Vol. 46 No. 1
SCIENCE IN CHINA (Series C)
February 2003
Analysis of nucleolar pre-rRNA processing sites in pea (Pisum sativum) LONG Hong (龙 鸿)1,2, ZENG Xianlu (曾宪录)1, JIAO Mingda (焦明大)1, HU Bo (胡 波)1, SUN Haijing (孙海晶)1, LIU Zhenlan (刘振兰)1, ZHANG Liyong (张立勇)1,3 & HAO Shui (郝 水)1 1. Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China; 2. College of Life Science, Nankai University, Tianjin 300071, China; 3. National Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China Correspondence should be addressed to Hao Shui (email:
[email protected]) Received May 8, 2002
Abstract The location of rRNA processing was analyzed by using in situ hybridization with ITS1 probe and immunolabeling of anti-fibrillarin mAb in pea (Pisum sativum) root pole cells. The results showed that rRNA processing sites were in dense fibrillar components (DFCs) and granular components (GCs), but not in fibrillar centers (FCs). Low doses of actinomycin D (AMD) treatment can selectively suppress pre-rRNA synthesis but cannot disturb the processing of preformed pre-rRNAs. With AMD treatment prolonged, the density of labeled signals gradually decreased, indicating the preformed pre-rRNAs were gradually processed. Keywords: Pisum sativum, nucleolus, rRNA, processing, fibrillar centres, dense fibrillar components, granular components.
Nucleolus is the organelle which is responsible for ribosome production in eukaryotic cells. It is the region of rDNA (the genes encoding three of the ribosomal RNA species: 18S, 5.8S, and 28S) transcription and precursor rRNA (pre-rRNA) processing[1,2]. Production of mature ribosomal RNAs involves an intricate series of pre-rRNA cleavages, which may occur via the simultaneous operation of alternative processing pathways[3]. The knowledge of the exact position of rRNA processing in nucleolus is essential for understanding the function of nucleolus[4]. rRNA gene transcription unit is transcribed by RNA polymerase Ⅰ, and a single 45S pre-rRNA precursor (about 13000 nucleotide) is generated after transcription. This precursor includes a 5′external transcribe spacer (5′ETS), preceding the 18S rRNA sequence; two internal transcribed spacer sequences (ITS1, ITS2) which flank the 5.8S rRNA sequence, and a short 3′external transcribe spacer (3′ETS), downstream the 28S rRNA sequence. rRNA processing can be divided into early events (producing 18S rRNA after precursor cleavage) and late events (producing 5.8S rRNA and 28S rRNA), and different splicing factors take part in the events respectively. The nucleolar ultrastructure is generally described as being composed of three types of structures, that is fibrillar centers (FCs), dense fibrillar components (DFCs), and granular components (GCs). Different
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studies have been focused on the processing sites of pre-rRNA in nucleolus with different methods. The early autoradiographic data showed that when rRNA was labeled with 3U-UTP for short times (<10 min), the signals were solely in DFC, and when labeling time extended, the signals appeared in GC[5
—9]
. But some later in situ hybridization experiments showed that 5′ETS signals were re-
stricted to DFC in mouse[10] and pea[11], but to FC and DFC in human[12], and the labeling signals of ITS1 and ITS2 were restricted to DFC in pea[11] but to DFC and GC in human[12]. So, it is still not clear on the exact pre-rRNA processing sites in nucleolus[1]. As an important rRNA splicing factor, fibrillarin is essential for pre-rRNA processing. Some experiments had showed that fibrillarin was involved in rRNA splicing[13], and nucleolar localization of fibrillarin can provide indirect evidence to rRNA processing. Unfortunately, it is also unclear on the subnucleolar distribution of fibrillarin[14,15]. In this paper we detect the pre-rRNA processing sites using in situ hybridization of ITS1 probe and immunolabeling of anti-fibrillarin monoclone antibody. 1 1.1
Materials and methods Materials The root pole cells of Pisum sativum were used as material.
1.2
Preparation of specimen Treatment with low doses of actinomycin D (AMD) can selectively suppress the synthesis of pre-rRNA according to Perry et al.[16] and Fetherston et al.[17]. Root pole cells of Pisum sativum were treated with 0.05 μg/mL AMD (Sigma, St. Louis, USA.) for 0.5 h, 1 h, 2 h, 20 h separately, while the control without AMD treatment. Then the root pole cells, with or without drug treatment, were fixed in 2.5% glutaraldehyde (in 0.1 mol/L phosphate buffer, pH 7.4) for 4 h and in 4% osmic acid for 2 h. The fixed pellets were dehydrated in ethanol and embedded in Lowicryl K4M (Chemische Werke Lowi GMBH & Co., Waldkraiburg, Germany) at −40℃. Ultrathin sections (60—80 nm) were made with a Reichert-Jung microtome and collected on Formvar-coated gold grids.
