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Biosynthesis of low molecular weight RNA in mouse myeloma cells
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Biochimica et Biophysica Acta, 521 (1978) 662--676 © Elsevier/North-Holland Biomedical Press
BBA 99319
BIOSYNTHESIS OF LOW MOLECULAR WEIGHT RNA IN MOUSE MYELOMA CELLS
ANNE BROWN and WILLIAM MARZLUFF
Department of Chemistry, Florida State University, Tallahassee, Fla. 32306 (U.S.A.) (Received March 17th, 1978)
Summary The low molecular weight RNAs in the nucleus and cytoplasm of mouse myeloma cells have been characterized. There are major nuclear species (other than 5-S and tRNA), four of which contain 'capped' 5' termini. There are three major small cytoplasmic RNAs, none of which contain 'caps'. The biosynthesis of the nuclear species when studied using [3H]adenosine as an RNA precursor is characterized by a rapid, transient appearance in the cytoplasm, followed by fixation in the nucleus. Within 15 min, the amount in the cytoplasm has reached a steady-state level maintained for 60 rain, while the accumulation in nuclei is linear after a short lag (less than 5 min). When biosynthesis is studied using [Me-~H]methionine as a precursor, much less labeled RNA is present in the cytoplasm, suggesting that methylation may immediately precede fixation into the nucleus.
Introduction
Several species of low molecular weight RNA have been reported to occur in eucaryotic cells. A diversity of cell types have been studied including Tetrahymena pyriformis [1], sea urchins [2] and several mammalian lines including HeLa cells [3] and Novikoff hepatoma cells [4,5]. These species, characterized primarily by gel electrophoresis, exhibit pattern similarity which appears to be correlated with the evolutionary relationships of the organisms. Thus, although Tetrahymena contains small molecular weight RNA species, their electrophoretic mobilities differ from the mobilities which are characteristic of mammalian small molecular weight species. Several small cytoplasrriic RNA species in addition to transfer RNA and ribosomal 5-S RNA, have been reported [6,7]. The most intensive characterizations of small RNA species, however, have been performed with those found in nuclei. Nearly identical mobility patterns on polyacrylamide gels have been
663 demonstrated for small nuclear species from HeLa cells, rat liver, Ehrlich acites cells, BHK cells and human l y m p h o c y t e nuclei [3,8--11]. Several species are methylated and show similar methylation patterns in mammalian cells [5,9,11,12]. The small molecular weight nuclear RNA species in HeLa cells [13], Novikoff hepatoma cell lines [4], and mouse m y e l o m a cells [14] have been analyzed in detail and show several interesting properties: they are extremely stable, with turnover rates comparable to transfer and ribosomal RNA; several species are found in very large numbers in the nucleus including two species estimated to be present in 2 • l 0 s and 2 • 106 molecules/cell [12,14]. Hybridization studies show that 100--200 complementary DNA sites exist for these species [19]. Three species are reported to contain 'capped' 5' termini similar to, b u t not identical to those found in viral and animal messenger RNA [15--17]. During mitosis, the nuclear species dissociate from the nucleus and are again associated with it when the membrane reforms [18,19]. No function has been discovered for this ubiquitous class of RNA molecules. We have examined the low molecular weight species in mouse m y e l o m a nuclei and cytoplasm. The species in each c o m p a r t m e n t are distinct. We have found species similar to those reported to occur in other mammalian cells. Several of these are methylated. Four nuclear species contain caps, three similar to those found in Novikoff hepatoma nuclear species, and there is in addition a capped, methylated species not previously reported. We have studied the metabolism of the RNAs and the data from the in vivo pulse labels indicate rapid synthesis (less than 5 min), and suggest a transient cytoplasmic appearance, followed b y fixation into the nuclear fraction. [Me-3H]RNA has a much shorter lifetime in the cytoplasm, suggesting that a methylation may be related to fixation of RNA into the nucleus. Materials and Methods
Labeling of cells. Mouse m y e l o m a 66-2 cells were used in all experiments and grown as previously described [20]. To label the RNA with ~2PO4, the cells were grown to a concentration of 5 - - 6 . 1 0 S / m l , pelleted gently, and resuspended in low phosphate medium. 32PO4 was added as H3PO4 to a final concentration of 80--100 uCi/ml. In most experiments, 120 ml cells were used. Cells were diluted with phosphate-free medium (0.5 vol.) after 12 h of labeling. After 24 h the cells were collected and the R N A was extracted. The cells normally double every 16 h but in phosphate-free medium the rate is decreased and only one doubling occurs. Preparation of RNA. All procedures were at 4°C except where noted. The cells were collected b y centrifugation and washed twice with cold 0.14 M KC1/0.01 M Tris, pH 7.5. They were then suspended in buffer A (0.32 M sucrose/2 mM Mg2+/3 mM Ca2*/10 mM Tris, pH 8/0.1% Triton X-100) at 2 • 10 T cells/ml and broken by homogenization in a Dounce homogenizer (20 strokes, B pestle). The nuclei were pelleted b y centrifugation at 1000 × g for 10 min. The nuclei were suspended in buffer B (0.88 M sucrose/3 mM Ca2+/ 2 mM Mg2÷/10 mM Tris/0.1% Triton X-100) and homogenized as above with 5 strokes. The nuclei were pelleted b y centrifugation (1000 ×g, 10 min).
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(1) Cytoplasm: To the combined supernatant fractions were added 0.05 vol. 10% sodium dodecyl sulphate (SDS) 0.05 vol. 2 M NaOAc, pH 5, 0.67 vol. 80% phenol, and 0.33 vol. CHCI3. The mixture was shaken for approximately 30 min at 25°C, and the RNA precipitated from the aqueous layer with 2.5 vol. 95% ethanol. The R N A was collected by centrifugation, redissolved in 0.5 ml 1% SDS-10 mM EDTA, layered over a 5--30% sucrose gradient (0.1% SDS, 0.1 M NaC1, 0.01 M EDTA, 10 mM Tris, pH 7.5) and centrifuged in the SW 27 rotor (Sorvall OTD-2) for 15 h, 25 000 rev./min, 25°C. The 4--8-S region was collected and the RNA precipitated with 95% ethanol. (2) Nuclei: The nuclear pellet was suspended in buffer A (5 ml for 100 ml cells) and to this was added an equal volume of 1% SDS, 10 mM EDTA, 0.20 vol. 2 M NaOAc, pH 5, 1.5 vols. 80% phenol, and 0.5 vols. CHC13. The mixture was incubated for 5--10 min at 55°C and then cooled on ice. The aqueous layer was removed and precipitated with 2.5 vols. 95% ethanol and the RNA was separated on gels as described below. Nuclear RNA for some experiments (indicated in Results) was prepared by first extracting the nuclear pellet with 0.35 M NaCI, 5 mM Mg 2+, 10 mM Tris, pH 8, centrifuging at 10 000 × g for 10 minutes to pellet the chromatin, and then separating the pellet from the supernatant. The RNA remaining with chromatin was removed by 55°C SDS-phenol treatment as above. The supernatant was layered over a 1 M sucrose pad (6 ml s u p e r n a t a n t / 2 . 5 ml sucrose) and centrifuged for 8 h at 35 K in the 65 rotor in a Beckman ultracentrifuge to remove ribosomal subunits and precursors. The RNA in the supernatant and in the pellet was then recovered b y SDS-phenol precedure described above. Separation and purification of low molecular weight RNA species. Cytoplasmic RNA from the 4--8-S region of the sucrose gradient was dissolved in gel buffer (20% glycerol/0.1% SDS/0.1 M EDTA/10 mM Tris, pH 7.5) and electrophoresed on 10% polyacrylamide gels at 80 V (13--20 MA) until the dye marker (bromphenol blue) reached the edge of the gel (20 cm). Nuclear R N A was electrophoresed as above or alternatively dissolved in 99% formamide and applied to 12% polyacrylamide-formamide gels [14]. The RNA bands were located b y autoradiography. The bands were cut from the gel, broken up, and placed in a plastic pipette with filter paper plugging the tip. The R N A was recovered b y electrophoresing it though the filter paper into dialysis bags attached to the pipette tips. This was done at 115 V and the elution was complete by 9--12 h as monitored with a Geiger counter. 28-S r R N A was added as carrier (0.5 A26onm) and the R N A was recovered by ethanol precipitation. Gel fluorography. Gels were soaked in 10% acetic acid/25% isopropanol overnight and processed for fluorography as described by Laskey and Mills [21] for 3H and 14C samples. The dried gel was placed against X-ray film (Kodak RPR-54 pre-exposed to Ass0nm of 0.15) at --70°C for an appropriate length of time. Digestion of RNA. The R N A was dissolved in 25 ~I 10 m M NaOAc/10 -4 M ZnOAc, p H 5.8. To this was added 2.5 pl P1 nuclease (Sankyo), (10.0 mg/ml in 100 m M NaOAc, p H 5.8). The digest was incubated in a closed capillary tube overnight. The digest was brought to p H 7 with approximately 2/zl 1 M Tris, p H 8.0, and 2/~I alkaline phosphatase (1 mg/ml) was added. The digest was incubated for 2--5 h in a capillary tube at 37°C. This procedure yields inorganic
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phosphate as the only radioactive product when 32po4-4-S tRNA is used. Analysis of 5'-termini. The RNA species were eluted from gels and digested with RNAase P1 and alkaline phosphatase as described above. The digest was spotted on a cellulose thin-layer plate or a DEAE-cellulose thin-layer plate and electrophoresed on a Shandon Flat-bed apparatus in 0.5% pyridine/5% acetic acid, pH 3.5, at 350--400 V until the inorganic phosphate was near the plate edge (monitored with a Geiger counter). The digest was run with dye markers [15]. The radioactivity on the plates was located by autoradiography and was eluted with either water (cellulose plates) or 2 M triethylammonium bicarbonate [22] when DEAE-cellulose plates were used. The sample was lyophilized and the residue was resuspended in a small a m o u n t of water and divided into appropriate aliquots depending upon the digestion procedures to be performed. Usually three aliquots were processed. (1) Nucleotide pyrophosphatase: the solution (30 p]) was adjusted with 2 ~l 1 M Tris, pH 8, to pH 7.5 and 5/A enzyme (0.32 mg/ml) was added. The digestion was incubated for 1 h at 37°C. (2) Alkaline phosphatase: after 1 h, 2 p] alkaline phosphatase was added to the above digest. (3) Redigestion with RNAase P1 and alkaline phosphatase: The solution (50 ~l) was made 10 mM NaOAc, pH 5.8, and 2 ~l RNAase P1 was added for 3--5 h at 37°C. The pH was brought to 7.5 with 1 M Tris, pH 8, and 2 ~l alkaline phosphatase was added and the mixture incubated for 2--3 h at 37°C. All digests were spotted on thin-layer cellulose plates and electrophoresed on a flat-bed apparatus as described above. RNA-DNA hybridization. Hybridization of RNA species N7 and C1 to m y e l o m a DNA was performed as described previously [14]. Incubations were performed using either C1 and N7 or both species simultaneously at a concentration of 0.04 pg/ml (of each RNA) in 0.9 M NaC1, 0.09 M Na3 citrate, 50% formamide at 55°C. Pulse labelling of RNA with [3H]adenosine. Cells were grown to a concentration of 5--6 • 10S/ml pelleted and resuspended in the same medium at a concentration of 4 . 106/ml. They were incubated for 1 h and then [2-3H]adenosine was added at a concentration of 50 ~Ci/ml. Aliquots were removed at the appropriate times, cells chilled by pipetting into 3 vols. ice-cold 0.14 M KC1, and the RNA extracted as described. For the pulse-label experiments with an actinomycin D chase, cells were treated as above and were incubated with [3H]adenosine for 15 min. At this time, actinomycin D was added to a final concentration of 20 pg/ml, and aliquots were removed at the appropriate times. The incorporation of [3H]adenosine into acid-isoluble material was stopped within 2 min. Pulse-labelling of RNA with [14C]methyl-methionine. 500 ml of cells were grown to a concentration of 5 . 1 0 S / m l . They were pelleted, washed once in methionine-free Dulbecco's modified Eagle's medium and resuspended in 60 ml of the same medium containing 20 wM adenosine, 20 p_M guanosine and 20 mM sodium formate. The cells were allowed 1 h to recover and then 50 ~Ci [Me-14C]methionine was added. Aliquots were removed at the appropriate times and the nuclear and cytoplasmic RNA were extracted as described for [ 3H]adenosine labelled cells.
