Molecular and Biochemical Parasitology, 44 (1991 ) 33-42
33
Elsevier MOLBIO 01427
Ultraviolet irradiation inhibits RNA decay and modifies ribosomal RNA processing in Trypanosoma brucei Hrl~ne Coquelet, Maurice Steinert and Etienne Pays Department of Molecular Biology, University of Brussels, Rhode-St.-Gendse, Belgium (Received 21 May 1990; accepted 19 July 1990)
A known effect of ultraviolet radiation on transcription is the arrest of RNA elongation. In Trypanosoma brucei, we show that UV also inhibits RNA decay, leading to specific accumulation of transcripts from the beginning of several transcription units. In addition, UV irradiation changes the pattern of ribosomal RNA processing, probably by altering the order in which the non-coding spacers are excised. These effects are still observed on RNA synthesized more than 2 h after irradiation, and do not depend on protein synthesis. Key words: Trypanosome; Ultraviolet irradiation; RNA decay; Ribosomal RNA processing
Introduction Ultraviolet irradiation is known to block RNA elongation through generation of pyrimidine dimers in the DNA. Transcription is inhibited as a function of both the distance from the initiation site and the dose of irradiation [1]. Except for a set of specific genes whose activity is enhanced by UV irradiation [2-4], no effect of UV on transcription initiation has been noted [1]. However, we recently reported that UV irradiation induces an apparent increase of transcription at the beginning of two trypanosome transcription units, those of the variant surface glycoprotein (VSG) and rDNA genes [5-7]. This paper deals with the characterization of the effects of UV on transcription in Trypanosoma brucei. T. brucei displays transcription characteristics which differ from those of many other eukaryotes, such as trans-splicing of the mRNAs [8-10], and Correspondence to: Etienne Pays, Department of Molecular Biology, University of Brussels, 67, rue des Chevaux, B-1640 Rhode-St-Gen~se, Belgium
Abbreviations: VSG ES, variant surface glycoprotein gene expression site; LSU, large ribosomal subunit; IVS, intervening spacer; ITS, internal transcribed spacer; LETS, leader external transcribed spacer.
polycistronic transcription [ 11-13]. These characteristics have so far precluded the characterization of transcription promoters, such as those of the genes encoding the major surface proteins, the VSG in bloodstream forms and procyclin in procyclic forms. Another unusual feature of T. brucei is the number of RNA molecules associated with the,large ribosomal subunit (LSU). This LSU indeed contains two large and four small RNA species in equimolecular amounts, in addition to the 5S and the 5.8S rRNAs [14]. Structural studies of these RNAs have shown that they are the homologous counterparts of the single 28S RNA from other eukaryotes [15]. Some insects and other protozoa also possess 28S RNA subunits divided into two molecules [16-18], resulting from the excision of an additional transcribed spacer in the rDNA unit. This sequence was called the 'intervening spacer' (IVS) to distinguish it from the 'internal transcribed spacers' (ITS), separating the 18S from the 5.8S and the 5.8S from the 28S rRNA subunits. The rRNA genes of trypanosomes are clustered in tandemly repeated units, as in other eukaryotes, and, within each unit, the six RNA species of the LSU are encoded separately [19, 20]. The synthesis of the LSU RNA thus requires
0166-6851/91/$03.50 © 1991 Elsevier science Publishers B.V. (Biomedical Division)
34 extensive post-transcriptional processing, involving the removal of five transcribed spacers or IVS. We here confirm and extend our observation that UV irradiation causes an apparent stimulation of transcription at the beginning of the trypanosome VSG and rDNA transcription units. We also show that the same effect is observed in the procyclin gene transcription unit. UV irradiation may thus provide a means of identifying transcription promoter regions. We present evidence that the specific enrichment of promoter-proximal transcripts is due to inhibition of RNA decay. In addition, we show that UV irradiation also alters the pathway of processing of primary ribosomal transcripts. Since cleavages of rRNA precursors are primarily dictated by the RNA secondary structure [16-18], it is possible that the effects of UV on RNA metabolism are through conformational alteration of RNA. Materials and Methods The procedures for DNA and RNA isolation, Southern and Northern blot hybridization as well as DNA cloning, were as described [21]. The T. brucei rDNA transcription unit was cloned from two genomic DNA libraries. The