1.3
Preparation of ITS1 probe ITS1 sequence contains 239 bp according to Beven et al.[11]. The ITS1 probe used in this paper is a double-strand DNA synthesized through PCR techniques. The two primers were designed by taking the advantage of DNAsis and Biopro software, and the sequence was as follows: Primer 1: 5′CGATGCCTTATATGC Primer 2: 5′GTGCTCAGAGCAAAG
3′ 3′
PCR amplification was conducted by using genome DNA as template. The probe sequence was as follows: 1 41
CGATGCCTTA TATGCAGTCC AACACGTGAA TTAGTTTGAA CACATGCGGT GGGCTTGAGG TGTTTCACAC CCCAACTTGC
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81 ATTGGCATCG GAGGGGAACG ACAAAATGCG TTCTCTTCTG 121 CCAAAACTCA AACCCCGACG CTGAATGCGT CAAGGAATTA 161 ACTTTGCTCT GAGCAC The sequence was labeled by biotin using nick translation method after PCR products were purified. 1.4
in situ hybridization of ITS1 probe For the detection of ITS1, 50 μL hybridization mixture (containing 25 μL deionized forma-
mide, 10 μL 50% dextran sulfate, 5 μL 20×SSC buffer, 2 μL of 10 mg/mL solution of E. coli DNA and 8 μL of the biotinylated ITS1 DNA at 10 μg/mL) was prepared in 1.5 mL Eppendorf tube. In order to denature the double-stranded DNA, the eppendorf tube was plunged into boiling water and then transferred to melting ice for 5—10 min. The grids bearing ultrathin sections of glutaraldehyde-fixed cells were coated with 1—2 μL hybridization mixture. The in situ hybridization was performed at 55℃ for 3.5 h. After hybridization, the grids were immediately washed 3 times with PBS. Immunocytological detection of biotin was carried out by incubating the grids in extravidin-gold (from Sigma, diluted at 1︰25 in PBS) at room temperature for 30 min. After labeling the grids were stained with 5% aqueous uranyl acetate for 10 min, examined and photographed under a Hitachi-600-B transmission electron microscope at 75 kV. For control samples, hybridization mixture was replaced by diluted probe solution. 1.5
Immunogold labelling The sections were treated with 1% saturated NaIO4 for 20 min, washed first by deionized water and then by 0.01 mol/L PBSTG (pH 7.4), then blocked with 1% BSA in 0.01 mol/L PBSTG (pH 7.4, containing 0.9% NaCl, 0.2% Tween-20 and 15 mmol/L glycine) for 20 min. The sections were subsequently incubated with anti-fibrillarin monoclone antibody mAb P2G3 (kindly gifted by Prof. M. Christensen, Georgetown College, USA) at 1︰500 dilution in 0.01 mol/L PBSTG (pH 7.4) containing 0.1% BSA at 4℃ for 4 h in wet box. Then the sections were washed in 0.01 mol/L PBSTG (pH 7.4 and pH 8.0), and incubated with 10 nm protein A-colloidal gold (Sigma) at 1︰25 dilution in 0.01 mol/L PBSTG (pH 8.0, containing 0.1% BSA) at room temperature for 2 h in a wet box, then washed with 0.01 mol/L PBS (first pH 8.0, then pH 7.4) and deionized water. The sections were stained with 5% uranyl acetate for 10 min before being observed and photographed with a Hitachi-600-B transmission electron microscope at 75 kV. Control sections were processed with the same methods except for using deionized water instead of mAb P2G3. 1.6
Statistic analysis of immunolabelling Statistic analysis of gold particles in photographs of transmission electron microscope was carried out by using an IBAS images system[18].