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Materials. RNAase P1 was obtained from Yamasa Shoyu Co. Tokyo, Japan. Nucleotide pyrophosphatase and alkaline phosphatase were from Sigma Chemical Co. Radioactive isotopes were obtained from Schwartz-Mann. Results
Analysis of nuclear low molecular weight RNA components: long term label 32p. There are seven major low molecular weight RNA species in the nucleus, six of which can be resolved on 10% polyacrylamide gels. These are shown in Fig. 1; the species shown were obtained by SDS-phenol deproteinization of unfractionated, detergent-washed nuclei. These are numbered N 1--7 in order of decreasing mobility. In addition to these, three minor bands, N8--N10, consistently appear after long term labelling of whole cells. The nomenclature for these RNAs is different from that which we used in our earlier work [14]. Here we use the following: nuclear RNAs are N1--N10 with N1 being the smallest (4 S) RNA. This will facilitate nomenclature as further studies are carried out on the larger RNAs. Cytoplasmic species are denoted C1--C3 with C1 being the smallest RNA larger than the wellcharacterized 4-S tRNA, 5-S rRNA and 5.8-S rRNA. Our species N5, N6, N7 presumably correspond to the U~, U2, U3 of Busch and co-workers [5] and to D, C, and A of Penman and co-workers [13]. The results obtained by this method of RNA preparation differ from those
NiO N9 Nil N7 N6 N5 N3, N4(O~ N2 (4.5S) NI (48)
Fig. 1. L o w m o l e c u l a r w e i g h t n u c l e a r R N A s . Left: R N A w a s p r e p a r e d f r o m purified n u c l e i b y t h e h o t p h e n o l p r o c e d u r e a n d s e p a r a t e d o n - 1 0 % p o l y a c r y l a m i d e gel. B a n d N1 is c o n t a m i n a t i n g t R N A . N 3 and N 4 (5-S R N A ) m i g r a t e as a single c o m p o n e n t w h i c h m a y be r e s o l v e d o n f o r m a m i d e - p o l y a c r y l a m i d e gels ( r i g h t ) o r b y e x t r a c t i o n o f n u c l e i w i t h 0 . 3 5 M salt. R i g h t : I n d i v i d u a l R N A s p e c i e s w e r e e l e c t r o p h o r e s e d o n a 12% p o l y a c r y t a m i d e - 7 0 % f o t - m a m i d e slab g e l L e f t to right: c y t o p l a s m i c 5 S, c y t o p l a s m i c 4 S. a mixt u r e o f N 6 a n d N7, N5, N 4 ( p r e p a r e d a f t e r e x t r a c t i o n w i t h 0 . 3 5 M KCI [ 1 4 ] ) a m i x t u r e of N3 a n d N 4 ( e l u t e d f r o m gel at l e f t ) .
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obtained when nuclei are extracted with low salt (0.35 M NaC1) in t w o important ways. First, the relative mobility of N6 is altered. It now runs closer to N5 than to N7 while, if it is extracted by salt without heating, it migrates closes to N7 [14]. Second, the 5-S rRNA is not resolved from species N3. It may be resolved b y extraction in 0.35 M NaC1 [14] or by electrophoresis in the presence of formamide (Fig. 1). Fig. 1 demonstrates that all of the RNAs may be resolved b y formamide-polyacrylamide gel electrophoresis. The 5-S RNA (N4) is not part of nascent ribosomal subunits as it remains in the supernatant fraction of a 100 000 × g centrifugation after extraction of nuclei in 0.35 M NaC1/5 mM Mg 2÷, a treatment which will n o t release 5-S rRNA from ribosomes (data not shown). Previously we have d o c u m e n t e d the sequence similarity of this RNA and 5-S rRNA [14].
Analysis of cytoplasmic low molecular weight R N A components: long term label 32p. The major low molecular weigth RNA species of the cytoplasm are 5-S ribosomal RNA and 4-S transfer RNA. The RNAs recovered from the cytoplasm following SDS-phenol deprotenization at 25°C are shown in Fig. 2. The most prominent species corresponds to transfer RNA. In addition to these, there are three lighter bands, C1, C2 and C3 in order of decreasing mobility, which are consistently present in the cytoplasm under long term label conditions. The C1 band has an electrophoretic mobility in 10% aqueous gels which corresponds closely to that of the nuclear species N7. In order to determine whether the C1 and N7 species are identical species, purified C1 and N7 R N A species were hybridized to mouse m y e l o m a DNA immobilized on nitrocellulose filters. The species were hybridized both separately and together. Fig. 3 shows the results of the hybridization reaction. The reaction in which both species are hybridized shows an additive effect equal to the sums of each c o m p o n e n t individually. We conclude that each species is a unique component, confined to a specific cellular compartment. This is verified by the differences in 5' termini (see below). Analysis of 5' termini of low molecular weight RNA species. Busch and co-workers have reported [15,16] that two species of low molecular weight nuclear R N A from rat hepatoma cell nuclei contain modified 5' termini similar to those subsequently found on eucaryotic messenger RNA species. Capped 5' termini have been reported to be present in nuclear RNA species U1 and U2. Both U1 and U2 RNAs have been sequenced. The 'caps' in these species were originally reported to contain a diphosphate bridge, b u t have now been shown to have a triphosphate bridge [23,24], a trimethyl guanosine linked to 2'-0methyl adenosine. The nuclear R N A species which were obtained from m y e l o m a nuclei were examined b y enzymatic analysis to ascertain whether a cap structure is found in any of the species. The R N A species which were examined were eluted from gels and purity monitored b y gel electrophoresis in formamide-polyacrylamide gels. They were digested with P1 nuclease; this enzyme does not form a 2'3' cyclic phosphate intermediate and can, therefore, hydrolyze the phosphodiester bond adjacent to a 2' ribosome-methylated nucleoside. Digestion of capped nuclear species should, therefore, result in 5' mononucleotides and the structure 22VmGpppXm in which the phosphate groups are labelled. The addi-
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(--)
3000 ~ , ~ . , ~
N7 + Cl
C3 C2 Cl
2000
CI ~E
o. r,3
5S lOCK; N7
4S
(+) 2
6 t(h)
Fig. 2. L o w m o l e c u l a r w e i g h t c y t o p l a s m i c R N A s . [ 3 2 p ] R N A was p r e p a r e d f r o m t h e c y t o p l a s m a n d analy z e d on 10% p o l y a c r y l a m i d e gels. B a n d s C ] , C2 and C3 are in o r d e r o f d e c r e a s i n g m o b i l i t y . Fig. 3. H y b r i d i z a t i o n of R N A species N7 a n d C1. R N A s w e r e e l e c t r o p h o r e s e d on 10% p o l y a c r y l a m i d e gels, e l u t e d , a n d r e - r u n o n 12% gels. T h e R N A s e l u t e d f r o m t h e 12% gels w e r e h y b r i d i z e d t o m o u s e D N A o n n i t r o c e l l u l o s e filters. Filters w e r e r e m o v e d at i n d i c a t e d t i m e s and w a s h e d as d e s c r i b e d u n d e r Materials a n d M e t h o d s . Specific a c t i v i t y w a s 5 • 105 cpm//Jg. R N A c o n c e n t r a t i o n s w e r e 0 . 0 4 ~ g / m l o f e a c h species.
tion of alkaline phosphatase to the products will result in labelled 'caps', unlabelled nucleosides, and labeled inorganic phosphate. The labelled products thus obtained can be readily separated from each other. Fig. 4 shows autoradiograms of cellulose thin-layer electropherograms of 32P-labelled RNA species which were digested with P1 nuclease and alkaline phosphatase. By this rapid procedure, the majority of the label is found as inorganic phosphate. However, in the nuclear RNAs (N3--N7) there was resistant material not present in tRNA and 5-S RNA (Fig. 4B). Often there were two spots, one of which (A) was also found in parallel digests of tRNA and 5-S RNA (Fig. 4A). We eluted each of the spots, A and B, running just behind the blue marker. The material was eluted from the cellulose thin-layer and was subjected to further enzymatic analysis. Redigestion with P1 nuclease and alkaline phosphatase converted the material in A (all lanes) to material electrophoresing with inorganic phosphate (Fig. 5A) indicating that this was a result of incomplete digestion. The material in B, lanes 2 - 4 , was resistant to further digestion by P1 nuclease and alkaline phosphatase. A and B material, eluted from the cellulose plate was digested with nucleotide pyrophosphatase. The results are shown in Fig. 5B. The A material remains in the original position upon cellulose thin-layer electrophoresis. B material was
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Fig. 4. T h i n - l a y e r e l e c t r o p h o r e t o g r a m o f R N A s p e c i e s d i g e s t e d w i t h P1 n u c l e a s e and alkaline p h o s p h a t a s e . [ 3 2 p ] R N A s p e c i e s w e r e s e p a r a t e d o n 10% p o l y a c r y l a m i d e gels and i n d i v i d u a l R N A s p e c i e s w e r e e l u t e d f r o m t h e gels, p r e c i p i t a t e d and r e s u s p e n d e d in a s m a l l v o l u m e o f buffer. T h e R N A w a s d i g e s t e d a n d the d i g e s t a p p l i e d to t h i n - l a y e r c e l l u l o s e s h e e t s . T h e s h e e t s w e r e e l e c t r o p h o r e s e d at p H 3 . 5 , d r i e d , a n d a u t o r a d i o g r a p h e d . A and B d e n o t e the first and s e c o n d r o w s o f resistant m a t e r i a l r u n n i n g b e h i n d t h e m a r k e r , (circles d e n o t e t h e p o s i t i o n s o f m a r k e r dyes). T h e s l o w e s t m o v i n g is the x y l e n e c y a n o l ( b l u e ) d y e . T h e s a m p l e s o n the right w e r e m o r e e x t e n s i v e l y d i g e s t e d , b u t t h e t y p i c a l result is the o n e o n the left.