beginning of the rDNA unit up to the gene of the first large fragment of the LSU (sometimes called the a-28S, or LS 1) was cloned from partial Sau3A digests of AnTat 1.1B DNA inserted into A 1059. Different fragments from this region were subcloned in pUC 18. The end of the rDNA unit (genes of the o~and fl-28S, or LS 1 and LS2, and of the four small RNAs) was cloned from a A gtwes EcoRI genomic library of the AnTat 1.1B variant, and subcloned directly in pUC 18. Run-on transcription assays were conducted as described by Murphy et al. [22]. The standard assay (1 ml) contained 500 #g of DNA in nuclei/12.5 % (v/v) glycerol/0.8 mg heparin/5 mM spermidine/5 mM MGC12/2.5 mM dithiothreitol/10 mM Tris-HC1 (pH 8)/0.5 mM ATP, UTP, CTP/1 mCi [~-32p]GTP (2000 Ci m m o l - l ) . The nuclei were usually incubated for 30 min at 30°C. UV irradiation was performed under the conditions defined by Johnson et al. [12]. Briefly, the blood containing trypanosomes was diluted in Baltz medium at 37°C to reach a parasite con-
centration of 1 - 4 × 1 0 7 ml - I . Samples (125 ml) were irradiated at 254 nm (1 J s -x m - 2 ) in sterile square dishes (22 × 22 cm) (Bio-Assay, Nunc, Roskilde, Denmark) usually for 1 min and with agitation. The irradiated cells were then transferred to culture flasks and kept in the dark, usually for 30 min, until centrifugation. Results UV irradiation of trypanosomes causes an apparent stimulation of initial transcription in isolated nuclei. We analyzed the effect of UV on the activity of several transcription units, by run-on assays with isolated nuclei from either control or irradiated cells. Two of these units have been characterized so far, the 9.5-kb rDNA unit [19] and the 45-kb AnTat 1.3A VSG gene expression site [5]. We also present data on the procyclin and actin genes. In isolated nuclei, transcription of the rDNA unit appears to start within a 600-bp RsaI fragment located 1.1 kb upstream from the 18S rRNA gene (Fig. 1A, fragment 3), as expected from previous studies [19]. If, prior to the isolation of the nuclei, the trypanosomes are exposed to increasing UV doses (254 nm; 1 J s -1 m - 2 for 0, 0.5, 1, 2 and 4 min), transcription of the 5' region of the rDNA unit appears to be stimulated by UV. As shown by the graph in Fig. 1, the increase is highest near the transcription start site, and seems to fade out in the ITS between the 18S and the 28S rRNA genes. In this experiment, the effect is optimal after 1 min irradiation, a 6-fold increase being observed near the transcription start site (fragment A3) under conditions where total transcription is inhibited by 60 %. After 4 min irradiation, the hybridization level decreases with increasing distance from the transcription start. Under similar conditions, an apparent stimulation of transcription by UV is also observed in the 5' part of the VSG gene expression site, as shown in Fig. 2A. The results are however less straightforward than in the rDNA case, for at least two reasons. First, the genomic region covered is partially repeated (ESAG 7 and 6 are almost identical; ref. 5), so that the distance-from-promoter effect, clearly shown in the preceding example, cannot be demonstrated in the case of fragments
35
®
©
!:i''
"
C
UV
E
D
.R
B
UV
Hd "18s"
-
........
C
D
C
I
::,
"58s"
2 ,
"
C
UV
"
uv/c
P "28s"
i
q~),;;::
UV
1 ,
2 ~Kb
3
U~
1
2
4rain
Fig. 1. Apparent stimulation of transcription by UV at the beginning of the rDNA transcription umt. RNA synthesized in nuclei from AnTat 1.3A bloodstream forms was hybridized to Southern blots of sequences cloned from the rDNA. In each panel, the first lane shows the ethidium bromide staining of the restriction digest, while lanes labeled 'c' and 'uv' show, from left to right, the hybridization pattern after UV irradiation for 0, 0.5, 1, 2 and 4 min respectively. The interpretation of the restriction digests is shown in the map, where the filled boxes refer to the rRNA genes. Plasmid vector sequences are marked by diamonds in each clone (A, B, C, D). The arrow under the restriction map indicates the transcription start site, as defined by White et al. [19]. The abbreviations for the restriction endonuclease sites are: B, BgIII; E, EcoRI; Hd, HindIII; P, PstI; R, Rsal. In the graph, the ratio of hybridization of 'uv' transcripts relative to the unirradiated control probes ('c') was determined by liquid scintillation counting and is presented on a logarithmic scale. The designation of fragments (A2 etc.) refers to those in the maps. The dotted curve shows the effect of UV irradiation on total RNA synthesis, as measured by liquid scintillation counting of trichloroacetic acid precipitates.
