supported by Cancer Center Grant CA-10893,
awarded
by the National Cancer Institute, Department of Health, Education, and Welfare;
the Bristol-Myers Foundation; and the Pauline Sterne Wolff Memorial Foundation.
2 The abbreviations used are: U-snRNA, uridine-rich small nuclear RNA;
hnRNA, heterogeneous nuclear RNA.
Received January 5, 1981 ; accepted March 4, 1981.
Fig. 1 shows an autoradiograph of the gel obtained with 32Plabeled snRNA's from HeLa cells and IMR-90 cells. It has been
reported already that snRNA's of Novikoff hepatoma cells
separate well under the conditions used (16, 17). snRNA's of
HeLa cells and IMR-90 cells also separated
well under the
JUNE 1981
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2215
K. Nohga et al.
cleotides. The RNase T, fingerprints
of U1 RNA's from HeLa
cells and IMR-90 cells were very similar to that of Novikoff
hepatoma cells except that the mobility of Fragment 17 was
clearly different.
RNase T, Catalog of U2 RNA. Fig. 3 shows the autoradiograph of uniformly labeled U2 RNA digested with RNase T,
and fractionated by electrophoresis and homochromatography.
The RNase Ti fingerprint of U2 RNA from HeLa cells was the
same as that of IMR-90 cells. When compared to the RNase T,
fingerprint of U2 RNA of Novikoff hepatoma cells, the RNase
Ti fingerprints of U2 RNA's from human cells had many simi
-ss
- ss
U3
—U3
larities. However, Fragment 7 of the Novikoff U2 RNA was
absent from U2 RNA's of the human cells. Fragment 6' was
-
U2
-
5 SS
found in the human cells but not in Novikoff hepatoma cells.
When digested further with RNase T2 and separated by the
Wyatt system (27), Fragment 6' yielded Ap, Up, Gp; it yielded
Ap, Up-Gp after digestion with RNase U2. These data show the
sequence of Fragment 6' in human U2 RNA is A-U-Gp. Thus,
there is a point mutation of the U-U-Gp (T-7) in the rat (24) to
A-U-Gp (T-6') (U -»A) in the human.
U2
—Ul
5.8S
DI
-
RNase T^ Catalog of U3 RNA. As shown previously (17, 18),
complete digestion of U3A and U3B RNA's of Novikoff hepa
U4
—U4
toma cells with RNase T, produced 27 fragments and 25
fragments, respectively, that were separated by electrophore
sis and homochromatography
(Fig. 4). The RNase Ti finger
prints of U3 RNA of HeLa cells and IMR-90 cells yielded 26
fragments (Fig. 4).
There was no difference between RNase T, fingerprints of
U3 RNA's from HeLa cells and IMR-90 cells. The RNase T,
SS
—SS
-US
US
fingerprint for U3 RNA from humans was similar to those of
U3A and U3B RNA's of Novikoff hepatoma cells, but somewhat
-U6
-
U6
different. Fragments B, C, E, F, G, and H in U3A RNA and
Fragments 17, 18, 22, and 25 in U3B RNA of Novikoff hepa
toma cells were absent from human U3 RNA. Fragments 17,
24', C, and H' in human U3 RNA were not found in Novikoff
hepatoma cells. There was only one detectable U3 RNA spe
cies in human cells which differs in some respects from both
rat U3A and U3B RNA's. Chart 1 shows the sequence of U3A
- 4S
RNA of Novikoff hepatoma cells (18); the underlined areas
show the few oligonucleotides which differ from those of human
U3 RNA.
-45
HeLa
IMR-90
1
DISCUSSION
Fig. 1. Electrophoretic pattern of 4 to 8S nuclear RNA of HeLa cells and IMR90 cells on 10% polyacrylamide gel. The RNA was visualized by autoradiography.
Conditions for electrophoresis are described under "Materials and Methods."
same conditions. Although U3 RNA of Novikoff hepatoma cells
separated into 3 distinct bands as shown previously (17), the
U3 RNA of HeLa cells and IMR-90 cells migrated as a single
band as shown in Fig. 1.
RNase TI Catalog of U1 RNA. Complete digestion of Novi
koff hepatoma U1 RNA with RNase T, and separation of these
fragments by electrophoresis and homochromatography
pro
duced 22 fragments (Fig. 2) as described previously (19).