sensitive to the enzyme yielding identical patterns for each RNA. In contrast, when B material was digested with nucleotide pyrophosphatase, P1 nuclease, and alkaline phosphatase, the material was completely converted to inorganic phosphate (Fig. 5C). The properties obtained above for line B material would be expected for capped structures. Such results have consistently been obtained for nuclear species N3, N5, N6 and N7. 1--2% of the 32PO4 was present in the 5' termini consistent with a unique 5' terminus for each molecule. While small amounts of caps were found in some preparations of N4 (5-S rRNA) (Figs. 4 and 5), this was almost certainly due to a small contamination by species N3, as the a m o u n t was only 0.1--0.2% of the total 32PO4. The products of nucleotide pyrophosphatase digestion have been tentatively identified as residual undigested material (which is completely sensitive on further digestion (Fig. 5C), PAre and ppAm. The modified guanosine monophosphate migrates in the other direction in this system and is in the buffer. This is consistent with a triphosphate linkage in the cap. Digestions with ribonuclease T2 followed by DEAE-cellulose chromatography has shown that each species (N3, N5, N6, N7) contain 2--3% of their radioactivity which elutes with a charge o f - - 6 , consistent with this cap structure {data not shown). N3 has not been previously reported to contain 'capped' ends. This type of assay was also applied to the low molecular weight RNA species C1--C3. The results of enzymatic analysis were similar to those seen for cytoplasmic 4-S and 5-S RNA which do not contain capped structures (Fig. 6), a further indication that C1 and N7 are different species. The material which was insensitive to P1 digestion was completely converted to inorganic phosphate on redigestion with P1 and alkaline phosphatase (not shown).
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Fig. 5 ( A ) A a n d B m a t e r i a l (see Fig. 4) w e r e e l u t e d f r o m t h e t h i n - l a y e r p l a t e a n d r e - d i g e s t e d w i t h P1 n u c l e a s e a n d a l k a l i n e p h o s p h a t a s e . 5A is a t h i n - l a y e r e l e c t r o p h o r e t o g r a m o f [ 3 2 p ] R N A d i g e s t i o n p r o d u c t s r e e x p o s e d to P1 n u c l e a s e a n d a l k a l i n e p h o s p h a t a s e . T h e o p e n circles i n d i c a t e t h e p o s i t i o n s o f t h e m a r k e r d y e s . T h e d i g e s t s w e r e a p p l i e d to cellulose t h i n - l a y e r s h e e t s a n d t h e s h e e t s w e r e e l e c t r o p h o r e s e d at p H 3.5, d r i e d a n d a u t o r a d i o g r a p h e d . (B) A a n d B m a t e r i a l ( F i g . 4) w e r e e l u t e d f r o m t h e t h i n - l a y e r p l a t e , d r i e d , r e s u s p e n d e d in b u f f e r a n d d i g e s t e d w i t h n u c l e o t i d e p y r o p h o s p h a t a s e . A n R N A a s e T 2 d i g e s t w a s u s e d as m a r k e r . (C) B m a t e r i a l (Fig. 4) w a s e l u t e d f r o m t h i n - l a y e r p l a t e s a n d d i g e s t e d w i t h e i t h e r P1, a l k a l i n e p h o s p h a t a s e , a n d n u c l e o t i d e p y r o p h o s p h a t a s e ( 1 ) or w i t h P I a n d a l k a l i n e p h o s p h a t a s e (2). T h e digests were electrophoresed on thin-layer sheets and the sheets were a u t o r a d i o g r a p h e d . (D) 32p-labelled n u c l e a r R N A s p e c i e s w e r e d i g e s t e d w i t h P1 n u c l e a s e a n d a l k a l i n e p h o s p h a t a s e . N7 w a s a n a l y z e d as t w o s e p a r a t e b a n d s since on f o r m a m i d e gels, t h e s e p a r a t i o n is r e p r o d u c i b l e . T h e d i g e s t w a s a p p l i e d to a thinl a y e r s h e e t a n d e l e c t r o p h o r e s e d at p H 3.5. T h e s h e e t w a s t h e n a u t o r a d i o g r a p h e d .
Biosynthesis of low molecular weight RNA. Fig. 7 shows a 12% formamide polyacrylamide gel of low molecular weight RNA (low Mr) species extracted from cells labelled for short periods with [3H]adenosine. The predominant species appearing in 5 min {lanes A and E) corresponds to 4.5-S t R N A precursor. However, small amounts of nuclear species N5, N6 and N7 were also found in the nucleus, after a short time. Lanes E, F, G and H contain the species made in 5, 15, and 30 min and 1 h, respectively. Species N6 and N7 increased at a constant rate in the nucleus after a short lag, as determined from quantitative plots of gel scans. (Fig. 8). Lanes A, B, C and D show the low Mr RNA species found in the cytoplasm at 5, 15 and 30 min and 1 h, respectively. The 4- and 5-S species accumulated in the cytoplasm linearly with time {Fig. 7}. A heavily
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Pi
b.:
e 4S
CI
CZ
C3
5S
Fig. 6. C y t o p l a s m i c l o w m o l e c u l a r w e i g h t R N A s are n o t c a p p e d . T h i n - l a y e r e l e c t r o p h o r e t o g r a m o f P1, a l k a l i n e p h o s p h a t a s e d i g e s t o f l o w m o l e c u l a r w e i g h t c y t o p l a s m i c R N A species l a b e l l e d w i t h 3 2 p . T h e r e s i s t a n t s p o t s m i g r a t e i d e n t i c a l l y w i t h t h o s e in l a n e A ( F i g . 4) a n d w e r e c o m p l e t e l y sensitive t o redigest i o n w i t h P1 ( n o t s h o w n ) .
labelled species corresponding to the cytoplasmic species C2 present in the long-term label was also present. However, due to incomplete resolution of this species from unstable RNAs present in the short pulse, we have been unable to quantitate its synthesis. In addition, the three nuclear species N5, N6, and N7 were found in the cytoplasm without a detectable lag. Unlike their accumulation in the nucleus, the amounts in the cytoplasm of N6 and N7 reached a constant level after a short time (Fig. 8). When cells labelled with [3H]adenosine for 15 min were then incubated with actinomycin D, we found that the nuclear species N5, N6 and N7 remained in the cytoplasmic fraction and failed to reassociate with the nuclei after chase times of 15 min. The 4 S increased while the 4.5-S band decreased, indicating that some tRNA processing occurs during the chase. The species N5, N6 and N7 remained constant in q u a n t i t y in the nucleus and cytoplasm over the chase period (Table I). Thus, the proper metabolism and processing of these RNAs may require continued RNA synthesis. Short term incubation o f cells labelled in vivo with [3H]methyl methionine. Fig. 9 shows the low Mr RNA obtained from whole cells incubated with
60 D
I5 0
30 C
5 A
5 E
-30 0
15 F
60 cl
6
A
Fig. 7. Pulse-labelled low molecular weight RNAs. RNA was analyzed on 12% polyacrylamide formamide gels and gels were fluorographed. RNA was derived from cells which has been incubated with [3H]adenosine as described in Materials and Methods. Aliquots were removed at 6, 16. 30 and 60 min (A, B, C, D, and E, F, G, H. respectively). Nuclei and cytoplasm were separated as described and RNA was prepared by phenol-SDS extraction of nuclei at 55’C (E, F, G, H) and of cytoplasm at 25’ (A. B. C. D). The exposure of the figure was chosen to show RNAs N6 and N7 clearly, particularly in the cytoplasmic samples. Lane H, being a much longer labelling time is overexposed in the figure but specific bands corresponding to each of the RNAs were present.
7
c
/
4.5s+4s
wo-‘) /A
N7
4
N6
NUC
NUC
RNA
TIME Fig. 8. The quantitative analysis of pulse label. The X-ray films were scanned in a densitometer and the areas under the peaks determined. The rates of synthesis of N6 and N7 in each fraction are shown. 5-S RNA assumulated at a linear rate as did tRNA (determined by sum of 4.5 S and 4 S).
673 TABLE I EFFECT OF ACTINOMYCIN D ON TRANSLOCATION
R N A species
Nucleus
Cytoplasm
T i m e after chase (rain)
T i m e after chase (rain)
0 <5 28
N7 N%5 + N6
OF RNA
5 <5 27
10 <5 31
15 <5 35
0 33 24
5 27 25
10 22 22
15 25 27
Cells w e r e p u l s e - l a b e l l e d f o r 10 m i n w i t h [ 3 H ] a d e n o s i n e a n d t h e a c t i n o m y c i n D a d d e d . A l i q u o t s w e r e r e m o v e d a t t h e i n d i c a t e d t i m e s , cells f r a c t i o n a t e d 0 R N A p r e p a r e d a n d a n a l y z e d o n a 1 0 % p o l y a c r y l a m i d e slab gel. T h e gel w a s f l u o r o g r a p h e d a n d t h e f l u o r o g ~ a p h w a s s c a n n e d in a d e n s l t o m e t e r . In t h i s e x p e r i m e n t little N7 w a s f o u n d in t h e n u c l e u s a f t e r 1 5 m i n a n d a b o u t e q u a l a m o u n t s o f N 5 a n d N 6 , a l t h o u g h t h e y w e r e n o t w e l l - e n o u g h r e s o l v e d in t h e d e n s i t o m e t e r s c a n to d e t e r m i n e e a c h s e p a r a t e l y . A n a l i q u o t o f t h e cells t h a t w e r e n o t t r e a t e d w i t h a c t l n o m y c i n s h o w e d t h e e x p e c t e d i n c r e a s e in n u c l e a r R N A s . T h e t o t a l a m o u n t o f 5 S R N A r e m a i n e d t h e s a m e d u r i n g t h e c h a s e (-*20%).