4-6 (data for these fragments have therefore been pooled in the corresponding graph). Second, it contains a RIME (R) element, copies of which exist and are transcribed elsewhere in the genome, clouding the data concerning fragment 2. Nevertheless, it is clear that UV induces an accumulation of transcripts from this initial region of the VSG expression site. The peak of stimulation is reached earlier for fragments more distant from the transcription start (fragments 4-6 as compared to 3, in panel A), as expected from the distancedependent inhibition of elongation. Beyond this region transcription is severely inhibited by UV irradiation, and the VSG gene, the most distal gene in this long transcription unit, is completely silenced [5, 6]. The procyclin genes are not expressed in the bloodstream form. However, transcription of these genes is observed in these forms in run-on assays,
strongly suggesting a post-transcriptional regulation (Fig. 2B). UV irradiation leads to a considerable apparent stimulation of this transcription (Fig. 2B), in sharp contrast with the inhibitory effect exerted on transcriptton of what is most probably the 3' end of an upstream transcription unit (fragment 1, Fig. 2B), and on transcription of the actin genes (Fig. 2C). The differential effect on the procyclin and actin genes is consistent with the available data regarding the distance of these genes to their respective promoters. Indeed, the procyclin genes appear to be positioned at a short distance from their promoter, whereas the actin genes appear to be about 5.5 kb downstream (E.P. et al., unpublished). Transcription of the actin genes is therefore more prone to elongation arrest following UV irradiation.
36
® UV :
0
®
.5
1
1-
2
0
©
.5
1
2
0
.5
1
2 rnin
. . . . . . .
4~
.°=
_
uv/c
10
5'
1,5- " ~ " ' e .
~ . -~~ .
.2
UV:
.5
1
2
.5
1
2
.5
1
2 rain
pES200.12
®T,,,
'
T
3
, ~ ? ~
pAP2
Z5
5 kb
Fig. 2. Apparent stimulation of transcription by UV at the beginning of the variant surface glycoprotein and procyclin gene transcription units. Run-on transcription assays have been conducted on nuclei from bloodstream forms, UV-irradiated for different periods (0, 0.5, 1 or 2 min, as indicated). The 32p-labeled transcripts were hybridized with stoichiometric amounts of restriction digests from different recombinant plasmids: pES200.12 (panel A) is specific to the beginning of the VSG expression site, and carries ESAGs 7 and 6 together with a RIME retroposon (R) [5], while pAP2 (panel B) contains a 4.7-kb PvuII fragment from the procyclin A locus of IITat 1.21, consisting of 3.7 kb of 5~ flanking sequence, the first of three tandem procyclin genes (P) and 0.3 kb of the 3' flanking sequence [30]. The plasmid in panel C contains a copy of the T. brucei actin gene, in a 1.5-kb SalI fragment (fragment 2). Plasmids in A and B were digested by PvuII + AvaIII, and the interpretation of the digests is shown in the maps, with black boxes for sequences from the plasmid vectors and open boxes for the genes and RIME. The data were quantitated by liquid scintillation counting, as shown in the corresponding graphs. The numbers in the graphs refer to the fragment numbers, indicated at the left of the ethidium bromide stainings. The effect of UV on overall transcription in isolated nuclei is shown by the dotted curve in the graph of panel A: this represents the percentage of acid-precipitable RNA synthesized after irradiation with different UV doses, taking RNA from unirradiated cells as 100%.
Analysis of the apparent stimulation in run-on assays. In the e x p e r i m e n t s d e s c r i b e d a b o v e , the app a r e n t stimulation o f t r a n s c r i p t i o n was o b s e r v e d in 3 0 - m i n i n c u b a t i o n a s s a y s at 30°C. In o r d e r to see if this effect is critically d e p e n d e n t on the length o f the in vitro incubation, w e m e a s u r e d the a p p a r ent s t i m u l a t i o n at the b e g i n n i n g o f the three tran-
scription units a l o n g with the kinetics o f o v e r a l l R N A synthesis. A s s h o w n in Fig. 3 (bottom), a s i m i l a r level o f s t i m u l a t i o n is o b t a i n e d in short (5 min) or p r o l o n g e d ( 6 0 - 9 0 min) i n c u b a t i o n periods. In this s y s t e m , the m a x i m u m i n c o r p o r a t i o n o f n u c l e o t i d e s is a c h i e v e d after 60 m i n in c o n t r o l nuclei, and 30 m i n in nuclei f r o m i r r a d i a t e d cells
37 (Fig. 3, top). We also tested if protein synthesis was required to achieve the apparent stimulation of run-on transcription following UV. No significant difference in the level of stimulation was observed if over 90% of protein synthesis was inhibited by performing UV irradiation and subsequent 30 minincubation of trypanosomes in the presence of 50 #g ml - ] cycloheximide and 25 # g ml - ] puromycin (Fig. 3, bottom).