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Results
In situ hybridization wih ITS1 The nucleoli of Pisum sativum consist of three basic components (fibrillar centers, dense fibrillar components, and granular components) under electron microscope. Fibrillar centers (FCs) are lightly stained regions, surrounded by dense fibrillar components. Dense fibrillar components (DFCs) are much more densely stained regions extending from the fibrillar centers. Granular components (GCs) are the regions containing more or less densely packed granules. Our results of ITS1 in situ hybridization showed that, in the group without AMD treatment, the labeled signals were observed in the regions of DFCs and GCs, but the regions of FCs were devoid of labeling (fig. 1(a)). Following a treatment with a low dose of AMD for 0.5 h, the labeled signals were distributed in DFCs and GCs, but not in FCs (fig. 1(b)). As for the AMD treatment for 1 h, gold particles still localized in DFCs and GCs, but the density of signals became low. Once again, FCs were devoid of labeling (fig. 1(c)). As for the AMD treatment for 2 h, the labeled signals were observed in DFCs and GCs, and the labeled signals were decreased (fig. 1(d)) compared with that above. There was no labeled signal in FCs either. As for the AMD treatment for 20 h, gold particles localized in DFCs and GCs. The density of gold particles was even lower than before (fig. 1(e)). No signal was observed in FCs (fig. 1(e)). Very few particles were detected in the regions outside nucleolus, showing specificity of our labeling experiments. In control group without ITS1 probe, very few gold particles were found in the nuclei (fig. 1(f)), also showing that the labeling system was specific. Statistical analysis of gold particles was performed by using IBAS images system. The average density of the gold particles in the without AMD treatment group was 65.51 2.1
particles/μm2, while the data of AMD treatment for 0.5 h, 1 h, 2 h, 20 h were 48.17, 21.68, 16.86, 5.78 particles/μm2 respectively (table 1). The results showed that as the time of AMD treatment increased, the density of the gold particles gradually decreased. Table 1 Density of gold particles by in situ hybridization of ITS1 probe with AMD treatment for different intervals (area: 0.346 μm2) Specimen 0h
1
gold (number) 2
density (number/μm ) 0.5 h
gold (number) density (number/μm2)
1h
gold (number) density (number/μm2)
2h
gold (number) density (number/μm2)
20 h
gold (number) density (number/μm2)
2
3
4
5
6
26
22
24
21
21
22
75.14
63.59
69.36
60.69
60.69
63.59
14
19
15
18
14
20
40.46
54.91
43.35
52.02
40.46
57.80
8
7
8
6
7
9
23.12
20.23
43.35
17.34
20.23
26.01
7
8
5
5
4
6
20.23
23.12
14.45
14.45
11.56
17.34
2
3
2
3
1
1
5.78
8.67
5.78
8.67
2.89
2.89
M
65.51±5.68 48.17±7.68 21.68±3.03 16.86±4.25 5.78±2.58
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Fig. 1. The distribution of in situ hybridization signals of ITS1 probe in the nucleolus of Pisum sativum treated with AMD for different intervals. (a) 0 h (no AMD) treatment, gold particles were distributed in DFC and GC with high density, while no signal was observed in FC. 51000×; (b) AMD treatment for 0.5 h, the distribution of gold particles was the same as above, but the density is lower. 51000×; (c) AMD treatment for 1 h, the distribution of gold particles was the same as above, the density of gold particles was even lower. 54000×; (d) AMD treatment for 2 h, the distribution of gold particles was the same as above, the density of gold particles was much lower. 54000×; (e) AMD treatment for 20 h, the distribution of gold particles was the same as above, there were very few gold particles observed. 54000×; (f) control, no labeled signal was observed in the nucleolus. 51000×.
2.2
Immunolabeling The results of immunolabeling of anti-fibrillain mAb were similar to that of in situ hybridiza-
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tion above. In the test group without AMD treatment (fig. 2(a)), low dose of AMD treatment for 0.5 h (fig. 2(b)), 1 h (fig. 2(c)), 2 h (fig. 2(d)), 20 h (fig. 2(e)), the labeled signals were solely detected in DFCs and GCs, not in FCs. After a prolonged AMD treatment, the density of labeled
Fig. 2. The distribution of immunolabeling signals of anti-fibrillarin antibody in the nucleolus of Pisum sativum treated with AMD for different intervals. (a) 0 h (no AMD) treatment, gold particles were distributed in DFC and GC with high density, while no signal was observed in FC; (b) AMD treatment for 0.5 h, the distribution of gold particles was the same as above, but the density is lower; (c) AMD treatment for 1 h, the distribution of gold particles was the same as above, the density of gold particles was even lower; (d) AMD treatment for 2 h, the distribution of gold particles was the same as above, the density of gold particles was much lower; (e) AMD treatment for 20 h, the distribution of gold particles was the same as above, there were very few gold particles observed; (f) control, no labeled signal was observed in the nucleolu (all figures, ×51000).