Complete RNase T, digestion of the U1 RNA from HeLa cells
and IMR-90 cells also yielded 22 fragments as shown in Fig. 2.
The RNase TI fingerprint of U1 RNA from HeLa cells was the
same as that of IMR-90 cells. The mobility of 3'-terminal Frag
ment 17 of HeLa cells was more variable than other oligonu-
2216
The RNase T, fingerprints
of U1, U2, and U3 RNA's from
HeLa cells were identical with those of human fibroblasts (IMR90 cells). This result indicates that the nucleotide sequences
of U1, U2, and U3 RNA's in human cancer cells are essentially
the same as those of normal human tissue.
The RNase T! fingerprint of U1 RNA from humans was very
similar to U1 RNA from Novikoff hepatoma cells except that
the mobilities of the 3'-terminal Fragment 17 were different.
Branlant ef al. (1) have shown recently that only 2 base sub
stitutions were observed from the rat to the human in nucleotide
sequences of U1A RNA's. One base substitution C—»U
was
observed at position 159 near the 3'-terminus. The difference
in the mobilities of Fragment 17 in U1 RNA's may result from
the base substitution at position 159. Earlier studies of Lerner
and Steitz (11 ) showed that the RNase T, fingerprint of mouse
U1 RNA was similar to that of the rat (19).
CANCER
RESEARCH
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VOL.
41
Comparison
, , ,
10
20
30
40
U3A
«3
U3A
CUG
U3A
IZO
130
140
150
G C C G G C U C C U A G U G U U G C U U C C U G CCAUUGCUAUUGGCAACUGAUGAUCGUCUUCGG
U3A
180
190
U C U U C AGGGGUGAGAGGGAGAGAArC
Chart 1. Comparison
of R/Vase T, Fingerprints
50
GpppABAGUGACIJA'iACUiUCAGGGAUCAUUUCUAUAGUUCGUUACUAGAGAAGUUUru
60
70
A C U G UGUAGAGCACCCGAAACCACGAGGAGGAG
of the U3A sequence
80
90
200
CAO
210
UCUGAGUGG
100
A C C GAGCGUUCUCUCCUGAGCGUGAA
160
110
170
UCT
216
U
of Novikoff hepatoma cells (18) with the U3 RNA of HeLa cells. The 32P-labeled U3 RNA from HeLa cells was
fingerprinted after digestion with RNase T, (Fig. 4). The oligonucleotides were subjected to pancreatic RNase or RNase U2. or to RNase T2 digestion, and the
resulting products were analyzed. The underlined oligonucleotides of the U3A RNA sequence of Novikoff hepatoma cells represent those few oligonucleotides which
differ from those in HeLa cells.
The RNase T! fingerprints
of U2 RNA's from HeLa cells and
the IMR-90 cells were also similar to that of Novikoff hepatoma
cells. Lerner and Steitz (11 ) have shown also that U2 RNA of
the mouse has a fingerprint similar to that of the rat (24).
However, Fragment 6' A-U-Gp was found in U2 RNA from
human cells instead of Fragment 7 U-U-Gp. Fragment 16 (A-AC-U-A-Gp) was found in lower yield in HeLa cell and IMR-90
U2 RNA compared to Novikoff hepatoma. The reason for this
is not clear. One possibility is that there is heterogeneity in U2
RNA resulting in submolar yields of Spot 16; however, no other
evidence is available to suggest heterogeneity of U2 RNA's.
The RNase T, fingerprints of U3 RNA from HeLa cells and
IMR-90 cells were somewhat different from those of U3A and
U3B RNA's for Novikoff hepatoma cells. It has been shown
(17) that there are 3 U3 RNA species in Novikoff hepatoma cell
nucleoli. However, the results obtained from RNase T, finger
prints of U3 RNA's of HeLa cells and IMR-90 cells indicate that
human U3 RNA is mainly one species. In addition to studies on
the mammalian U3 RNA, Wise and Weiner (26) noted recently
that there are large regions of homology of the D2 RNA of
Dictyostelium and the Novikoff hepatoma U3B RNA (17) in
which over 40% of the sequences were 75% or more homolo
gous. These results strongly support earlier suggestions that
U1, U2, and U3 RNA's are conserved through evolution (8).
However, there appears to be less conservation of the U3 RNA
sequence inasmuch as there were more differences between
the oligonucleotides of the U3 RNA of the HeLa and Novikoff
species.