UV irradiation causes the accumulation of steadystate transcripts from genes located at a short distance from the promoter. The apparent stimulation, following exposure to UV, of transcription of the 5 ~ part of the VSG expression site and of cpm (x 1 0 4) 20
/ 10 ~
'
A~
'
3o
A
'
io
UV --A
'
~)
min
UVIC rDNA: VSG: PRO:
3.2
4.7
4.4[~4
5.04.5 4.8
4.4
4.2
4.7
4.9~5
4.2
3.1
4.8
Fig. 3. Kinetics of run-on transcription in nuclei from control and UV-irradiated trypanosomes. Run-on transcription assays have been conducted on nuclei from either unirradiated ('c') or UV-treated (60 J m -2) ('uv') bloodstream forms, for different periods at 30°C. At each time interval, a 5-/zl sample was precipitated in 10% trichloroaceticacid and counted, while the remaining (995 #1) was processed for hybridizationwith DNA sequences from the beginning of the rDNA, VSG and procyclin gene transcription units (fragments 3 in Figs. 1 and 2). The ratio of apparent stimulation for each transcription unit is indicated for different incubation periods below the graph. The second columnof data for the 30 min-assay (cyclo/puro)shows results obtained if UV irradiation and subsequent 30 min incubation of trypanosomesin vitro are performed in the presence of 50 #g ml-1 cycloheximide and 25 #g ml-I puromycin. Under these conditions,the inhibition of protein synthesis was more than 90%, as determined by incubation of l-ml samples from control and UV-treated trypanosomes in the presence of 50 #Ci of [35S]methionine, followed by trichloroacetic acid precipitation. This experiment also showed that the UV treatment itself inhibits protein synthesis by approximately 50%.
the procyclin genes is reflected in Northern blots of steady-state transcripts from these transcription units (Fig. 4A, lanes 'uv'). The first panel shows the accumulation of transcripts characteristic of the 5' part of the VSG gene expression site (essentially ESAGs 6 and 7), and a similar increase in steady-state transcripts is seen for the procyclin genes (third panel). On the contrary, no such UV-dependent accumulation is observed for VSG and actin genes (panels 2 and 4), which are more distant from their promoter. In addition, the experiment also illustrates the changes in transcriptional patterns induced in the bloodstream trypanosomes by the temperature shift (from 37°C to 20°C) applied at time 0 (lanes 'c'): one sees a progressive down regulation of the VSG gene expression site (panels 1 and 2), a stimulation of the procyclin genes (panel 3), while transcription of the actin genes is not significantly affected (panel 4). The reasons for this temperature shift are presented in the next section.
The accumulation of transcripts from the beginning of the VSG and procyclin gene transcription units is due to inhibition of RNA decay. The apparent stimulation of transcription by UV is most probably due to inhibition of RNA degradation, as demonstrated in the second panel of Fig. 4A. This panel shows the amount of VSG mRNA present in cells at different time intervals after the VSG gene has been inactivated. This inactivation was achieved by shifting the incubation temperature from 37°C to 20°C, conditions which are known to trigger the transformation of bloodstream into procyclic forms and completely suppress the production of run-on transcripts of the VSG gene [5]. The progressive decrease of the VSG mRNA amount in unirradiated trypanosomes (lanes 'c') thus represents the rate of degradation of the preexisting VSG mRNA. It clearly appears that following UV irradiation, the VSG mRNA decay is delayed (lanes 'uv'). This observation is extended in Fig. 4B, showing results of pulse chase RNA labelling experiments, which allow the measurement of RNA decay irrespective of the actual transcriptional activity of a gene. The inhibiting effect of UV on RNA degradation is observed for transcripts from both the beginning and the end of the VSG transcription unit, as well as for procyclin
38
c . , c u cu
c u cU cU kb , ', - - I
I
~ I
3-
il
I ]
kb
',I I I
i I
I
c~lcU cU
cUcUcU
2.2-
1 . 6 - ~
,-',
I I I I
~,
,,
! I I
I
I I
I
0
2
I
0
4h
2
cpm (xlOO) 10
40
120 1 801
C
33
Elsevier MOLBIO 01427
Ultraviolet irradiation inhibits RNA decay and modifies ribosomal RNA processing in Trypanosoma brucei Hrl~ne Coquelet, Maurice Steinert and Etienne Pays Department of Molecular Biology, University of Brussels, Rhode-St.-Gendse, Belgium (Received 21 May 1990; accepted 19 July 1990)
