- Email: [email protected]
Alternative use of a polyadenylation signal and of a downstream 3′ splice site
J. Mol. Biol. (1988)
204, 1031-1040
Alternative
Use of a Polyadenylation Signal and of a Downstream 3’ Splice Site
Effect of 5,6-Dichloro-1-fi-D-Ribofuranosylbenzimidazole H&he
Gallinaro, Pascale Vincendon, Annie Sittler and Monique Jacob?
Laboratoire de Ghnkttique Mol~culaire des Eucaryotes du CNRS Unite 184 de Biologic MoEt%daire et de GEnie Ge%kttiquede 1’INSERM Institut de Chimie Biologique, Faculte’ de Mkdecine 11 rue Humann - 67085 Strasbourg-CMex, France (Received
25 April
1988,
and in revisedform
19 July
1988)
A minor pathway for the processing of transcripts from the early region 3 transcription unit of adenovirus 2 is described. It results from the selection of a promoter-proximal polyadenylation site Pl instead of the major promoter-distal sites P2 and P4. 5,6-DichloroI-/3-n-ribofuranosylbenzimidazole (DRB) considerably reduces the use of the major pathways and enhances the minor one. The characterization of three novel nuclear RNAs whose amount increases in DRB-treated cells leads us to propose the following steps for the selection of Pl. The nascent transcript is first, cleaved in the branch point-3’ splice site region of the first E3 IVS (intervening sequence), generating a promoter-proximal RNA with a heterogeneous 3’ end (F-RNA) and a promoter-distal RNA with a heterogeneous 5’ end (G-RNA). This cleavage prevents the formation of the premessenger RNAs ending at P2 and P4. The 3’ end of F-RNA is about 130 nucleotides downstream from Pl and F-RNA contains the signals required for cleavage-polyadenylation. It may thus generate CPI, which is the transcript ending at Pl. The cleavage of the nascent transcript in a region crucial for spliceosome assembly suggests that defective assembly may render RNA sequences at the 3’ end of IVS accessible to intracellular nucleases and trigger the use of an upstream polyadenylation site. Such a mechanism may explain the alternative use of neighbouring, mutually exclusive, splice and polyadenylation sites.
1. Introduction The diversity of products of a gene is mostly the consequence of the alternative use of several initiation, splicing and polyadenylation sites (for reviews, see Leff et al., 1986; Breitbart et al., 1987). Usage of the various sites often changes according to the cell type or the stage of development. The early transcription unit 3 (E3$) from adenovirus 2 (Ad2) that we are currently studying produces series of mRNAs with the same initiation site (C) but with different polyadenylation and splice sites (Chow et al., 1979; H&is& et al., 1980; Stllhandske et al., 1983; Cladaras & Wold, 1985). As is t Author to whom all correspondence should be sent. $ Abbreviations used: E3, early transcription unit 3: Ad2, adenovirus 2; DRB, 5,6-dichloro-l-b-nribofuranosyl-benzimidazole; bp, base-pair(s); IVS, intervening sequence; snRNP, small nuclear ribonucleoprotein; hmRNP, heterogeneous nuclear ribonucleoprotein.
frequently the case in viruses, a late transcription unit overlaps early gene 3. In particular, a polyadenylation site (Pl) used for the formation of the 3’ end of a late family of transcripts (Le Moullec et al., 1983; Bhat & Wold, 1986) is found in the promoter-proximal region of E3. We recently detected an early transcript (CPI) with its 5’ end at the E3 capsite C and with its 3’ end at the late polyadenylation site Pl (Gallinaro et aZ., 1986). This finding suggested the existence of a yet-undescribed minor early pathway for the formation of E3 transcripts. The switch to this pathway implies the selection of a polyadenylation site located within an intron of the major early transcripts. A similar situation
occurs in several
other genes (for reviews,
see Leff et al., 1986; Breitbart et aZ., 1987). The mechanism of selection of such sites might be different from that used for polyadenylation sites located, for instance, in the 3’ untranslated region of mRNAs, and our system provides an opportunity to study this process. 1031
0 1988 Academic Press Limited
H. Gallinaro
1032
Our approach to this problem was to search for possible intermediate RNAs that would be related to the formation of CPl. To facilitate such a study, it was of interest to find a way to stimulate the use of the minor, relative to the pathway. We found that 5,6-dichloro-1-/I-n-ribofuranosylbenzimidazole (DRB) stimulated the minor pathway, by enhancing a cleavage event at a specific site of the nascent E3 transcript. The switch to the minor pathway includes this cleavage event; then, the 5’ product of cleavage is used as a precursor for the formation of the polyadenylated RNA CPl. Early work on the effect of DRB on the expression of more or less well-characterized genes suggested that this drug provoked premature transcription termination, pausing or attenuation primarily because small-size nuclear RNA accumulated in the presence of the drug (Sehgal et al., 1979; Laub et al., 1980; Tamm et al., 1980). These results were in contradiction with other in vivo and also with in vitro studies where DRB was shown to inhibit transcription initiation (Sehgal et al., 1976a,b; Winicov & Button, 1982; Egyhazi et al., 1983; Zandomeni et al., 1983; Holst & Egyhazi, 1985). On the other hand, the recent work of Mukherjee & Molloy (1987), who, like us, studied the products of a specific gene demonstrate that DRB inhibits initiation of transcription of the /I-globin gene in vivo and in vitro. In our case which is, as will be discussed, a particular one, we are able to demonstrate a yet-unrecognized effect of DRB; the drug enhances a cleavage event normally used in the cell for the regulation of the adenovirus E3 transcription unit. Therefore, its final effect may be post-transcriptional in particular genes.