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signals decreased. In control group, no signal was detected in the nucleolus (fig. 2(f)). Statistical analysis of gold particles was performed (table 2). The average density of the gold particles in the without AMD treatment was 72.53 particles/μm2, while the data of AMD treatment for 0.5 h, 1 h, 2 h, 20 h were 49.61, 18.30, 10.12, 7.71 particles/μm2 respectively (table 2). The results also showed that after a prolonged AMD treatment (0.5 h, 1 h, 2 h, 20 h), the density of the gold particles gradually decreased. Table 2 Density of gold particles by immunolabeling of anti-fibrillarin antibody with AMD treatment for different intervals (area: 0.346 μm2) 0h 0.5 h 1h 2h 20 h
3
Specimen gold (number) density (number/µm2) gold (number) density (number/µm2) gold (number) density (number/µm2) gold (number) density (number/µm2) gold (number) density (number/µm2)
1 24 69.36 13 37.57 6 17.34 3 8.67 3 8.67
2 21 60.69 20 57.80 5 14.45 4 11.56 2 5.78
3 28 80.92 17 49.13 6 17.34 5 14.45 2 5.78
4 26 75.14 18 52.02 7 20.23 3 8.67 2 5.78
5 53 85.48 15 43.35 6 17.34 2 5.78 3 8.67
6 22 63.59 20 57.80 8 23.12 4 11.56 4 11.56
M 72.53±9.75 49.61±8.05 18.30±2.98 10.12±3.03 7.71±2.36
Discussion
The activity of nucleolus involves rDNA transcription, pre-rRNA processing, and synthesis of 40S and 60S ribosome subunits. It is still controversial on the subnucleolus localization of these events. Early pulse-chase experiments showed that the intermediate products of rRNA processing transported among the components of nucleolus[19,20], but later, a number of experiments demonstrated that active rDNA were localized at the boundary between FC and DFC[21
—23]
, although
some immunoelectron microscopic cytochemistry studies showed that rDNA and RNA polymerase I exists in FC[24]. There are also some experiments supporting the results above, including 5′EST in situ hybridization, showing detected signals in FCs[25] and DFCs[12,26,27] but very few in GCs[26]. In situ hybridizations with ITS1 and ITS2 revealed that the signals were distributed in DFCs and GCs first, and then disappeared after AMD treatment[26]. Confocal microscopy showed that ITS1 was distributed in the corresponding regions of DFCs[11]. However, confocal microscopy cannot conduct a precise subnucleolar localization because of its optical limitation. In the present study, we show the results of in situ hybridization at electron microscope level with high resolving power. Fibrillarin is a basic protein of Mr 34000—38000, localized in the nucleolus, and showed to be much evolutionary conservation[28]. The main feature of fibrillarin is that it has a motif which is rich in glycine and dimethylarginine (called the GAR domain) and a RNA recognition sequence
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(RRM)[29]. Based on these sequences, fibrillarin can associate with other proteins and several species of snoRNAs including U3, U8, U13, U14 and U15 through its specific sites [box C (UGAUGA) and box D (CUGA)][30]. The splicing complex composed of proteins and RNAs plays an important role in pre-rRNA processing. Some experiments showed that fibrillarin was involved in 5′ETS and 18 S rRNA processing[31
—34]
. Ochs et al. detected fibrillarin in FC and DFC by the
[35]
method of human autoimmune sera , while others detected it in DFC[36,37]. Our results showed that in the without AMD treatment group, labeled signals were observed in the regions of DFCs and GCs with high density, implying the DFCs and GCs were the localization of the cleavage of ITS1, as well as 5′ETS and 18 S rRNA, which was associated with fibrillarin. The regions of FCs were devoid of labeling, indicating no processing events occurred in the regions. Following a time interval treatment with a low dose of AMD for 0.5 h, 1 h, 2 h, 20 h, the density of labeling signals decreased, showing that low dose of AMD can selectively suppress pre-rRNA, and allow processing of preformed pre-rRNAs. Our results from the nucleolus of Pisum sativum demonstrated that the early events of rRNA processing (producing 5′ETS, 18 S rRNA, and ITS1) happened in DFCs and GCs, but not in FCs. Acknowledgements
This work was supported by the Major State Basic Research Program (973) of China.
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