A known effect of ultraviolet radiation on transcription is the arrest of RNA elongation. In Trypanosoma brucei, we show that UV also inhibits RNA decay, leading to specific accumulation of transcripts from the beginning of several transcription units. In addition, UV irradiation changes the pattern of ribosomal RNA processing, probably by altering the order in which the non-coding spacers are excised. These effects are still observed on RNA synthesized more than 2 h after irradiation, and do not depend on protein synthesis. Key words: Trypanosome; Ultraviolet irradiation; RNA decay; Ribosomal RNA processing
Introduction Ultraviolet irradiation is known to block RNA elongation through generation of pyrimidine dimers in the DNA. Transcription is inhibited as a function of both the distance from the initiation site and the dose of irradiation [1]. Except for a set of specific genes whose activity is enhanced by UV irradiation [2-4], no effect of UV on transcription initiation has been noted [1]. However, we recently reported that UV irradiation induces an apparent increase of transcription at the beginning of two trypanosome transcription units, those of the variant surface glycoprotein (VSG) and rDNA genes [5-7]. This paper deals with the characterization of the effects of UV on transcription in Trypanosoma brucei. T. brucei displays transcription characteristics which differ from those of many other eukaryotes, such as trans-splicing of the mRNAs [8-10], and Correspondence to: Etienne Pays, Department of Molecular Biology, University of Brussels, 67, rue des Chevaux, B-1640 Rhode-St-Gen~se, Belgium
Abbreviations: VSG ES, variant surface glycoprotein gene expression site; LSU, large ribosomal subunit; IVS, intervening spacer; ITS, internal transcribed spacer; LETS, leader external transcribed spacer.
polycistronic transcription [ 11-13]. These characteristics have so far precluded the characterization of transcription promoters, such as those of the genes encoding the major surface proteins, the VSG in bloodstream forms and procyclin in procyclic forms. Another unusual feature of T. brucei is the number of RNA molecules associated with the,large ribosomal subunit (LSU). This LSU indeed contains two large and four small RNA species in equimolecular amounts, in addition to the 5S and the 5.8S rRNAs [14]. Structural studies of these RNAs have shown that they are the homologous counterparts of the single 28S RNA from other eukaryotes [15]. Some insects and other protozoa also possess 28S RNA subunits divided into two molecules [16-18], resulting from the excision of an additional transcribed spacer in the rDNA unit. This sequence was called the 'intervening spacer' (IVS) to distinguish it from the 'internal transcribed spacers' (ITS), separating the 18S from the 5.8S and the 5.8S from the 28S rRNA subunits. The rRNA genes of trypanosomes are clustered in tandemly repeated units, as in other eukaryotes, and, within each unit, the six RNA species of the LSU are encoded separately [19, 20]. The synthesis of the LSU RNA thus requires
0166-6851/91/$03.50 © 1991 Elsevier science Publishers B.V. (Biomedical Division)
34 extensive post-transcriptional processing, involving the removal of five transcribed spacers or IVS. We here confirm and extend our observation that UV irradiation causes an apparent stimulation of transcription at the beginning of the trypanosome VSG and rDNA transcription units. We also show that the same effect is observed in the procyclin gene transcription unit. UV irradiation may thus provide a means of identifying transcription promoter regions. We present evidence that the specific enrichment of promoter-proximal transcripts is due to inhibition of RNA decay. In addition, we show that UV irradiation also alters the pathway of processing of primary ribosomal transcripts. Since cleavages of rRNA precursors are primarily dictated by the RNA secondary structure [16-18], it is possible that the effects of UV on RNA metabolism are through conformational alteration of RNA. Materials and Methods The procedures for DNA and RNA isolation, Southern and Northern blot hybridization as well as DNA cloning, were as described [21]. The T. brucei rDNA transcription unit was cloned from two genomic DNA libraries. The beginning of the rDNA unit up to the gene of the first large fragment of the LSU (sometimes called the a-28S, or LS 1) was cloned from partial Sau3A digests of AnTat 1.1B DNA inserted into A 