2. Materials and Methods (a) Cell culture, viral infection and preparaticm of nuclear poly(A)+ and poly(A)RNA HeLa S3 cells were grown in suspension culture and infected with Ad2 (100 plaque-forming units/cell). Cycloheximide (25 pg/ml) was added to the suspension 2.25 h post-infection and the cells were harvested by lowspeed centrifugation at 3 h post-infection. Cells were resuspended at lo7 cells/ml in fresh medium, 10% (v/v) calf serum, cycloheximide (25 pg/ml) and DRB (25 pg/ml) and then incubated for 20 min at 37°C. For control cells, DRB was omitted. It has been shown that cycloheximide increases the amount of E3 transcripts without affecting the polyadenylation-splicing pattern (Chow et al., 1979; Gallinaro et al., 1986; Sittler et al., 1986). DRB-treated and control cells were labelled for 20 min with 50 PCi [3H]uridine/ml (10 Ci/mmol). For treated cells, the labelling and the drug-treatment were performed simultaneously. They were stopped by transferring the cells on to frozen isotonic buffer. Cell harvesting, purification of nuclei and extraction of RNA were as described (Gallinaro et al., 1983; Sittler et al., 1986). Contaminating DNA was digested by RNase-free DNase and the DNA fragments were removed by chromatography on a Sephadex G-75 column. Poly(A)+ RNA was selected by 3 passages over an oligo(dT)-cellulose column (Collaborative Research Inc.) and the nuclear RNA that did not bind was designated as poly(A)RNA. In order to
et al. remove possible contaminating poly(A)- RNA, poly(A)+ RNA was denatured in 80% (v/v) formamide and rechromatographed on oligo(dT)-cellulose after lowering the formamide concentration to 5%. (b) Preparation
of probes and labelling
The probes used for Northern blotting analysis are shown in Fig. l(b) and were labelled by nick-translation. Probes Da and Db were previously cloned in the SmaI site of pHp34 (Sittler et al., 1986). The probes used for S, mapping analysis as shown in Figs 3 and 4 were all prepared from the EcoRI-Hind111 DNA fragment of 1286 bp (positions -236 to + 1049). For preparation of the TaqI-RaaI probe, it was first digested by RsaI. The largest fragment was purified by centrifugation in a 5% to 20% (w/v) sucrose gradient and then digested by TaqI. End-labelling was performed without further purification. For preparation of the DdeI probe, the 1286 bp fragment was cut by DdeI and the probe was purified by electroelution after electrophoresis on a 5% polyacrylamide gel. For preparation of the AvaI-RsaI probe, the 1286 bp DNA fragment was digested by the 2 enzymes and the probe was purified by sucrose gradient centrifugation. For 3’ end-labelling, the probes were filled in at the 3’ end using the Klenow fra ment of DNA polymerase I in the presence of [a- f2 P]dCTP or [a-32P]dTTP (800 Ci/mmol), respectively, and the other cold dNTPs. 5’ End-labelling was as described by Maxam & Gilbert (1980) in the presence of [Y-~*P]ATP (5000 Ci/mmol; Amersham). The samples were electrophoresed on a strand separation gel and the single-stranded DNA probes were electroeluted. (c) Agarose/formaldehyde
gel electrophoresis and blotting of RNA
RNA was dissolved in 50% formamide, 1.8 Mformaldehyde, 25 mlcl-Mops, denatured by heating for 10 min at 65°C and separated by electrophoresis through horizontal 1.5% (w/v) agarose gels cast in 25 m&r-Mops, 5 mhr-sodium acetate, 1 mM-EDTA (final pH 7.0), and 2.2 M-formaldehyde. The migration was at 7.5 V/cm for 4 h. The gel was placed in 20 x SSC (SSC is 0.15 M-NaCl, 0.015 M-trisodium citrate, pH 7) for 0.75 h and the RNAs were transferred to nitrocellulose sheets (BA 85 Schleicher and Schiill) by blotting overnight. The prehybridization and hybridization with nick-translated probes were as described (Gallinaro et al., 1983). (d) 8, nuclease mapping S, nuclease analysis of RNA was carried out as described by Weaver & Weissmann (1979). An excess of the end-labelled DNA probes (8000 to 11,000 cts/min) and the RNA to be analyzed were coprecipitated by ethanol. They were dissolved in 10 ~1 of FNPE buffer (80% formamide, 0.4 M-NaCl, 40 mnr-Pipes (pH 6*4), 1 m&r-EDTA), denatured by heating for 10 min at 85°C and hybridized at 53°C for 1.5 h, then at 50°C for another 1.5 h and, finally, at 48°C for 0.5 h. The samples were diluted in 0.4 ml of cold digestion buffer and treated with 160 units of S, nuclease (Appligene) at 20°C for 1 h. Digestion with different concentrations of enzyme or for longer periods of time did not change the pattern of protected bands significantly. Digestion was stopped and the hybrids were precipitated with ethanol. After denaturation of the hybrids by heating in 80% formamide, 5 mw-Tris-borate (pH 8.3), 0.1 mM-EDTA for
10 min at 85”C, the protected DNA fragments were analysed by electrophoresis on an So,0 polyacrylamide/ 8 M-urea sequencing gel. To determine precisely the end of the DNA fragments, the homologous 5’ end-labelled single-stranded probes were cleaved according to Maxam & Gilbert (1980) and the reaction products were run in parallel. (e) Primer extension analysis The RNA sample was coprecipitated with an excess of the 5’ end-labelled primers p26 or p57 (Sittler et al., 1986). The pellet was dissolved in 10 ~1 of 250 mM-NaC1, 40 mMPipes (pH 6.4), 5 mM-EDTA, 0.2% (w/v) SDS, heated at 65°C for 5 min and hybridized by slow cooling to 30°C overnight. The hybrids were precipitated with ethanol and dissolved in 10 ~1 of 31 mw-Tris. HCl (pH 8.3), 25 mM-KCl, 3.75 mM-MgCl,, 5 mM-dithiothreitol, 0.2 mM of each dNTP and 2 units of RNasin (Promega Biotec). cDNA was synthesized by addition of 4 units of AMV reverse transcriptase and incubation at 41°C for 30 min. For sequence control, we used the same 5’ end-labelled primer and, as template. a single-stranded non-coding DNA fragment that overlaps the RNA extremities (positions 603 to 1189); cDNA synthesis was performed as above but in the presence of 1 of the 4 ddNTPs (50 PM for ddATP. 60 PM for ddCTP and ddGTP, 80 PM for ddTTP). The elongation products were analyzed on an 8% polyacrylamide/8 M-urea sequencing gel.