[Me-14C]methionine for 15 and 30 min and 1 h. Lanes A, B and C show the species extracted from the nucleus. There was an accumulation in the nucleus of several methylated species b u t not in the cytoplasm. Nuclear species N4, N5, N6 and N7 were methylated after short incubation periods and the majority of
:!.i'CI
A
B
C
M
D
E
Fig. 9. M e t h y l a t i o n o f p u l s e - l a b e l l e d R N A s . G e l e l e c t r o p h o r e s i s p a t t e r n o f l o w m o l e c u l a r w e i g h t R N A f r o m m y e l o m a n u c l e i a n d c y t o p l a s m o f cells p u l s e - l a b e l l e d w i t h [Me-14C]methionine. R N A w a s a n a l y z e d on 12% p o l y a c r y l a m i d e f o r m a m i d e gels a n d t h e gels w e r e f l u o r o g r a p h e d . A l i q u o t s o f labelled cells w e r e r e m o v e d at 15, 30 a n d 60 m i n . T h e n u c l e i a n d c y t o p l a s m w e r e s e p a r a t e d as d e s c r i b e d a n d R N A w a s p r e p a r e d b y p h e n o l - S D S e x t r a c t i o n o f n u c l e i at 5 5 ° C (C, B, A) a n d o f c y t o p l a s m at 25 ° ( D , E, F ) . M d e n o t e s n u c l e a r R N A m a r k e r f r o m cells labelled o v e r n i g h t w i t h [ 3 H ] a d e n o s i n e .
674 label was found in the nuclear fraction rather than being distributed between the nucleus and cytoplasm in amounts comparable with [3H]adenosine. For example, after 30 min incubation with [Me-3H]methionine, there was at least 5 times as much N4, N5, N6 and N7 in the nucleus as the cytoplasm compared to only twice as much when [3H]adenosine was used (Fig. 7). This is consistent with methylation in the cytoplasm occurring prior to transport back into the nucleus, or with methylation being essential for fixing the RNA in the nucleus. Discussion We have identified several species of low molecular weight RNA in mouse m y e l o m a cells. The patterns on polyacrylamide gels are consistent with those seen when other mammalian cell lines are examined. Capped 5' termini were found in four nuclear RNA species, N3, N5, N6 and N7. Three of these correspond to RNA species, two o f which have been sequenced, which were reported to contain capped termini [15,16,24,25]. The fourth species, N3, has not been previously reported as being capped. These species were shown to be methylated. These data are consistent with previous reports of methylations of the 5' terminus and internal methylations [15,16]. There are three cytoplasmic species detectable which are slightly larger than the nuclear species. The smallest had an electrophoretic mobility close to N7, but is distinct from it as judged by RNA-DNA hybridization. It is present at about 10% the level of N7. The other two, C2 and C3, are larger than N7 but smaller than the mRNA for sea urchin histone H4 (400 nucleotides [26], data not shown). Estimates in 6% polyacrylamide gels give about 250 and 300 nucleotides, respectively. Species C2 is predominant, present in similar a m o u n t to the low Mr nuclear RNAs {about 3 • 10 s molecules/cell) while species C3 is a minor species. None of these are methylated or 'capped', C2 probably corresponds to '7-S' RNA reported to be associated with cytoplasmic membranes
[71. The situation is quite different when comparisons of nuclear and cytoplasmic small RNA species are made from cells which were labelled for short time periods. N5, N6 and N7 appear in both the nuclear and cytoplasmic fractions. At the earliest times (pulse of 5 m i n ) n e a r l y equal amounts of each appear in the nucleus and in the cytoplasm. There follows a nearly linear accumulation in the nucleus while the a m o u n t in the cytoplasm reaches a constant level rapidly. This may indicate that there is time after transcription during which newly made species leak from the nucleus upon fractionation because they have not yet become permanently associated with the nucleus. This has been shown to occur with 5-S RNA which requires approximately 30 min to become associated with nuclear ribosomal precursors and which, therefore, appears in the cytoplasm for 30 min after a short label [27]. Alternatively, the data may truly represent an in vivo phenomenon. N5, N6 and N7 have been reported to contain cap II structures and Perry and Kelley [28] have shown with m R N A that only Cap I structures are formed in the nucleus. The second methylation (ym) occurs in the cytoplasm. Therefore, the appearance of the species in the cytoplasm may very well represent a transient in vivo cytoplasmic appearance of the species.
675 Results obtained with methionine-pulsed cells differ from those obtained when R N A is extracted from cells which have been labelled for short time periods with [3H] adenosine. In this case, very small amounts of labelled species are found in the cytoplasmic fractions while there is a relatively large accumulation in the nucleus. It appears as if the species in the cytoplasm are undermethylated compared to those associated with the nuclear fraction. It is possible that the nuclear species N5, N6 and N7 require a methylation event in order to become 'fixed' into the nucleus. This situation would be reflected by the appearance of very little label in cytoplasmic fractions when cells are labelled for brief periods with methionine. Thus, undermethylated species may be found associated with the cytoplasm and subsequent cytoplasmic methylation may be necessary for re-entry and fixation into the nucleus. Chase experiments with actinomycin D were performed to attempt to follow the kinetics of reincorporation of the species into the nucleus. Unexpectedly, we found that after 15 min the species remained in the cytoplasm and there was no accumulation in the nucleus. The amounts in nucleus and cytoplasm remained constant. Possibly continued RNA synthesis is necessary for efficient processing of RNA. Actinomycin D is known to interfere with H n R N A processing [29]. We did not detect any obvious precursors for the species N5-N7. Their appearance is as rapid as 4.5 and 5-S RNA. The data here do not establish the primary transcript from which these RNAs are derived. However, the primary transcript, if much larger, must be rapidly processed to the length of the final RNA (within 3 min). Busch and co-workers [23] have established that the cap species in 2 of these is GpppA so that the primary transcript could possibly start with p p p A and this may subsequently become capped. Experiment with in vitro system deficient in processing may help define the true primary transcript. We find in these cells essentially total restriction of several small RNAs to the nucleus, as reported earlier by Busch and co-workers [5]. It has definitely been established that these RNAs are not associated with the chromosomes during mitosis [19] but that they reassociate with the nucleus on formation of t h e nuclear envelope. In these cultures after the long-term label in 32PO4 and reduced phosphate medium the growth rate has slowed and there are a few mitotic cells. In contrast, Zieve and Penman [13] have reported substantial amounts of these RNAs in the cytoplasmic fraction of HeLa cells. However, in their nuclear preparation they used an ionic detergent, deoxycholate, which has been shown previously [14,31] to extract these RNAs from chromatin. Whether the structural differences noted here (the nuclear species are all 'capped' while the cytoplasmic species are unmodified) is related to their subcellular localization is not known. However, the 'cap' structure itself is unlikely to be responsible as the cytoplasmic mRNAs are all capped. The rates of synthesis of each of the species N5, N6, and N7 are 20--25% the rate o f synthesis of 5-S rRNA on a molar basis and 5% the rate of synthesis of the 4.5-S t R N A presursor. In contrast, species N3 appears to be made at a lower rate, a b o u t 5--10% the rate of 5 S, and is present in about half the concentration of N5, N6, and N7. Since there are about 100 copies of each of the genes for N5, N6, and N7 compared to 500 copies of 5-S genes, this implies the genes are transcribed at the same relative rate, which is probably close to the
676
maximal possible rate, in exponentially growing cells. All of these genes are probably transcribed by RNA polymerase III (Marzluff and Pan, submitted for publication) as are the 5-S and tRNA genes. The other RNA, N3, may well be transcribed at a lower rate as it is present in a larger number of copies in the genome, close to that of 5 S [14]. Study of the pathway of biosynthesis will require cell-free systems capable of carrying out RNA processing reactions as well as transcription.