1059. Different fragments from this region were subcloned in pUC 18. The end of the rDNA unit (genes of the o~and fl-28S, or LS 1 and LS2, and of the four small RNAs) was cloned from a A gtwes EcoRI genomic library of the AnTat 1.1B variant, and subcloned directly in pUC 18. Run-on transcription assays were conducted as described by Murphy et al. [22]. The standard assay (1 ml) contained 500 #g of DNA in nuclei/12.5 % (v/v) glycerol/0.8 mg heparin/5 mM spermidine/5 mM MGC12/2.5 mM dithiothreitol/10 mM Tris-HC1 (pH 8)/0.5 mM ATP, UTP, CTP/1 mCi [~-32p]GTP (2000 Ci m m o l - l ) . The nuclei were usually incubated for 30 min at 30°C. UV irradiation was performed under the conditions defined by Johnson et al. [12]. Briefly, the blood containing trypanosomes was diluted in Baltz medium at 37°C to reach a parasite con-
centration of 1 - 4 × 1 0 7 ml - I . Samples (125 ml) were irradiated at 254 nm (1 J s -x m - 2 ) in sterile square dishes (22 × 22 cm) (Bio-Assay, Nunc, Roskilde, Denmark) usually for 1 min and with agitation. The irradiated cells were then transferred to culture flasks and kept in the dark, usually for 30 min, until centrifugation. Results UV irradiation of trypanosomes causes an apparent stimulation of initial transcription in isolated nuclei. We analyzed the effect of UV on the activity of several transcription units, by run-on assays with isolated nuclei from either control or irradiated cells. Two of these units have been characterized so far, the 9.5-kb rDNA unit [19] and the 45-kb AnTat 1.3A VSG gene expression site [5]. We also present data on the procyclin and actin genes. In isolated nuclei, transcription of the rDNA unit appears to start within a 600-bp RsaI fragment located 1.1 kb upstream from the 18S rRNA gene (Fig. 1A, fragment 3), as expected from previous studies [19]. If, prior to the isolation of the nuclei, the trypanosomes are exposed to increasing UV doses (254 nm; 1 J s -1 m - 2 for 0, 0.5, 1, 2 and 4 min), transcription of the 5' region of the rDNA unit appears to be stimulated by UV. As shown by the graph in Fig. 1, the increase is highest near the transcription start site, and seems to fade out in the ITS between the 18S and the 28S rRNA genes. In this experiment, the effect is optimal after 1 min irradiation, a 6-fold increase being observed near the transcription start site (fragment A3) under conditions where total transcription is inhibited by 60 %. After 4 min irradiation, the hybridization level decreases with increasing distance from the transcription start. Under similar conditions, an apparent stimulation of transcription by UV is also observed in the 5' part of the VSG gene expression site, as shown in Fig. 2A. The results are however less straightforward than in the rDNA case, for at least two reasons. First, the genomic region covered is partially repeated (ESAG 7 and 6 are almost identical; ref. 5), so that the distance-from-promoter effect, clearly shown in the preceding example, cannot be demonstrated in the case of fragments
35
®
©
!:i''
"
C
UV
E
D
.R
B
UV
Hd "18s"
-
........
C
D
C
I
::,
"58s"
2 ,
"
C
UV
"
uv/c
P "28s"
i
q~),;;::
UV
1 ,
2 ~Kb
3
U~
1
2
4rain
Fig. 1. Apparent stimulation of transcription by UV at the beginning of the rDNA transcription umt. RNA synthesized in nuclei from AnTat 1.3A bloodstream forms was hybridized to Southern blots of sequences cloned from the rDNA. In each panel, the first lane shows the ethidium bromide staining of the restriction digest, while lanes labeled 'c' and 'uv' show, from left to right, the hybridization pattern after UV irradiation for 0, 0.5, 1, 2 and 4 min respectively. The interpretation of the restriction digests is shown in the map, where the filled boxes refer to the rRNA genes. Plasmid vector sequences are marked by diamonds in each clone (A, B, C, D). The arrow under the restriction map indicates the transcription start site, as defined by White et al. [19]. The abbreviations for the restriction endonuclease sites are: B, BgIII; E, EcoRI; Hd, HindIII; P, PstI; R, Rsal. In the graph, the ratio of hybridization of 'uv' transcripts relative to the unirradiated control probes ('c') was determined by liquid scintillation counting and is presented on a logarithmic scale. The designation of fragments (A2 etc.) refers to those in the maps. The dotted curve shows the effect of UV irradiation on total RNA synthesis, as measured by liquid scintillation counting of trichloroacetic acid precipitates.