3. Results (a) The steady-state profoundly
E3 transcript
pattern
is
in DRB-treated cells
modi$ed
The structure and nucleotide sequence of the early region 3 as well as t,he major hallmarks for (0)
.I
1033
Polyadenylation-splicing
Alternative
synthesis and processing of transcripts have been described (Chow et al., 1979; H&is& et al., 1980; H&is& & Galibert, 1981; St&lhandske et al., 1983; Cladaras & ,Wold, 1985) and are summarized in Figure l(b). Of the four polyadenylation sites, only P2 and P4 are actively used at the early phase of infection, generat’ing the two major primary transcripts pre-c and pre-a (Fig. l(a)). Pl is mostly used at the late phase of infection for the formation of the 3’ end of RNAs from the L4 family (Le Moullec et al., 1983; Bhat & Wold, 1986). However, at the early phase of infection we found a small amount of an RNA (CPl) starting at the E3 capsite and terminating at Pl (Gallinaro et al., 1986). As for P3 (not shown in Fig. l), its existence was suggested from electron microscopic studies of transcripts (Chow et al., 1979) but it has never been detected by other methods. To study the effect of DRB on the production of the E3 transcripts ending at PI, P2 and P4. we first analysed the nuclear poly(A)+ RNA from untreated and DRB-treated cells by Northern blotting. The optimal conditions for DRB treatment were found to be 25 pg/ml for 20 minutes. The effect of the drug did not change upon doubling its concentration. The maximum effect was reached between 10 and 15 minutes, and persisted up to 30 minutes (data not shown). In poly(A)+ RNA from untreated cells, probe Db, specific to IVSl sequences (Fig. l(b)), displays the two precursors pre-a and pre-c, and the two related intermediates TVSl-exon 2-poly(A) (Fig. 2, lanes 1 and 3). These RNAs have been fully
.
h
Late
L4
pre-maNA
I
CPl
IVSl
EXl (b)
,----C I 1
Da
Ex2
,------,-------Dl I 372
PlA2 ‘L’ 620 768
02 951
A3 1740
A4 2157
L
P2
A6
P4
2190
2830
3255
.
Db
Figure 1. (a) The Ad-2 genome and the E3 and L4 primary transcripts. The r strand of the genome is divided into 100 units. Above is shown the late L4 pre-mRNA that terminates at Pl. The black squares represent the 3 leaders common to all major late families of RNA. Below is shown the early transcription unit 3 with its 3 polyadenylation sites. (b) The landmarks of the E3 transcription unit (from Stllhandske et al., 1983; Cladaras t Wold, 1985). C is the capsite, Ds are 5’ splice sites, As are 3’ splice sites and Ps are polyadenylation signals. The probes Da and Db used for Northern blot analysis are shown below (for isolation, see Materials and Methods).
1034
H. Gallinaro et al.
F
1234
5
6
7
8
Figure 2. Northern blot analysis of nuclear E3 RNA. The probes used to display the RNAs are indicated at the top and shown in Fig. l(b). Lanes marked + indicate the DRB-treated cells and lanes marked - indicate the control cells: 1.8% of either the poly(A)+ RNA fraction or the poly(A)- fraction was analysed. This represented: for poly(A)+ RNA (lanes 1 to 4), 1.35 pg (lanes - ) and 1.40 pg (lanes +); for poly(A)- RNA (lanes 5 to 8), 18 pg (lanes -) and 16pg (lanes +). Lanes 3 and 4 show overexposure of lanes 1 and 2. The E3 RNAs were previously characterized by other methods (Gallinaro et al., 1986; Sittler et al., 1986, 1987). characterized (Sittler et al., 1986, 1987). An RNA of about 800 nucleotides (indicated as CPl) is also detected and is more clearly visualized after overexposure of the gel (lane 3). This RNA is a very minor species when compared to the transcripts terminating at P2 and P4. In DRB-treated cells, we observe an almost complete disappearance of pre-c, a considerable decrease of pre-a and the disappearance of the two IVSl-exon 2-poly(A) RNAs (lanes 2 and 4). In contrast, the amount of the small RNA CPl increases, as well as its apparent complexity (lane 4). This increased complexity is not due to an increased heterogeneity of the 3’ ends of the core of CPl. This was shown by an S, nuclease protection experiment (Fig. 4(a)). The number of protected bands is not modified by treatment with DRB; only their intensity increases. Therefore, we assume that the heterogeneity observed by Northern blotting is due to a heterogeneity of the length of the poly(A) tail and we conclude that DRB provokes a marked increase of the proportion of WI relative to that of pre-a and pre-c. By using an exon l-specific probe, we verified that the amount of nuclear E3 mRNAs did not change much during the 20 minute treatment with DRB (data not shown). This apparent stability of mRNA is due to its long half-life and has been described (Harlow & Molloy, 1980; Nilsen et al., 1983; Edwards et al., 1985). Nuclear non-polyadenylated RNA was studied. It consists primarily of growing transcripts and of