Acknowledgements This proposal was supported by grants from NSF and NIH Ag-00413. W.F. M. is a recipient of a Research Career Development Award CA00178 from the National Cancer Institute.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
L a r s e n , P . H . , F r e d e r i k s e n , S. a n d P l e s n e r , P. ( 1 9 7 1 ) B i o c h i m . B i o p h y s . A c t a 2 5 4 , 7 8 - - 9 0 F r e d e r i k s c n , S., H e l l u n g - L a r s c n , P. a n d E n g b e r g , J. ( 1 9 7 3 ) E x p . Cell Res. 78, 2 8 7 - - 2 9 4 W e i n b e r g , R . A . a n d P e n m a n , S. ( 1 9 6 8 ) J. MoL Biol. 3 8 , 2 8 9 - - 3 0 4 R o - C h o i , T.S. a n d B u s c h , H. ( 1 9 7 4 ) T h e Cell N u c l e u s V o l . V . p p . 1 5 1 - - 2 0 8 , A c a d e m i c Press, N e w York Busch, H . R o - C h o i , T . S . , P r e s t a y k o , A.W., S h i b a t a , H., C r o o k e , S.T., E I - K h a t i b , S.M., C h o i , Y . C . a n d M a u r i t z e n , C.M. ( 1 9 7 1 ) P e r s p e c t . Biol. Med. 15, 1, 1 1 7 - - 1 3 9 Elicieri, G . L . ( 1 9 7 4 ) Cell 3, 1 1 - - 1 4 Elieieri, G , L . ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 2 5 , 2 0 2 - - 2 0 7 P r e s t a y k o , A.W. a n d B u s c h , H. ( 1 9 6 8 ) B i o c h i m . B i o p h y s . A c t a 1 6 9 , 3 2 7 - - 3 3 7 L a r s e n , P.H. a n d F r e d e r i k s e n , S. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 6 2 , 2 9 0 - - 3 0 7 F r e d e r i k s e n , S.. P e d e r s o n , T . R . , L a r s e n , P.H. a n d E n g b e r g , J. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 4 0 , 64-76 L a r s e n , P.H., T y r s t e d , G., E n g b e r g , J. a n d F r e d e r i k s e n , S. ( 1 9 7 4 ) E x p . Cell Res. 8,5, 1 - - 1 7 W e i n b e r g , R . A . a n d P e n m a n , S. ( 1 9 6 9 ) B i o c h i m . B i o p h y s . A c t a 1 9 0 , 10---29 Zievc, G. a n d P e n m a n , S. ( 1 9 7 6 ) Cell 8, 1 9 - - 3 1 M a r z l u f f , W . F . , W h i t e , E . L . , B e n j a m i n , R. a n d H u a n g , R . C . C . ( 1 9 7 5 ) B i o c h e m i s t r y 14, 3 7 1 5 - - 3 7 2 4 R o - C h o i , T.S., C h o i , Y.C.C., H e n n i n g , D., M c C l o s k e y , J.M. a n d B u s c h , H. ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 3921--3928 R e d d y , R., R o - C h o i , T.S., H e n n i n g , F. a n d B u s c h , H. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 6 4 8 6 - - 6 4 9 4 C o r y , S. a n d A d a m s , J.M. ( 1 9 7 6 ) J. Mol. Biol. 9 9 , 5 1 9 - - 5 4 6 R e i n , A. a n d P e n m a n , S. ( 1 9 6 9 ) B i o c h i m . B i o p h y s . A c t a 1 9 0 , 1 - - 1 9 G o l d s t e i n , L. ( 1 9 7 6 ) N a t u r e 2 6 1 , 5 1 9 - - 5 2 1 M a r z l u f f , W . F . , M u r p h y , E. a n d H u a n g , R . C . C . ( 1 9 7 3 ) B i o c h e m i s t r y 12, 3 4 4 0 - - 3 4 4 6 L a s k e y . R . A . a n d Mills, A . D . ( 1 9 7 5 ) E u r . J. B i o c h e m . 56, 3 3 5 - - 3 4 1 B r o w n l e e , G . F . ( 1 9 7 3 ) D e t e r m i n a t i o n o f S e q u e n c e s in R N A , Elsevier, A m s t e r d a m R o - C h o i , T.S., R e d d y , R . , C h o i , Y.C., R a j , N.B. a n d H e n n i n g , D. ( 1 9 7 4 ) F e d . P r o c . 33, 1 5 4 8 A d a m s , J. a n d C o r y , S. ( 1 9 7 5 ) Mol. Biol. R . P . 2 (4), 2 8 7 - - 2 9 4 R o - C h o i , T.S. a n d H e n n i n g , D. ( 1 9 7 7 ) J. Biol. C h e m . 2 5 2 , N o . 11, 3 8 1 4 - - 3 8 2 0 G r u n s t e i n , M. a n d S c h e d l , P.L. ( 1 9 7 6 ) J. Mol. Biol. 1 0 4 , 3 2 3 - - 3 4 9 L e i b o w i t z , R . D . , W e i n b e r g , R . A . a n d P e n m a n . S. ( 1 9 7 3 ) J. Mol. Biol. 73, 1 3 9 - - 1 4 4 P e r r y , R.P. a n d K e l l e y , D,E. ( 1 9 7 6 ) Cell 8, 4 3 3 - - 4 4 2 Levis, R . a n d P e n m a n , S. ( 1 9 7 7 ) Cell 1 1 , 1 0 5 - - 1 1 3 P e r r y , R.P. ( 1 9 7 6 ) A n n u . Rev. B i o c h e m . 4 5 , 6 0 5 - - 6 2 9 M a y f i e l d , J . E . a n d B o n n e r , J. ( 1 9 7 2 ) P r o c . N a t l . A c a d . Sci. U.S. 6 9 , 7 - - 1 0 S k l a r , V . E . F . a n d R o e d e r , R . G . ( 1 9 7 7 ) Cell 1 0 , 4 0 5 - - 4 1 4
Biochimica et Biophysica Acta, 521 (1978) 662--676 © Elsevier/North-Holland Biomedical Press
BBA 99319
BIOSYNTHESIS OF LOW MOLECULAR WEIGHT RNA IN MOUSE MYELOMA CELLS
ANNE BROWN and WILLIAM MARZLUFF
Department of Chemistry, Florida State University, Tallahassee, Fla. 32306 (U.S.A.) (Received March 17th, 1978)
Summary The low molecular weight RNAs in the nucleus and cytoplasm of mouse myeloma cells have been characterized. There are major nuclear species (other than 5-S and tRNA), four of which contain 'capped' 5' termini. There are three major small cytoplasmic RNAs, none of which contain 'caps'. The biosynthesis of the nuclear species when studied using [3H]adenosine as an RNA precursor is characterized by a rapid, transient appearance in the cytoplasm, followed by fixation in the nucleus. Within 15 min, the amount in the cytoplasm has reached a steady-state level maintained for 60 rain, while the accumulation in nuclei is linear after a short lag (less than 5 min). When biosynthesis is studied using [Me-~H]methionine as a precursor, much less labeled RNA is present in the cytoplasm, suggesting that methylation may immediately precede fixation into the nucleus.
Introduction
Several species of low molecular weight RNA have been reported to occur in eucaryotic cells. A diversity of cell types have been studied including Tetrahymena pyriformis [1], sea urchins [2] and several mammalian lines including HeLa cells [3] and Novikoff hepatoma cells [4,5]. These species, characterized primarily by gel electrophoresis, exhibit pattern similarity which appears to be correlated with the evolutionary relationships of the organisms. Thus, although Tetrahymena contains small molecular weight RNA species, their electrophoretic mobilities differ from the mobilities which are characteristic of mammalian small molecular weight species. Several small cytoplasrriic RNA species in addition to transfer RNA and ribosomal 5-S RNA, have been reported [6,7]. The most intensive characterizations of small RNA species, however, have been performed with those found in nuclei. Nearly identical mobility patterns on polyacrylamide gels have been
663 demonstrated for small nuclear species from HeLa cells, rat liver, Ehrlich acites cells, BHK cells and human l y m p h o c y t e nuclei [3,8--11]. Several species are methylated and show similar methylation patterns in mammalian cells [5,9,11,12]. The small molecular weight nuclear RNA species in HeLa cells [13], Novikoff hepatoma cell lines [4], and mouse m y e l o m a cells [14] have been analyzed in detail and show several interesting properties: they are extremely stable, with turnover rates comparable to transfer and ribosomal RNA; several species are found in very large numbers in the nucleus including two species estimated to be present in 2 • l 0 s and 2 • 106 molecules/cell [12,14]. Hybridization studies show that 100--200 complementary DNA sites exist for these species [19]. Three species are reported to contain 'capped' 5' termini similar to, b u t not identical to those found in viral and animal messenger RNA [15--17]. During mitosis, the nuclear species dissociate from the nucleus and are again associated with it when the membrane reforms [18,19]. No function has been discovered for this ubiquitous class of RNA molecules. We have examined the low molecular weight species in mouse m y e l o m a nuclei and cytoplasm. The species in each c o m p a r t m e n t are distinct. We have found species similar to those reported to occur in other mammalian cells. Several of these are methylated. Four nuclear species contain caps, three similar to those found in Novikoff hepatoma nuclear species, and there is in addition a capped, methylated species not previously reported. We have studied the metabolism of the RNAs and the data from the in vivo pulse labels indicate rapid synthesis (less than 5 min), and suggest a transient cytoplasmic appearance, followed b y fixation into the nuclear fraction. [Me-3H]RNA has a much shorter lifetime in the cytoplasm, suggesting that a methylation may be related to fixation of RNA into the nucleus. Materials and Methods
Labeling of cells. Mouse m y e l o m a 66-2 cells were used in all experiments and grown as previously described [20]. To label the RNA with ~2PO4, the cells were grown to a concentration of 5 - - 6 . 1 0 S / m l , pelleted gently, and resuspended in low phosphate medium. 32PO4 was added as H3PO4 to a final concentration of 80--100 uCi/ml. In most experiments, 120 ml cells were used. Cells were diluted with phosphate-free medium (0.5 vol.) after 12 h of labeling. After 24 h the cells were collected and the R N A was extracted. The cells normally double every 16 h but in phosphate-free medium the rate is decreased and only one doubling occurs. Preparation of RNA. All procedures were at 4°C except where noted. The cells were collected b y centrifugation and washed twice with cold 0.14 M KC1/0.01 M Tris, pH 7.5. They were then suspended in buffer A (0.32 M sucrose/2 mM Mg2+/3 mM Ca2*/10 mM Tris, pH 8/0.1% Triton X-100) at 2 • 10 T cells/ml and broken by homogenization in a Dounce homogenizer (20 strokes, B pestle). The nuclei were pelleted b y centrifugation at 1000 × g for 10 min. The nuclei were suspended in buffer B (0.88 M sucrose/3 mM Ca2+/ 2 mM Mg2÷/10 mM Tris/0.1% Triton X-100) and homogenized as above with 5 strokes. The nuclei were pelleted b y centrifugation (1000 ×g, 10 min).