4-6 (data for these fragments have therefore been pooled in the corresponding graph). Second, it contains a RIME (R) element, copies of which exist and are transcribed elsewhere in the genome, clouding the data concerning fragment 2. Nevertheless, it is clear that UV induces an accumulation of transcripts from this initial region of the VSG expression site. The peak of stimulation is reached earlier for fragments more distant from the transcription start (fragments 4-6 as compared to 3, in panel A), as expected from the distancedependent inhibition of elongation. Beyond this region transcription is severely inhibited by UV irradiation, and the VSG gene, the most distal gene in this long transcription unit, is completely silenced [5, 6]. The procyclin genes are not expressed in the bloodstream form. However, transcription of these genes is observed in these forms in run-on assays,
strongly suggesting a post-transcriptional regulation (Fig. 2B). UV irradiation leads to a considerable apparent stimulation of this transcription (Fig. 2B), in sharp contrast with the inhibitory effect exerted on transcriptton of what is most probably the 3' end of an upstream transcription unit (fragment 1, Fig. 2B), and on transcription of the actin genes (Fig. 2C). The differential effect on the procyclin and actin genes is consistent with the available data regarding the distance of these genes to their respective promoters. Indeed, the procyclin genes appear to be positioned at a short distance from their promoter, whereas the actin genes appear to be about 5.5 kb downstream (E.P. et al., unpublished). Transcription of the actin genes is therefore more prone to elongation arrest following UV irradiation.
36
® UV :
0
®
.5
1
1-
2
0
©
.5
1
2
0
.5
1
2 rnin
. . . . . . .
4~
.°=
_
uv/c
10
5'
1,5- " ~ " ' e .
~ . -~~ .
.2
UV:
.5
1
2
.5
1
2
.5
1
2 rain
pES200.12
®T,,,
'
T
3
, ~ ? ~
pAP2
Z5
5 kb
Fig. 2. Apparent stimulation of transcription by UV at the beginning of the variant surface glycoprotein and procyclin gene transcription units. Run-on transcription assays have been conducted on nuclei from bloodstream forms, UV-irradiated for different periods (0, 0.5, 1 or 2 min, as indicated). The 32p-labeled transcripts were hybridized with stoichiometric amounts of restriction digests from different recombinant plasmids: pES200.12 (panel A) is specific to the beginning of the VSG expression site, and carries ESAGs 7 and 6 together with a RIME retroposon (R) [5], while pAP2 (panel B) contains a 4.7-kb PvuII fragment from the procyclin A locus of IITat 1.21, consisting of 3.7 kb of 5~ flanking sequence, the first of three tandem procyclin genes (P) and 0.3 kb of the 3' flanking sequence [30]. The plasmid in panel C contains a copy of the T. brucei actin gene, in a 1.5-kb SalI fragment (fragment 2). Plasmids in A and B were digested by PvuII + AvaIII, and the interpretation of the digests is shown in the maps, with black boxes for sequences from the plasmid vectors and open boxes for the genes and RIME. The data were quantitated by liquid scintillation counting, as shown in the corresponding graphs. The numbers in the graphs refer to the fragment numbers, indicated at the left of the ethidium bromide stainings. The effect of UV on overall transcription in isolated nuclei is shown by the dotted curve in the graph of panel A: this represents the percentage of acid-precipitable RNA synthesized after irradiation with different UV doses, taking RNA from unirradiated cells as 100%.
Analysis of the apparent stimulation in run-on assays. In the e x p e r i m e n t s d e s c r i b e d a b o v e , the app a r e n t stimulation o f t r a n s c r i p t i o n was o b s e r v e d in 3 0 - m i n i n c u b a t i o n a s s a y s at 30°C. In o r d e r to see if this effect is critically d e p e n d e n t on the length o f the in vitro incubation, w e m e a s u r e d the a p p a r ent s t i m u l a t i o n at the b e g i n n i n g o f the three tran-
scription units a l o n g with the kinetics o f o v e r a l l R N A synthesis. A s s h o w n in Fig. 3 (bottom), a s i m i l a r level o f s t i m u l a t i o n is o b t a i n e d in short (5 min) or p r o l o n g e d ( 6 0 - 9 0 min) i n c u b a t i o n periods. In this s y s t e m , the m a x i m u m i n c o r p o r a t i o n o f n u c l e o t i d e s is a c h i e v e d after 60 m i n in c o n t r o l nuclei, and 30 m i n in nuclei f r o m i r r a d i a t e d cells
37 (Fig. 3, top). We also tested if protein synthesis was required to achieve the apparent stimulation of run-on transcription following UV. No significant difference in the level of stimulation was observed if over 90% of protein synthesis was inhibited by performing UV irradiation and subsequent 30 minincubation of trypanosomes in the presence of 50 #g ml - ] cycloheximide and 25 # g ml - ] puromycin (Fig. 3, bottom).