non-polyadenylated splicing intermediates or products. Probes Da and Db specific to exon 1 and IVSI sequences, respectively (see Fig. l(b)), display a background signal due to the heterogeneous growing transcripts (Fig. 2, lanes 5 and 7). Over this background, we systematically detect a novel RNA or F-RNA of about 750 nucleotides. In addition, free IVSl is detected (lane 7). In DRBtreated cells, the general pattern of poly(A)- RNA is strikingly modified. The amount of large transcripts and of free IVSl decreases, while that of the small transcripts, and in particular of F-RNA, increases (lanes 6 and 8). Thus, while minor in untreated cells, F-RNA becomes a relatively abundant component in DRB-treated ceils. These results show that DRB considerably modifies the pattern of the E3 nuclear transcripts. It affects the selection of the polyadenylation sites, as shown by the changes of distribution of the primary transcripts terminating at Pl, P2 and P4 (Fig. 2). The decrease of the precursors terminating at P2 and P4 leads to a decrease of the related splicing intermediates and products (IVSI-exon 2 and free IVSI). On the other hand, the amount of the small promoter-proximal RNAs termed F-RNA and CPI increases. As can be seen in Figure I, the use of Pl and the excision of the first E3 TVS (DlA2) are mutally exclusive. In the following sections, we shall try to establish whether, and how, the increase of CPl and F-RNA is related to the mechanism of selection of Pl or excision of TVSI.
(b) Possible mechanism of selection of the polyadenylation site Pl The Northern blot analyses in Figure 2 suggest that F-RNA is a promoter-proximal RNA with a 3 end close to the 3’ end of TVSl. S, nuclease mapping and primer extension experiments were performed in order to determine its extremities more precisely. For the determination of its 5’ end, F-RNA from untreated cells was first hybridselected with the IVSl-specific probe Db. The selected RNAs were then electrophoresed in polyacrylamide gels. RNA 2, which has the size of FRNA and which can be revealed by hybridization with the exon l-specific probe Da (data not shown), was eluted (Fig. 3(a)). A primer, ~57 (Fig. 3(b), bottom) was then annealed to RNA 2 and extended. A 627 nucleotide cDNA was obtained (Fig. 3(b), lane 2), clearly different from the 255 nucleotide product synthesized from free lariat IVSl (lane l), which is the major hybrid-selected RNA species (Fig. 3(a)). The results indicate that the 5’ end of F-RNA is at the E3 capsite. This conclusion was confirmed by an S, mapping experiment using the 5’ end-labelled probe pr756 overlapping the capsite (Fig. 3(c)). The 3’ end of F-RNA was determined on the hybrid-selected RNA 2, by S, nuclease protection experiments, and was shown to be heterogeneous and located slightly upstream from the A2 3’ splice
Alternative (.a 1
1035
Polyadenylation-splicing (c) M
(b) Ml23
627
527-
:
.255
242238217-
* -p57 180-
AvuI C
Le2
Lel Dl
I-W
Le2
Lel
RSUI
I
721
-34
,+
Fok I
I 372
571 627
768
--Y
P57
-
pr756 721nt
255nt 627nf
Figure 3. Determination of the 5’ end of the F-RNA. (a) Hybrid selection. The method was as described (Sittler et al., 1986). The in viwo ‘H-labelled nuclear poly(A)- RNAs of 399 to 1996 nucleotides from untreated cells were hybridized to probe Db covalently linked to diazobenzyloxymethyl (DBM) paper. The hybrid-selected RNAs were fractionated on 5% polyacrylamide/&3 ~-urea gels and displayed by fluorography. RNA 2 was electroeluted and used for the experiments in (b) and (c). RNAs 1, 3 and 4 are different IVSl forms (Sittler et al., 1987). (b) Primer extension. Primer p57 was hybridized to eluted RNA 1 (lane 1) and RNA 2 (lane 2) and extended. Lane 3 shows a control experiment (probe treated in the absence of RNA). (c) S1 nuclease protection experiment. The 5’ end-labelled single-stranded AvaIRsaI probe was hybridized to RNA 2. The protected DNA fragment (721 nucleotides) and the residual probe (756 M-urea sequencing gel. Size markers: pBR322 digested by Mspl nucleotides) were analysed on a 5% polyacrylamide/8 (lanes M) or by FokI (lane Ml). nt, nucleotides.
site (results not shown). This heterogeneity, as well as the precise location of the 3’ ends, is illustrated in Figure 4(b), which shows that the same pattern of
bands is obtained in untreated and DRB-treated cells. The data indicate that DRB enhances the formation of F-RNA but that it is not responsible for its heterogeneity. By comparing the mobility of the protected DNA bands with that of the products of sequencing reactions and by combining the results of three we determined that the separate experiments, predominant 3’ termini of F-RNA were between positions 746 and 753 with minor termini up to positions 743 upstream and 759 downstream. As
shown in Figure 6, the sequence overlapping
the 3’
end region of F-RNA
a low
stability
is A + U-rich,
suggesting
of the hybrids and a possible trimming
S1 nuclease.