664
(1) Cytoplasm: To the combined supernatant fractions were added 0.05 vol. 10% sodium dodecyl sulphate (SDS) 0.05 vol. 2 M NaOAc, pH 5, 0.67 vol. 80% phenol, and 0.33 vol. CHCI3. The mixture was shaken for approximately 30 min at 25°C, and the RNA precipitated from the aqueous layer with 2.5 vol. 95% ethanol. The R N A was collected by centrifugation, redissolved in 0.5 ml 1% SDS-10 mM EDTA, layered over a 5--30% sucrose gradient (0.1% SDS, 0.1 M NaC1, 0.01 M EDTA, 10 mM Tris, pH 7.5) and centrifuged in the SW 27 rotor (Sorvall OTD-2) for 15 h, 25 000 rev./min, 25°C. The 4--8-S region was collected and the RNA precipitated with 95% ethanol. (2) Nuclei: The nuclear pellet was suspended in buffer A (5 ml for 100 ml cells) and to this was added an equal volume of 1% SDS, 10 mM EDTA, 0.20 vol. 2 M NaOAc, pH 5, 1.5 vols. 80% phenol, and 0.5 vols. CHC13. The mixture was incubated for 5--10 min at 55°C and then cooled on ice. The aqueous layer was removed and precipitated with 2.5 vols. 95% ethanol and the RNA was separated on gels as described below. Nuclear RNA for some experiments (indicated in Results) was prepared by first extracting the nuclear pellet with 0.35 M NaCI, 5 mM Mg 2+, 10 mM Tris, pH 8, centrifuging at 10 000 × g for 10 minutes to pellet the chromatin, and then separating the pellet from the supernatant. The RNA remaining with chromatin was removed by 55°C SDS-phenol treatment as above. The supernatant was layered over a 1 M sucrose pad (6 ml s u p e r n a t a n t / 2 . 5 ml sucrose) and centrifuged for 8 h at 35 K in the 65 rotor in a Beckman ultracentrifuge to remove ribosomal subunits and precursors. The RNA in the supernatant and in the pellet was then recovered b y SDS-phenol precedure described above. Separation and purification of low molecular weight RNA species. Cytoplasmic RNA from the 4--8-S region of the sucrose gradient was dissolved in gel buffer (20% glycerol/0.1% SDS/0.1 M EDTA/10 mM Tris, pH 7.5) and electrophoresed on 10% polyacrylamide gels at 80 V (13--20 MA) until the dye marker (bromphenol blue) reached the edge of the gel (20 cm). Nuclear R N A was electrophoresed as above or alternatively dissolved in 99% formamide and applied to 12% polyacrylamide-formamide gels [14]. The RNA bands were located b y autoradiography. The bands were cut from the gel, broken up, and placed in a plastic pipette with filter paper plugging the tip. The R N A was recovered b y electrophoresing it though the filter paper into dialysis bags attached to the pipette tips. This was done at 115 V and the elution was complete by 9--12 h as monitored with a Geiger counter. 28-S r R N A was added as carrier (0.5 A26onm) and the R N A was recovered by ethanol precipitation. Gel fluorography. Gels were soaked in 10% acetic acid/25% isopropanol overnight and processed for fluorography as described by Laskey and Mills [21] for 3H and 14C samples. The dried gel was placed against X-ray film (Kodak RPR-54 pre-exposed to Ass0nm of 0.15) at --70°C for an appropriate length of time. Digestion of RNA. The R N A was dissolved in 25 ~I 10 m M NaOAc/10 -4 M ZnOAc, p H 5.8. To this was added 2.5 pl P1 nuclease (Sankyo), (10.0 mg/ml in 100 m M NaOAc, p H 5.8). The digest was incubated in a closed capillary tube overnight. The digest was brought to p H 7 with approximately 2/zl 1 M Tris, p H 8.0, and 2/~I alkaline phosphatase (1 mg/ml) was added. The digest was incubated for 2--5 h in a capillary tube at 37°C. This procedure yields inorganic
665
phosphate as the only radioactive product when 32po4-4-S tRNA is used. Analysis of 5'-termini. The RNA species were eluted from gels and digested with RNAase P1 and alkaline phosphatase as described above. The digest was spotted on a cellulose thin-layer plate or a DEAE-cellulose thin-layer plate and electrophoresed on a Shandon Flat-bed apparatus in 0.5% pyridine/5% acetic acid, pH 3.5, at 350--400 V until the inorganic phosphate was near the plate edge (monitored with a Geiger counter). The digest was run with dye markers [15]. The radioactivity on the plates was located by autoradiography and was eluted with either water (cellulose plates) or 2 M triethylammonium bicarbonate [22] when DEAE-cellulose plates were used. The sample was lyophilized and the residue was resuspended in a small a m o u n t of water and divided into appropriate aliquots depending upon the digestion procedures to be performed. Usually three aliquots were processed. (1) Nucleotide pyrophosphatase: the solution (30 p]) was adjusted with 2 ~l 1 M Tris, pH 8, to pH 7.5 and 5/A enzyme (0.32 mg/ml) was added. The digestion was incubated for 1 h at 37°C. (2) Alkaline phosphatase: after 1 h, 2 p] alkaline phosphatase was added to the above digest. (3) Redigestion with RNAase P1 and alkaline phosphatase: The solution (50 ~l) was made 10 mM NaOAc, pH 5.8, and 2 ~l RNAase P1 was added for 3--5 h at 37°C. The pH was brought to 7.5 with 1 M Tris, pH 8, and 2 ~l alkaline phosphatase was added and the mixture incubated for 2--3 h at 37°C. All digests were spotted on thin-layer cellulose plates and electrophoresed on a flat-bed apparatus as described above. RNA-DNA hybridization. Hybridization of RNA species N7 and C1 to m y e l o m a DNA was performed as described previously [14]. Incubations were performed using either C1 and N7 or both species simultaneously at a concentration of 0.04 pg/ml (of each RNA) in 0.9 M NaC1, 0.09 M Na3 citrate, 50% formamide at 55°C. Pulse labelling of RNA with [3H]adenosine. Cells were grown to a concentration of 5--6 • 10S/ml pelleted and resuspended in the same medium at a concentration of 4 . 106/ml. They were incubated for 1 h and then [2-3H]adenosine was added at a concentration of 50 ~Ci/ml. Aliquots were removed at the appropriate times, cells chilled by pipetting into 3 vols. ice-cold 0.14 M KC1, and the RNA extracted as described. For the pulse-label experiments with an actinomycin D chase, cells were treated as above and were incubated with [3H]adenosine for 15 min. At this time, actinomycin D was added to a final concentration of 20 pg/ml, and aliquots were removed at the appropriate times. The incorporation of [3H]adenosine into acid-isoluble material was stopped within 2 min. Pulse-labelling of RNA with [14C]methyl-methionine. 500 ml of cells were grown to a concentration of 5 . 1 0 S / m l . They were pelleted, washed once in methionine-free Dulbecco's modified Eagle's medium and resuspended in 60 ml of the same medium containing 20 wM adenosine, 20 p_M guanosine and 20 mM sodium formate. The cells were allowed 1 h to recover and then 50 ~Ci [Me-14C]methionine was added. Aliquots were removed at the appropriate times and the nuclear and cytoplasmic RNA were extracted as described for [ 3H]adenosine labelled cells.
666
Materials. RNAase P1 was obtained from Yamasa Shoyu Co. Tokyo, Japan. Nucleotide pyrophosphatase and alkaline phosphatase were from Sigma Chemical Co. Radioactive isotopes were obtained from Schwartz-Mann. Results
Analysis of nuclear low molecular weight RNA components: long term label 32p. There are seven major low molecular weight RNA species in the nucleus, six of which can be resolved on 10% polyacrylamide gels. These are shown in Fig. 1; the species shown were obtained by SDS-phenol deproteinization of unfractionated, detergent-washed nuclei. These are numbered N 1--7 in order of decreasing mobility. In addition to these, three minor bands, N8--N10, consistently appear after long term labelling of whole cells. The nomenclature for these RNAs is different from that which we used in our earlier work [14]. Here we use the following: nuclear RNAs are N1--N10 with N1 being the smallest (4 S) RNA. This will facilitate nomenclature as further studies are carried out on the larger RNAs. Cytoplasmic species are denoted C1--C3 with C1 being the smallest RNA larger than the wellcharacterized 4-S tRNA, 5-S rRNA and 5.8-S rRNA. Our species N5, N6, N7 presumably correspond to the U~, U2, U3 of Busch and co-workers [5] and to D, C, and A of Penman and co-workers [13]. The results obtained by this method of RNA preparation differ from those
NiO N9 Nil N7 N6 N5 N3, N4(O~ N2 (4.5S) NI (48)
Fig. 1. L o w m o l e c u l a r w e i g h t n u c l e a r R N A s . Left: R N A w a s p r e p a r e d f r o m purified n u c l e i b y t h e h o t p h e n o l p r o c e d u r e a n d s e p a r a t e d o n - 1 0 % p o l y a c r y l a m i d e gel. B a n d N1 is c o n t a m i n a t i n g t R N A . N 3 and N 4 (5-S R N A ) m i g r a t e as a single c o m p o n e n t w h i c h m a y be r e s o l v e d o n f o r m a m i d e - p o l y a c r y l a m i d e gels ( r i g h t ) o r b y e x t r a c t i o n o f n u c l e i w i t h 0 . 3 5 M salt. R i g h t : I n d i v i d u a l R N A s p e c i e s w e r e e l e c t r o p h o r e s e d o n a 12% p o l y a c r y t a m i d e - 7 0 % f o t - m a m i d e slab g e l L e f t to right: c y t o p l a s m i c 5 S, c y t o p l a s m i c 4 S. a mixt u r e o f N 6 a n d N7, N5, N 4 ( p r e p a r e d a f t e r e x t r a c t i o n w i t h 0 . 3 5 M KCI [ 1 4 ] ) a m i x t u r e of N3 a n d N 4 ( e l u t e d f r o m gel at l e f t ) .
667
obtained when nuclei are extracted with low salt (0.35 M NaC1) in t w o important ways. First, the relative mobility of N6 is altered. It now runs closer to N5 than to N7 while, if it is extracted by salt without heating, it migrates closes to N7 [14]. Second, the 5-S rRNA is not resolved from species N3. It may be resolved b y extraction in 0.35 M NaC1 [14] or by electrophoresis in the presence of formamide (Fig. 1). Fig. 1 demonstrates that all of the RNAs may be resolved b y formamide-polyacrylamide gel electrophoresis. The 5-S RNA (N4) is not part of nascent ribosomal subunits as it remains in the supernatant fraction of a 100 000 × g centrifugation after extraction of nuclei in 0.35 M NaC1/5 mM Mg 2÷, a treatment which will n o t release 5-S rRNA from ribosomes (data not shown). Previously we have d o c u m e n t e d the sequence similarity of this RNA and 5-S rRNA [14].
Analysis of cytoplasmic low molecular weight R N A components: long term label 32p. The major low molecular weigth RNA species of the cytoplasm are 5-S ribosomal RNA and 4-S transfer RNA. The RNAs recovered from the cytoplasm following SDS-phenol deprotenization at 25°C are shown in Fig. 2. The most prominent species corresponds to transfer RNA. In addition to these, there are three lighter bands, C1, C2 and C3 in order of decreasing mobility, which are consistently present in the cytoplasm under long term label conditions. The C1 band has an electrophoretic mobility in 10% aqueous gels which corresponds closely to that of the nuclear species N7. In order to determine whether the C1 and N7 species are identical species, purified C1 and N7 R N A species were hybridized to mouse m y e l o m a DNA immobilized on nitrocellulose filters. The species were hybridized both separately and together. Fig. 3 shows the results of the hybridization reaction. The reaction in which both species are hybridized shows an additive effect equal to the sums of each c o m p o n e n t individually. We conclude that each species is a unique component, confined to a specific cellular compartment. This is verified by the differences in 5' termini (see below). Analysis of 5' termini of low molecular weight RNA species. Busch and co-workers have reported [15,16] that two species of low molecular weight nuclear R N A from rat hepatoma cell nuclei contain modified 5' termini similar to those subsequently found on eucaryotic messenger RNA species. Capped 5' termini have been reported to be present in nuclear RNA species U1 and U2. Both U1 and U2 RNAs have been sequenced. The 'caps' in these species were originally reported to contain a diphosphate bridge, b u t have now been shown to have a triphosphate bridge [23,24], a trimethyl guanosine linked to 2'-0methyl adenosine. The nuclear R N A species which were obtained from m y e l o m a nuclei were examined b y enzymatic analysis to ascertain whether a cap structure is found in any of the species. The R N A species which were examined were eluted from gels and purity monitored b y gel electrophoresis in formamide-polyacrylamide gels. They were digested with P1 nuclease; this enzyme does not form a 2'3' cyclic phosphate intermediate and can, therefore, hydrolyze the phosphodiester bond adjacent to a 2' ribosome-methylated nucleoside. Digestion of capped nuclear species should, therefore, result in 5' mononucleotides and the structure 22VmGpppXm in which the phosphate groups are labelled. The addi-
668
(--)
3000 ~ , ~ . , ~
N7 + Cl
C3 C2 Cl
2000
CI ~E
o. r,3
5S lOCK; N7
4S
(+) 2
6 t(h)
Fig. 2. L o w m o l e c u l a r w e i g h t c y t o p l a s m i c R N A s . [ 3 2 p ] R N A was p r e p a r e d f r o m t h e c y t o p l a s m a n d analy z e d on 10% p o l y a c r y l a m i d e gels. B a n d s C ] , C2 and C3 are in o r d e r o f d e c r e a s i n g m o b i l i t y . Fig. 3. H y b r i d i z a t i o n of R N A species N7 a n d C1. R N A s w e r e e l e c t r o p h o r e s e d on 10% p o l y a c r y l a m i d e gels, e l u t e d , a n d r e - r u n o n 12% gels. T h e R N A s e l u t e d f r o m t h e 12% gels w e r e h y b r i d i z e d t o m o u s e D N A o n n i t r o c e l l u l o s e filters. Filters w e r e r e m o v e d at i n d i c a t e d t i m e s and w a s h e d as d e s c r i b e d u n d e r Materials a n d M e t h o d s . Specific a c t i v i t y w a s 5 • 105 cpm//Jg. R N A c o n c e n t r a t i o n s w e r e 0 . 0 4 ~ g / m l o f e a c h species.