Ringuette et al. (20) have found that one snRNA purified
from the chromatin of SV40-transformed WI-38 human fibroblasts stimulated the transcription of chromatin in homologous
isolated nuclei as well as in nuclei of untransformed human and
monkey cells. It seems possible that some RNA's may play
some role in differentiation or development (4, 20), but their
precise function requires more definitive studies.
ACKNOWLEDGMENTS
We wish to express our appreciation to William H. Spohn for his helpful advice.
We also wish to express our thanks to Dr. Wen-Yu Li for the supply of radioactive
Novikoff nuclear U1. U2, and U3 RNA's.
REFERENCES
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1981
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2217
K. Nohga et al.
IMR-90
Ul Ti
C.A.-
HeLa
Ul Ti
21
C. A.-
22
22
b
19
19
Ji
13
18 20
13
14
18
15
15
12
12
10
17
11
S
10 17
7
698
5
2
11
3
7
<* «r
*
2A
Novikoff
Ul Ti
CA.-
2B
HeLa
U2Ti
C A26
24
22
A
19*
16
13
25
14
22
•
A
19
21
4
20
*
12
15
27
16
17
10
11
<*
17
5
7
15
698
6.6
3
2
2C
Fig. 2. Autoradiograph
of a 2-dimensional
3A
separation of a complete RNase T, digest of 32P-labeled U1 RNA's from HeLa cells W), IMR-90 cells (B), and Novikoff
hepatoma cells (C). Electrophoresis was carried out in the first dimension on cellulose acetate (C. A.), pH 3.5 (5% acetic acid-0.01 % pyridine-7 M urea). The second
dimension was homochromatography
(H. C.) on PEI-cellulose sheets.
Fig. 3. Autoradiograph of a 2-dimensional separation of a complete RNase T, digest of 32P-labeled U2 RNA's from HeLa cells 04), IMR-90 cells (6). and Novikoff
hepatoma cells (C). Electrophoresis and homochromatography
were performed as described in the legend to Fig. 2. Fragments 10 and 11. and Fragments 12 to 14
comigrated on PEI-cellulose sheets. The identity of these fragments was determined by digestion of these fragments with RNase U2 or pancreatic RNase. C. A.,
cellulose acetate; H. C., homochromatography.
2218
CANCER
RESEARCH
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VOL. 41
Comparison
IMR-90
U2Ti
I
25
7"i Fingerprints
C./L-
Novikoff
U2 Ti
21
22
of RNase
26
19
20
27
17
16
13. H». 11.12
16
18
27
IS
17
18
3
13.14jO.ll42
IS
2
3B
3C
JUNE 1981
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2219
K. Nohga et al.
C.A.-
HeLa
U3 Ti
IMR-90
U3 Ti
23
24
24
I
24
C
C. A.-
23
20
H
20
24
19
* ** m*
UUTCUAGp
17
"
^^^
CCAÃœUCp
»
^
^»
12
21
15
C
19
IO
^^
CAt
CACCCGp
ÄAACGp
13
11
5
11
12
A*Cp
UUGp
567
CCGp
2
(UA)Gp
10
17
7
6
15
13
8 . 9
A
3
89
(CU)Gp
AGp
CGp
l
UGn
4A
Novikoff
U3A Ti
C.A..
23
24
4B
Novikoff
U3B
C.A.-
Ti
25
24
23
22
20
20
15
19 E F
21
b
AAACCACG,,
15
1918
21
Ib
13
C.B
17
10
10
11
11
567
12
567
12
8
2
9
3
2
3
4D
Fig. 4. Autoradiograph
of a 2-dimensional
hepatoma cells (C, D). Electrophoresis
2220
separation of a complete RNase T, digest of "P-labeled
and homochromatography
U3 RNA's from HeLa cells (A), IMR-90 cells (B), and Novikoff
(H. C.) were performed as described in the legend to Fig. 2. C. A., cellulose acetate.
CANCER
RESEARCH
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VOL.
41
Comparison of RNase T1 Fingerprints of U1, U2, and U3 Small
Nuclear RNA's of HeLa Cells, Human Normal Fibroblasts, and
Novikoff Hepatoma Cells
Katsuhiko Nohga, Ramachandra Reddy and Harris Busch
Cancer Res 1981;41:2215-2220.
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