However,
the pattern
by
of bands did not
change when the nuclease concentration was modified (Fig. 4(d)), suggesting that nuclease trimming may be responsible for minor quantitative changes but it is not the major cause of the heterogeneity, which is likely to reflect the situation in vivo. Thus, F-RNA is an RNA starting at the E3 capsite and extending up to the region between the IVSl branch point and 3’ splice site. It contains the entire sequence of the non-polyadenylated core of
(b)
(a)
(c)
MM+
-I
M
premRNA 242 238 217 201 _, ws"QO 160
CPl4 A2
Q Bi
RNA c 14’.
G-RNAC
C
01
Pl
El
A2
C
Bl
A2
Ode1 pr Ode
Bl
A2
740
768
DC?*1
OdeL * 912
pr Ode 593
+-----r-
323nt
/VSl
17snt
F-RNA
372
Ode I
s-2
Tr CPl
Dl
C I I
A2
TI G-RNA
----cI=c32Ool \\~147/140nl
159/,71”,
-1 Cd) -
31 RNA
2
1 3 1.5 III /
F-RNA
1234GA -IQ
Figure 4. S1 nuclease mapping experiments. The 5’ or 3’ end-labelled (*) probes are shown at the bottom of each panel as well as the positions of the major protected fragments; - and + refer to the absence or presence of DRB. Lanes M, 5’ end-labelled Ms$ cleavage products of pBR322. Lane 0, probe treated in the absence of RNA. Lanes G, A + G, T + C and C, products of sequencing reactions. The sites of interest (A2, Bl) are indicated on these gels. So as to precisely localize the extremity of the fragments protected by the probe, we took into account the difference of migration of 2 nucleotides between the sequencing products and S, nuclease-treated fragments (Contreras et al., 1982; Gallinaro et al., 1986). (a) The 3’ extremity of CPl; 1.54pg (lane -) and 15Opg (lane +) of poly(A)+ RNA, representing 2% of the total poly(A)+ fraction, were analysed. (b) The 3’ extremities of F-RNA: (c) The 5’ extremities of G-RNA. In both (b) and (c), 18.8 pg (lanes - ) and 20.0 pg (lanes + ) of poly(A) - RNA representing 2% of the total fraction were analysed. Tr, transcripts that protect the full-length probe. (d) S, nuclease control for F-RNA. RNA was analysed as in (b), except that the ratio of S1 nuclease to RNA was changed as indicated at the top. Lanes 2 and 4 contain twice as much RNA as lanes 1 and 3. Note that each band is detected in all lanes and that only its intensity changes from one lane to another. nt, nucleotides.
1037
Alternative Polyadenylation-splicing CPl, as previously characterized (Gallinaro et al., 1986), plus about 130 nucleotides at the 3’ end. In fact, it has all the characteristics required for a substrate of the cleavage-polyadenylation reactions leading to the formation of CPl (for a review, see Birnstiel et al., 1985). Unfortunately, the precursorproduct relationship is difficult to demonstrate by pulse-chase experiments in vivo, since equilibration of the nucleotide pool takes about one minute, while CPl is already fully labelled two minutes after addition of a labelled nucleoside (Gallinaro et al., 1986). This makes interpretation of chase experiments rather hazardous. Nevertheless, the parallel stimulation of the formation of F-RNA and CPl in DRB-treated cells (Figs 2 and 4) supports the idea of a precursor-product relationship. In addition, recent experiments in vitro demonstrate that an RNA with its 3’ end close to that of F-RNA is faithfully cleaved and polyadenylated at Pl (Gallinaro, Meyer & Jacob, unpublished results). Note that the formation of F-RNA prevents the formation of pre-a and pre-c, while allowing that of CPl. Therefore, the formation or non-formation of F-RNA might be a crucial step in the selection of the E3 polyadenylation sites.
G-RNA were found between positions 764 and 770, whereas minor termini were found up to positions 752 and 775 (Fig. 6). As for F-RNA (see above), we were able to exclude the possibility that the major termini were artifactually produced by S, nuclease trimming. This conclusion was further confirmed by a primer extension analysis. An oligonucleotide p26 complementary to an exon 2 sequence was labelled at its 5’ end, annealed to nuclear poly(A)RNA of untreated and DRB-treated cells and extended (Fig. 5). A group of extension products ending in the region of the 3’ splice site between positions 763 and 768 was predominant and more intense in DRB-treated cells than in normal cells. This 763768 sequence is very close to the 764-770 sequence determined by S, nuclease mapping (Fig. 6). Thus, the two methods give comparable results, as far as the major 5’ ends are concerned, but not for the M
AGC
T
(c) Formation of F-RNA by cleavage of the primary transcript If F-RNA is involved in the selection of the E3 polyadenylation sites, then it is of importance to understand how it is formed. F-RNA might be either a product of transcription termination or a product of cleavage of the primary transcript. Both possibilities may be envisaged. The active use of Pl at the late phase of infection (Fig. l(a)) indicates that transcription termination may occur at some site downstream from Pl; at the early phase, the same type of event may provoke the formation of F-RNA. On the other hand, endonucleolytic cleavages might be important events in the mechanisms regulating pre-RNA processing as shown, for instance for the Ll ribosomal protein gene of Xenopus laevis (Caffarelli et al., 1987). To decide between these alternatives, we considered that an RNA with its 5’ end in the branch point-3’ splice site region should obligatorily exist in the case of post-transcriptional cleavage but not in the case of transcription termination. In addition, if this RNA is generated by cleavage, its amount should vary in parallel with that of F-RNA in the presence of DRB. We have looked for such an RNA in the nuclear non-polyadenylated RNAs. For this purpose, S, nuclease protection experiments were performed using the same probe as for the determination of the 3’ end of F-RNA but labelled at the 5’ end instead of the 3’ end. Figure 4(c) shows that an RNA with a heterogeneous 5’ end around the A2 3’ splice site is detected. This RNA is designated as G-RNA. Its amount increases in DRB-treated cells, as expected if it is formed simultaneously with F-RNA by a cleavage of the primary transcript. The predominant 5’ termini of
Bl
G-RNA
A2
Lel
871 -I+ e
G-RNA Tr
Figure
p
P26 104/109nl 871nl
5. Primer extension analysis of G-RNA. RNA from control (lane -) and DRB-treated
Poly(A)cells (lane +) were used as templates. The localization of primer p26 and of its extension products is indicated at the bottom. Lane M, size markers as in Fig. 4. Lanes A, G, C and T, extension products made with p26 using a single-stranded non-coding DNA fragment as template and 1 of the 4 ddNTPs. The positions of A2 and Bl are indicated. nt, nucleotides.