tion of alkaline phosphatase to the products will result in labelled 'caps', unlabelled nucleosides, and labeled inorganic phosphate. The labelled products thus obtained can be readily separated from each other. Fig. 4 shows autoradiograms of cellulose thin-layer electropherograms of 32P-labelled RNA species which were digested with P1 nuclease and alkaline phosphatase. By this rapid procedure, the majority of the label is found as inorganic phosphate. However, in the nuclear RNAs (N3--N7) there was resistant material not present in tRNA and 5-S RNA (Fig. 4B). Often there were two spots, one of which (A) was also found in parallel digests of tRNA and 5-S RNA (Fig. 4A). We eluted each of the spots, A and B, running just behind the blue marker. The material was eluted from the cellulose thin-layer and was subjected to further enzymatic analysis. Redigestion with P1 nuclease and alkaline phosphatase converted the material in A (all lanes) to material electrophoresing with inorganic phosphate (Fig. 5A) indicating that this was a result of incomplete digestion. The material in B, lanes 2 - 4 , was resistant to further digestion by P1 nuclease and alkaline phosphatase. A and B material, eluted from the cellulose plate was digested with nucleotide pyrophosphatase. The results are shown in Fig. 5B. The A material remains in the original position upon cellulose thin-layer electrophoresis. B material was
669
Fig. 4. T h i n - l a y e r e l e c t r o p h o r e t o g r a m o f R N A s p e c i e s d i g e s t e d w i t h P1 n u c l e a s e and alkaline p h o s p h a t a s e . [ 3 2 p ] R N A s p e c i e s w e r e s e p a r a t e d o n 10% p o l y a c r y l a m i d e gels and i n d i v i d u a l R N A s p e c i e s w e r e e l u t e d f r o m t h e gels, p r e c i p i t a t e d and r e s u s p e n d e d in a s m a l l v o l u m e o f buffer. T h e R N A w a s d i g e s t e d a n d the d i g e s t a p p l i e d to t h i n - l a y e r c e l l u l o s e s h e e t s . T h e s h e e t s w e r e e l e c t r o p h o r e s e d at p H 3 . 5 , d r i e d , a n d a u t o r a d i o g r a p h e d . A and B d e n o t e the first and s e c o n d r o w s o f resistant m a t e r i a l r u n n i n g b e h i n d t h e m a r k e r , (circles d e n o t e t h e p o s i t i o n s o f m a r k e r dyes). T h e s l o w e s t m o v i n g is the x y l e n e c y a n o l ( b l u e ) d y e . T h e s a m p l e s o n the right w e r e m o r e e x t e n s i v e l y d i g e s t e d , b u t t h e t y p i c a l result is the o n e o n the left.
sensitive to the enzyme yielding identical patterns for each RNA. In contrast, when B material was digested with nucleotide pyrophosphatase, P1 nuclease, and alkaline phosphatase, the material was completely converted to inorganic phosphate (Fig. 5C). The properties obtained above for line B material would be expected for capped structures. Such results have consistently been obtained for nuclear species N3, N5, N6 and N7. 1--2% of the 32PO4 was present in the 5' termini consistent with a unique 5' terminus for each molecule. While small amounts of caps were found in some preparations of N4 (5-S rRNA) (Figs. 4 and 5), this was almost certainly due to a small contamination by species N3, as the a m o u n t was only 0.1--0.2% of the total 32PO4. The products of nucleotide pyrophosphatase digestion have been tentatively identified as residual undigested material (which is completely sensitive on further digestion (Fig. 5C), PAre and ppAm. The modified guanosine monophosphate migrates in the other direction in this system and is in the buffer. This is consistent with a triphosphate linkage in the cap. Digestions with ribonuclease T2 followed by DEAE-cellulose chromatography has shown that each species (N3, N5, N6, N7) contain 2--3% of their radioactivity which elutes with a charge o f - - 6 , consistent with this cap structure {data not shown). N3 has not been previously reported to contain 'capped' ends. This type of assay was also applied to the low molecular weight RNA species C1--C3. The results of enzymatic analysis were similar to those seen for cytoplasmic 4-S and 5-S RNA which do not contain capped structures (Fig. 6), a further indication that C1 and N7 are different species. The material which was insensitive to P1 digestion was completely converted to inorganic phosphate on redigestion with P1 and alkaline phosphatase (not shown).
670
Fig. 5 ( A ) A a n d B m a t e r i a l (see Fig. 4) w e r e e l u t e d f r o m t h e t h i n - l a y e r p l a t e a n d r e - d i g e s t e d w i t h P1 n u c l e a s e a n d a l k a l i n e p h o s p h a t a s e . 5A is a t h i n - l a y e r e l e c t r o p h o r e t o g r a m o f [ 3 2 p ] R N A d i g e s t i o n p r o d u c t s r e e x p o s e d to P1 n u c l e a s e a n d a l k a l i n e p h o s p h a t a s e . T h e o p e n circles i n d i c a t e t h e p o s i t i o n s o f t h e m a r k e r d y e s . T h e d i g e s t s w e r e a p p l i e d to cellulose t h i n - l a y e r s h e e t s a n d t h e s h e e t s w e r e e l e c t r o p h o r e s e d at p H 3.5, d r i e d a n d a u t o r a d i o g r a p h e d . (B) A a n d B m a t e r i a l ( F i g . 4) w e r e e l u t e d f r o m t h e t h i n - l a y e r p l a t e , d r i e d , r e s u s p e n d e d in b u f f e r a n d d i g e s t e d w i t h n u c l e o t i d e p y r o p h o s p h a t a s e . A n R N A a s e T 2 d i g e s t w a s u s e d as m a r k e r . (C) B m a t e r i a l (Fig. 4) w a s e l u t e d f r o m t h i n - l a y e r p l a t e s a n d d i g e s t e d w i t h e i t h e r P1, a l k a l i n e p h o s p h a t a s e , a n d n u c l e o t i d e p y r o p h o s p h a t a s e ( 1 ) or w i t h P I a n d a l k a l i n e p h o s p h a t a s e (2). T h e digests were electrophoresed on thin-layer sheets and the sheets were a u t o r a d i o g r a p h e d . (D) 32p-labelled n u c l e a r R N A s p e c i e s w e r e d i g e s t e d w i t h P1 n u c l e a s e a n d a l k a l i n e p h o s p h a t a s e . N7 w a s a n a l y z e d as t w o s e p a r a t e b a n d s since on f o r m a m i d e gels, t h e s e p a r a t i o n is r e p r o d u c i b l e . T h e d i g e s t w a s a p p l i e d to a thinl a y e r s h e e t a n d e l e c t r o p h o r e s e d at p H 3.5. T h e s h e e t w a s t h e n a u t o r a d i o g r a p h e d .
Biosynthesis of low molecular weight RNA. Fig. 7 shows a 12% formamide polyacrylamide gel of low molecular weight RNA (low Mr) species extracted from cells labelled for short periods with [3H]adenosine. The predominant species appearing in 5 min {lanes A and E) corresponds to 4.5-S t R N A precursor. However, small amounts of nuclear species N5, N6 and N7 were also found in the nucleus, after a short time. Lanes E, F, G and H contain the species made in 5, 15, and 30 min and 1 h, respectively. Species N6 and N7 increased at a constant rate in the nucleus after a short lag, as determined from quantitative plots of gel scans. (Fig. 8). Lanes A, B, C and D show the low Mr RNA species found in the cytoplasm at 5, 15 and 30 min and 1 h, respectively. The 4- and 5-S species accumulated in the cytoplasm linearly with time {Fig. 7}. A heavily
671
Pi
b.:
e 4S
CI
CZ
C3
5S
Fig. 6. C y t o p l a s m i c l o w m o l e c u l a r w e i g h t R N A s are n o t c a p p e d . T h i n - l a y e r e l e c t r o p h o r e t o g r a m o f P1, a l k a l i n e p h o s p h a t a s e d i g e s t o f l o w m o l e c u l a r w e i g h t c y t o p l a s m i c R N A species l a b e l l e d w i t h 3 2 p . T h e r e s i s t a n t s p o t s m i g r a t e i d e n t i c a l l y w i t h t h o s e in l a n e A ( F i g . 4) a n d w e r e c o m p l e t e l y sensitive t o redigest i o n w i t h P1 ( n o t s h o w n ) .
labelled species corresponding to the cytoplasmic species C2 present in the long-term label was also present. However, due to incomplete resolution of this species from unstable RNAs present in the short pulse, we have been unable to quantitate its synthesis. In addition, the three nuclear species N5, N6, and N7 were found in the cytoplasm without a detectable lag. Unlike their accumulation in the nucleus, the amounts in the cytoplasm of N6 and N7 reached a constant level after a short time (Fig. 8). When cells labelled with [3H]adenosine for 15 min were then incubated with actinomycin D, we found that the nuclear species N5, N6 and N7 remained in the cytoplasmic fraction and failed to reassociate with the nuclei after chase times of 15 min. The 4 S increased while the 4.5-S band decreased, indicating that some tRNA processing occurs during the chase. The species N5, N6 and N7 remained constant in q u a n t i t y in the nucleus and cytoplasm over the chase period (Table I). Thus, the proper metabolism and processing of these RNAs may require continued RNA synthesis. Short term incubation o f cells labelled in vivo with [3H]methyl methionine. Fig. 9 shows the low Mr RNA obtained from whole cells incubated with
60 D
I5 0
30 C
5 A
5 E
-30 0
15 F
60 cl
6
A
Fig. 7. Pulse-labelled low molecular weight RNAs. RNA was analyzed on 12% polyacrylamide formamide gels and gels were fluorographed. RNA was derived from cells which has been incubated with [3H]adenosine as described in Materials and Methods. Aliquots were removed at 6, 16. 30 and 60 min (A, B, C, D, and E, F, G, H. respectively). Nuclei and cytoplasm were separated as described and RNA was prepared by phenol-SDS extraction of nuclei at 55’C (E, F, G, H) and of cytoplasm at 25’ (A. B. C. D). The exposure of the figure was chosen to show RNAs N6 and N7 clearly, particularly in the cytoplasmic samples. Lane H, being a much longer labelling time is overexposed in the figure but specific bands corresponding to each of the RNAs were present.