UV irradiation causes the accumulation of steadystate transcripts from genes located at a short distance from the promoter. The apparent stimulation, following exposure to UV, of transcription of the 5 ~ part of the VSG expression site and of cpm (x 1 0 4) 20
/ 10 ~
'
A~
'
3o
A
'
io
UV --A
'
~)
min
UVIC rDNA: VSG: PRO:
3.2
4.7
4.4[~4
5.04.5 4.8
4.4
4.2
4.7
4.9~5
4.2
3.1
4.8
Fig. 3. Kinetics of run-on transcription in nuclei from control and UV-irradiated trypanosomes. Run-on transcription assays have been conducted on nuclei from either unirradiated ('c') or UV-treated (60 J m -2) ('uv') bloodstream forms, for different periods at 30°C. At each time interval, a 5-/zl sample was precipitated in 10% trichloroaceticacid and counted, while the remaining (995 #1) was processed for hybridizationwith DNA sequences from the beginning of the rDNA, VSG and procyclin gene transcription units (fragments 3 in Figs. 1 and 2). The ratio of apparent stimulation for each transcription unit is indicated for different incubation periods below the graph. The second columnof data for the 30 min-assay (cyclo/puro)shows results obtained if UV irradiation and subsequent 30 min incubation of trypanosomesin vitro are performed in the presence of 50 #g ml-1 cycloheximide and 25 #g ml-I puromycin. Under these conditions,the inhibition of protein synthesis was more than 90%, as determined by incubation of l-ml samples from control and UV-treated trypanosomes in the presence of 50 #Ci of [35S]methionine, followed by trichloroacetic acid precipitation. This experiment also showed that the UV treatment itself inhibits protein synthesis by approximately 50%.
the procyclin genes is reflected in Northern blots of steady-state transcripts from these transcription units (Fig. 4A, lanes 'uv'). The first panel shows the accumulation of transcripts characteristic of the 5' part of the VSG gene expression site (essentially ESAGs 6 and 7), and a similar increase in steady-state transcripts is seen for the procyclin genes (third panel). On the contrary, no such UV-dependent accumulation is observed for VSG and actin genes (panels 2 and 4), which are more distant from their promoter. In addition, the experiment also illustrates the changes in transcriptional patterns induced in the bloodstream trypanosomes by the temperature shift (from 37°C to 20°C) applied at time 0 (lanes 'c'): one sees a progressive down regulation of the VSG gene expression site (panels 1 and 2), a stimulation of the procyclin genes (panel 3), while transcription of the actin genes is not significantly affected (panel 4). The reasons for this temperature shift are presented in the next section.
The accumulation of transcripts from the beginning of the VSG and procyclin gene transcription units is due to inhibition of RNA decay. The apparent stimulation of transcription by UV is most probably due to inhibition of RNA degradation, as demonstrated in the second panel of Fig. 4A. This panel shows the amount of VSG mRNA present in cells at different time intervals after the VSG gene has been inactivated. This inactivation was achieved by shifting the incubation temperature from 37°C to 20°C, conditions which are known to trigger the transformation of bloodstream into procyclic forms and completely suppress the production of run-on transcripts of the VSG gene [5]. The progressive decrease of the VSG mRNA amount in unirradiated trypanosomes (lanes 'c') thus represents the rate of degradation of the preexisting VSG mRNA. It clearly appears that following UV irradiation, the VSG mRNA decay is delayed (lanes 'uv'). This observation is extended in Fig. 4B, showing results of pulse chase RNA labelling experiments, which allow the measurement of RNA decay irrespective of the actual transcriptional activity of a gene. The inhibiting effect of UV on RNA degradation is observed for transcripts from both the beginning and the end of the VSG transcription unit, as well as for procyclin
38
c . , c u cu
c u cU cU kb , ', - - I
I
~ I
3-
il
I ]
kb
',I I I
i I
I
c~lcU cU
cUcUcU
2.2-
1 . 6 - ~
,-',
I I I I
~,
,,
! I I
I
I I
I
0
2
I
0
4h
2
cpm (xlOO) 10
40
120 1 801
C