1038
H. Gallinaro Branch point
%Cyw
3’ Spliie
IVS 1
4
734
et al.
750
740
760
~~UUUA~CG~~~UCUUC~UUUGUAAUU~ACAA~AGUU~~AG~G~ F-RNA G-RNA G-RNA
l
junction I
Ex 2
770
777
.000~*~~~*e~000000~~~
* *
SI lluctoaeo
. . . . . ..*.00000000000oeo*~mo*ooooo~ q . . . . ..o...*......~...clo.....
ooovTlllll~uvtl~vv
6
lz *I3
wmnebn
vPvllTv~vv~~v~oo
Figure 6. The branch point-3’ splice site region. The sequence of the region (H&s& et al., 1980) is presented. The extremities of the fragments as determined by S, nuclease mapping (average of 3 experiments) is shown below the sequence. (e) Strong bands; (0) weak bands; (0) faint bands. The 5’ extremities of G-RNA as determined by primer extension are also indicated. (a) Strong bands; (0) weak bands; (0) faint bands. The intensity of all these bands increases in DRB-treated cells. Others, whose intensitv decreases, are marked by a star. A and B indicate the 2 possible interpretations of the results (see the text).
minor bands. It is not possible to decide whether these minor bands are artifactually generated by S,
nuclease trimming or whether they are not, displayed in the primer extension experiments for some reason(s) to be determined. On the other hand, the DRB-induced increase was apparently lower when studied by primer extension than by S1 nuclease mapping or Northern blotting. We have no explanation for this discrepancy. Nevertheless, we would like to emphasize that a DRB-induced increase was systematically observed in all experiments with all methods. In conclusion, we have characterized a family of G-RNAs with a heterogeneous 5’ end around the 3’ splice site of the first IVS. It is likely to be generated simultaneously with F-RNA by a cleavage of the primary transcript and the possible modalities of such a cleavage will be examined in the next section. Note that the production of both F-RNA and G-RNA is similarly enhanced in the presence of DRB (Fig. 4), suggesting that the drug is involved in cleavage stimulation. (d) Generation
of F-RNA
and
G-RNA
The demonstration of two families of RNAs, F-RNA with a heterogeneous 3’ end in the branch point-3’ splice site region of IVSl and G-RNA with a heterogeneous 5’ end around the 3’ splice site indicates that a nuclease cleavage has occurred in this region. The inspection of the precise localization of the major 3’ termini of F-RNA and of the major 5’ termini of G-RNA (Fig. 6) shows that these ends do not coincide, as would b;e’ expected if single internucleotide bonds were cleaved. Rather, there is a gap of about ten nucleotides between the major extremities, suggesting that whole segments of the sequence were removed. Due to the fact that minor bands may be indicative either of high efficiency of cleavage (little material left) or of low efficiency of cleavage (most material uncleaved), it is not
possible to decide as to the exact primary cleavage sites (A or B, Fig. 6). Nevertheless, the accessibility of a short sequence (A) or sequences (B) to intranuclear nucleases suggest that one or several of the factors that normally bind the branch point-3’ splicing region do not bind a fraction of the nascent RNA molecules. This fraction is small in untreated cells and much larger in DRB-treated cells. It is well-known that the branch point-3’ splice site region is crucial for the assembly of the splicing complex (Green, 1986; Padgett et al., 1986; Aebi & Weissmann, 1987; Maniatis & Reed, 1987) and, therefore, we may assume that assembly is inhibited for a fraction of the molecules and that DRB accentuates this inhibition. 4. Discussion The demonstration of novel E3 nuclear RNAs in adenovirus 2-infected HeLa cells allows us to describe a novel early pathway for the formation of E3 transcripts and to propose a mechanism for the switch from the major pathways to the minor one (Fig. 7). It should be emphasized that the minor pathway is functional in infected HeLa cells in the absence of both DRB and cycloheximide. This was shown by the detection of CPl (Gallinaro et al., 1986) and F-RNA (Sittler, 1986). Cycloheximide, which allows a relative increase of all E3 RNA, and DRB, which enhances the minor pathway, and has helped us to specify the transcripts involved in the regulation process. The major event in the switch seems to be a cleavage of the growing transcript. If we examine the alternative possibility of generation of F-RNA by transcription termination, we observe that it implies the formation of G-RNA by re-initiation immediately downstream from the termination site. The DRB
data allow this possibility
to be excluded;
indeed, the drug that enhances the formation of both RNAs would thus enhance both termination and initiation of transcription, which is unlikely
Alternative
Polyadenylation-splicing
Naacsnt C t
4
Dl I 372
II
Transcript
pxiiiiq I 507
p1 I, 620
j7iiFiq
Bl 1 740
CI.w8go ,,
F-RNA
/I
1039
A2 I 768
l
I Hlll G-RNA + 1111
CIw48g8 polyrdonylrllon
I
CPl I,
r,
1 “n
Figure 7. Proposed mechanism for Pl selection at the early phase of infection.