7
c
/
4.5s+4s
wo-‘) /A
N7
4
N6
NUC
NUC
RNA
TIME Fig. 8. The quantitative analysis of pulse label. The X-ray films were scanned in a densitometer and the areas under the peaks determined. The rates of synthesis of N6 and N7 in each fraction are shown. 5-S RNA assumulated at a linear rate as did tRNA (determined by sum of 4.5 S and 4 S).
673 TABLE I EFFECT OF ACTINOMYCIN D ON TRANSLOCATION
R N A species
Nucleus
Cytoplasm
T i m e after chase (rain)
T i m e after chase (rain)
0 <5 28
N7 N%5 + N6
OF RNA
5 <5 27
10 <5 31
15 <5 35
0 33 24
5 27 25
10 22 22
15 25 27
Cells w e r e p u l s e - l a b e l l e d f o r 10 m i n w i t h [ 3 H ] a d e n o s i n e a n d t h e a c t i n o m y c i n D a d d e d . A l i q u o t s w e r e r e m o v e d a t t h e i n d i c a t e d t i m e s , cells f r a c t i o n a t e d 0 R N A p r e p a r e d a n d a n a l y z e d o n a 1 0 % p o l y a c r y l a m i d e slab gel. T h e gel w a s f l u o r o g r a p h e d a n d t h e f l u o r o g ~ a p h w a s s c a n n e d in a d e n s l t o m e t e r . In t h i s e x p e r i m e n t little N7 w a s f o u n d in t h e n u c l e u s a f t e r 1 5 m i n a n d a b o u t e q u a l a m o u n t s o f N 5 a n d N 6 , a l t h o u g h t h e y w e r e n o t w e l l - e n o u g h r e s o l v e d in t h e d e n s i t o m e t e r s c a n to d e t e r m i n e e a c h s e p a r a t e l y . A n a l i q u o t o f t h e cells t h a t w e r e n o t t r e a t e d w i t h a c t l n o m y c i n s h o w e d t h e e x p e c t e d i n c r e a s e in n u c l e a r R N A s . T h e t o t a l a m o u n t o f 5 S R N A r e m a i n e d t h e s a m e d u r i n g t h e c h a s e (-*20%).
[Me-14C]methionine for 15 and 30 min and 1 h. Lanes A, B and C show the species extracted from the nucleus. There was an accumulation in the nucleus of several methylated species b u t not in the cytoplasm. Nuclear species N4, N5, N6 and N7 were methylated after short incubation periods and the majority of
:!.i'CI
A
B
C
M
D
E
Fig. 9. M e t h y l a t i o n o f p u l s e - l a b e l l e d R N A s . G e l e l e c t r o p h o r e s i s p a t t e r n o f l o w m o l e c u l a r w e i g h t R N A f r o m m y e l o m a n u c l e i a n d c y t o p l a s m o f cells p u l s e - l a b e l l e d w i t h [Me-14C]methionine. R N A w a s a n a l y z e d on 12% p o l y a c r y l a m i d e f o r m a m i d e gels a n d t h e gels w e r e f l u o r o g r a p h e d . A l i q u o t s o f labelled cells w e r e r e m o v e d at 15, 30 a n d 60 m i n . T h e n u c l e i a n d c y t o p l a s m w e r e s e p a r a t e d as d e s c r i b e d a n d R N A w a s p r e p a r e d b y p h e n o l - S D S e x t r a c t i o n o f n u c l e i at 5 5 ° C (C, B, A) a n d o f c y t o p l a s m at 25 ° ( D , E, F ) . M d e n o t e s n u c l e a r R N A m a r k e r f r o m cells labelled o v e r n i g h t w i t h [ 3 H ] a d e n o s i n e .
674 label was found in the nuclear fraction rather than being distributed between the nucleus and cytoplasm in amounts comparable with [3H]adenosine. For example, after 30 min incubation with [Me-3H]methionine, there was at least 5 times as much N4, N5, N6 and N7 in the nucleus as the cytoplasm compared to only twice as much when [3H]adenosine was used (Fig. 7). This is consistent with methylation in the cytoplasm occurring prior to transport back into the nucleus, or with methylation being essential for fixing the RNA in the nucleus. Discussion We have identified several species of low molecular weight RNA in mouse m y e l o m a cells. The patterns on polyacrylamide gels are consistent with those seen when other mammalian cell lines are examined. Capped 5' termini were found in four nuclear RNA species, N3, N5, N6 and N7. Three of these correspond to RNA species, two o f which have been sequenced, which were reported to contain capped termini [15,16,24,25]. The fourth species, N3, has not been previously reported as being capped. These species were shown to be methylated. These data are consistent with previous reports of methylations of the 5' terminus and internal methylations [15,16]. There are three cytoplasmic species detectable which are slightly larger than the nuclear species. The smallest had an electrophoretic mobility close to N7, but is distinct from it as judged by RNA-DNA hybridization. It is present at about 10% the level of N7. The other two, C2 and C3, are larger than N7 but smaller than the mRNA for sea urchin histone H4 (400 nucleotides [26], data not shown). Estimates in 6% polyacrylamide gels give about 250 and 300 nucleotides, respectively. Species C2 is predominant, present in similar a m o u n t to the low Mr nuclear RNAs {about 3 • 10 s molecules/cell) while species C3 is a minor species. None of these are methylated or 'capped', C2 probably corresponds to '7-S' RNA reported to be associated with cytoplasmic membranes
[71. The situation is quite different when comparisons of nuclear and cytoplasmic small RNA species are made from cells which were labelled for short time periods. N5, N6 and N7 appear in both the nuclear and cytoplasmic fractions. At the earliest times (pulse of 5 m i n ) n e a r l y equal amounts of each appear in the nucleus and in the cytoplasm. There follows a nearly linear accumulation in the nucleus while the a m o u n t in the cytoplasm reaches a constant level rapidly. This may indicate that there is time after transcription during which newly made species leak from the nucleus upon fractionation because they have not yet become permanently associated with the nucleus. This has been shown to occur with 5-S RNA which requires approximately 30 min to become associated with nuclear ribosomal precursors and which, therefore, appears in the cytoplasm for 30 min after a short label [27]. Alternatively, the data may truly represent an in vivo phenomenon. N5, N6 and N7 have been reported to contain cap II structures and Perry and Kelley [28] have shown with m R N A that only Cap I structures are formed in the nucleus. The second methylation (ym) occurs in the cytoplasm. Therefore, the appearance of the species in the cytoplasm may very well represent a transient in vivo cytoplasmic appearance of the species.
675 Results obtained with methionine-pulsed cells differ from those obtained when R N A is extracted from cells which have been labelled for short time periods with [3H] adenosine. In this case, very small amounts of labelled species are found in the cytoplasmic fractions while there is a relatively large accumulation in the nucleus. It appears as if the species in the cytoplasm are undermethylated compared to those associated with the nuclear fraction. It is possible that the nuclear species N5, N6 and N7 require a methylation event in order to become 'fixed' into the nucleus. This situation would be reflected by the appearance of very little label in cytoplasmic fractions when cells are labelled for brief periods with methionine. Thus, undermethylated species may be found associated with the cytoplasm and subsequent cytoplasmic methylation may be necessary for re-entry and fixation into the nucleus. Chase experiments with actinomycin D were performed to attempt to follow the kinetics of reincorporation of the species into the nucleus. Unexpectedly, we found that after 15 min the species remained in the cytoplasm and there was no accumulation in the nucleus. The amounts in nucleus and cytoplasm remained constant. Possibly continued RNA synthesis is necessary for efficient processing of RNA. Actinomycin D is known to interfere with H n R N A processing [29]. We did not detect any obvious precursors for the species N5-N7. Their appearance is as rapid as 4.5 and 5-S RNA. The data here do not establish the primary transcript from which these RNAs are derived. However, the primary transcript, if much larger, must be rapidly processed to the length of the final RNA (within 3 min). Busch and co-workers [23] have established that the cap species in 2 of these is GpppA so that the primary transcript could possibly start with p p p A and this may subsequently become capped. Experiment with in vitro system deficient in processing may help define the true primary transcript. We find in these cells essentially total restriction of several small RNAs to the nucleus, as reported earlier by Busch and co-workers [5]. It has definitely been established that these RNAs are not associated with the chromosomes during mitosis [19] but that they reassociate with the nucleus on formation of t h e nuclear envelope. In these cultures after the long-term label in 32PO4 and reduced phosphate medium the growth rate has slowed and there are a few mitotic cells. In contrast, Zieve and Penman [13] have reported substantial amounts of these RNAs in the cytoplasmic fraction of HeLa cells. However, in their nuclear preparation they used an ionic detergent, deoxycholate, which has been shown previously [14,31] to extract these RNAs from chromatin. Whether the structural differences noted here (the nuclear species are all 'capped' while the cytoplasmic species are unmodified) is related to their subcellular localization is not known. However, the 'cap' structure itself is unlikely to be responsible as the cytoplasmic mRNAs are all capped. The rates of synthesis of each of the species N5, N6, and N7 are 20--25% the rate o f synthesis of 5-S rRNA on a molar basis and 5% the rate of synthesis of the 4.5-S t R N A presursor. In contrast, species N3 appears to be made at a lower rate, a b o u t 5--10% the rate of 5 S, and is present in about half the concentration of N5, N6, and N7. Since there are about 100 copies of each of the genes for N5, N6, and N7 compared to 500 copies of 5-S genes, this implies the genes are transcribed at the same relative rate, which is probably close to the
676
maximal possible rate, in exponentially growing cells. All of these genes are probably transcribed by RNA polymerase III (Marzluff and Pan, submitted for publication) as are the 5-S and tRNA genes. The other RNA, N3, may well be transcribed at a lower rate as it is present in a larger number of copies in the genome, close to that of 5 S [14]. Study of the pathway of biosynthesis will require cell-free systems capable of carrying out RNA processing reactions as well as transcription.
Acknowledgements This proposal was supported by grants from NSF and NIH Ag-00413. W.F. M. is a recipient of a Research Career Development Award CA00178 from the National Cancer Institute.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
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