(Mukherjee & Molloy, 1987). The particular cleavage of the E3 pre-RNA occurs in the branch point-3’ splice site region of IVSl. This region is of the utmost importance for the formation of the splicing complex in vitro (for reviews, see Green, 1986; Padgett et al., 1986; Aebi & Weissmann, 1987; Maniatis & Reed, 1987) and probably also in vivo. Our data suggest that, for a fraction of the transcripts, one or several constituents of the splicing complex (snRNPs or other factors) do not bind their specific sites on the RNA, leaving the way open to intranuclear nucleases. We observed that the average size of hnRNP decreased upon treatment with DRB and that it became more sensitive to degradation (data not shown). This is compatible with perturbations of assembly but obviously does not prove it. In vitro investigations are in progress to solve this problem. Once the primary transcript has been cleaved,
of polyadenylation and splice sites (Bovenberg et al., 1986; Leff et al., 1986, 1987; Peterson & Perry, 1986; Breitbart et al., 1987; Galli et al., 1987; Tsurushita et al., 1987). That DRB does not cleave any intervening sequence is proven by the absence in other E3 IVS of detectable cleavages similar to those found in IVSl. On the other hand, the fact that DRB may act post-transcriptionally at the level of premRNA processing underlies some earlier observations. For instance, it has been shown that DRB severely inhibits only RNAs that require extensive processing for their formation (Harlow & Molloy, 1980). Moreover, the synthesis of unspliced RNAs such as Ad-2 polypeptide IX mRNA mRNA (Vennstr6m et al., 1979) or interferon (Sehgal & Tamm, 1979) is insensitive to the drug. Studies of the effect of DRB on the expression of other selected genes might be helpful for our
the major early precursors longer be formed, whereas
understanding of the molecular mechanism underlying the events observed in DRB-treated cells.
pre-a and pre-c can no the promoter-proximal
cleavage product F-RNA may generate CPl (Fig. 7). Thus, a defect in the assembly of the splicing complex at the level of IVSl might trigger the use of an upstream polyadenylation signal and thus
interfere
with
polyadenylation
site selection.
An alternative to this mechanism is that DRB may affect the recognition of the polyadenylation site Pl, and that this event may provoke a cleavage at the far 3’ end of IVSl. Though this alternative cannot be excluded, we rather favour the first possibility, which follows the canonical rules of 3’ end formation of mRNA (Birnstiel et al., 1985).
Our results give some indications
as to the mode
those whose regulation
& Molloy,
1987) in agreement
concern a large number particular
cleavage
probably
restricted
event
of genes, if not all, the that
we
describe
is
to a few genes; for instance,
use
References
with
earlier studies (Sehgal et al., 1976a, b; Winicov & Button, 1982; Egyhazi et al., 1983; Zandomeni et al., 1983; Holst & Egyhazi, 1985). Using another selected gene, we show that DRB enhances a posttranscriptional cleavage event. In contrast to the inhibition of transcription initiation, which may
the alternative
We are grateful to Mrs L. Kister for her invaluable technical assistance throughout this work, and to Dr G. Richards for a critical reading of the manuscript. We thank J. L. Weickert and M. Gilbert for growing HeLa cells, C. Kutschis, C. Werl& and B. Boulay for assistance in the preparation of the manuscript. This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la San6 et de la Recherche MBdicale, the Fondation pour la Recherche MBdicale Franpaise, the Association pour la Recherche sur le Cancer and the Ligue Nationale Franpaise contre le Cancer.
of action of DRB. Investigations on the transcripts of the /?-globin gene demonstrate that DRB inhibits initiation in vivo and transcription in vitro
(Mukherjee
implies
Aebi, M. & Weissmann, C. (1987). Trends Genet. 3, 102107. Bhat, B. M. 6 Wold, W. S. M. (1986). J. Viral. 60, 54-63. Birnstiel, M. L., Busslinger, M. & &rub, K. (1985). Cell, 41, 349-359.
Bovenberg, R. A. L., van de Meerendonk, W. P. M., Baas, P. D., Steenbergh, P. H., Lips, C. J. M. & Jansz, H. S. (1986). Nucl. Acids Res. 14, 8785-8803. Breitbart, R. E., Andreadis, A. & Nadal-Ginard. B. (1987). Annu. Rev. Biochem. 56, 467495.
1040
H. Gallinaro
Caffarelli, E., Fragapane, P., Gehring, C. & Bozzoni, I. (1987). EMBO J. 6, 3493-3498. Chow, L. T., Broker, T. R. & Lewis, J. B. (1979). J. Mol. Biol. 134, 265-303. Cladaras, C. & Wold, W. S. M. (1985). Virology, 140, 2% 43. J., van de Contreras, R., Gheysen, D., Knowland, Voorde, A. & Fiers, W. (1982). Nature (London), 390, X%505. Edwards, D. R., Parfett, C. L. & Denhardt, D. T. (1985). Mol. Cell. Biol. 5, 32863288. Egyhazi, E., Holst, M. & Tayip, U. (1983). Eur. J. Biochem. 130, 223-226. Galli, G., Guise, J. W., McDevitt, M. A., Tucker, P. W. & Nevins, J. R. (1987). Genes Develop. 1, 471481. Gallinaro, H., Puvion, E., Kister, L. Jacob, M. (1983). EMBO J. 2, 953-960. Gallinaro, H., Sittler, A., Kister, L. & Jacob, M. (1986). Biochimie, 68, 1009-1017. Green, M. R. (1986). Annu. Rev. Genet. 20, 671-708. Harlow, P. & Molloy, G. (1980). Arch. Biochem. Biophys. 203, 764-773. H&is&, J. & Galibert, F. (1981). Nucl. Acids Res. 9, 12291240. H&is&, J., Courtois, G. & Galibert, F. (1980). Nucl. Acids Res. 8, 2173-2192. Holst, M. & Egyhazi, E. (1985). J. Cell. Biochem. 29, 115126. Laub, O., Jakobovits, E. B. & Aloni, Y. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 3297-3301. Leff, S. E., Rosenfeld, M. G. & Evans, R. M. (1986). Annu. Rev. B&hem. 55, 1091-1117. Leff, S. E., Evans, R. M. & Rosenfeld, M. G. (1987). CeZE, 48, 517-524. Le Moullec, J. M., Akusjiirvi, G., Stalhandske, P., Pettersson, U., Chambraud, B., Gilardi, P., Nasri, M. & Perricaudet, M. (1983). J. Viral. 48, 127-134. Maniatis, T. & Reed, R. (1987). Nature (London), 325, 673-678.
Edited
et al. Maxam, A. M. & Gilbert, W. (1980). Methods Enzymol. 65, 499-560. Mukherjee, R. & Molloy, G. R. (1987). J. BioE. Chem. 262, 13697-13705. Nilsen, T. W., Maroney, P. A. & Baglioni, C. (1983). Mol. Cell. Biol. 3, 64-69. Padgett. R. A., Grabowski, 1’. J., Konarska, M. M., Seiler, S. & Sharp, P .A. (1986). Annu. Rev. Biochcwb. 55, 1119-1150. Peterson, M. L. & Perry, R. P. (1986). Proc. Nat. Acad. Sci., U.S.A. 83, 8883-8887. Sehgal, P. B. & Tamm, I. (1979). Virology, 92, 240-244. Sehgal, P. B., Darnell, J. E., Jr & Tamm, I. (1976u). Cell. 9, 473480. Sehgal, P. B., Derman, E., Molloy, 0. R., Tamm, I. & Darnell, J. E. (19763). Science, 194, 431433. Sehgal, P. B., Fraser, N. W. & Darnell, J. E.. Jr (1979). Virology, 94, 185-191. Sittler, A. (1986). Ph.D. thesis. University Louis Pasteur, Strasbourg. Sittler, A., Gallinaro, H. & Jacob, M. (1986). Nucl. Acids Res. 14, 1187-1207. Sittler, A., Gallinaro, H., Kister, L. & Jacob, M. (1987). J. Mol. Biol. 197, 737-742. Stblhandske, P., Persson, H., Perricaudet, M., Philipson, L. & Pettersson, U. (1983). Gene, 22, 157-165. Tamm, I., Kikuchi, T.. Darnell, J. E., Jr & SaldittGeorgieff, M. (1980). Biochemistry, 19, 2743-2748. Tsurushita, N., Avdalovic, N. M. & Korn, L. J. (1987). Nucl. Acids Res. 15, 46034615. Vennstriim, B., Persson, H., Pettersson, U. & Philipson, L. (1979). Nucl. Acids Res. 7, 1405-1418. Weaver, R. F. & Weissmann, C. (1979). Nucl. Acids Res. 7, 117551193. Winicov, I. & Button, D. (1982). Eur. J. Biochem. 124, 239244. Zandomeni, R., Bunick, D., Ackerman, S., Mittleman, B. & Weinmann, R. (1983). J. Mol. Biol. 167, 561-574.
by B. Mach
204, 1031-1040
Alternative
Use of a Polyadenylation Signal and of a Downstream 3’ Splice Site
Effect of 5,6-Dichloro-1-fi-D-Ribofuranosylbenzimidazole H&he
Gallinaro, Pascale Vincendon, Annie Sittler and Monique Jacob?
Laboratoire de Ghnkttique Mol~culaire des Eucaryotes du CNRS Unite 184 de Biologic MoEt%daire et de GEnie Ge%kttiquede 1’INSERM Institut de Chimie Biologique, Faculte’ de Mkdecine 11 rue Humann - 67085 Strasbourg-CMex, France (Received
25 April
1988,
and in revisedform
19 July
1988)
A minor pathway for the processing of transcripts from the early region 3 transcription unit of adenovirus 2 is described. It results from the selection of a promoter-proximal polyadenylation site Pl instead of the major promoter-distal sites P2 and P4. 5,6-DichloroI-/3-n-ribofuranosylbenzimidazole (DRB) considerably reduces the use of the major pathways and enhances the minor one. The characterization of three novel nuclear RNAs whose amount increases in DRB-treated cells leads us to propose the following steps for the selection of Pl. The nascent transcript is first, cleaved in the branch point-3’ splice site region of the first E3 IVS (intervening sequence), generating a promoter-proximal RNA with a heterogeneous 3’ end (F-RNA) and a promoter-distal RNA with a heterogeneous 5’ end (G-RNA). This cleavage prevents the formation of the premessenger RNAs ending at P2 and P4. The 3’ end of F-RNA is about 130 nucleotides downstream from Pl and F-RNA contains the signals required for cleavage-polyadenylation. It may thus generate CPI, which is the transcript ending at Pl. The cleavage of the nascent transcript in a region crucial for spliceosome assembly suggests that defective assembly may render RNA sequences at the 3’ end of IVS accessible to intracellular nucleases and trigger the use of an upstream polyadenylation site. Such a mechanism may explain the alternative use of neighbouring, mutually exclusive, splice and polyadenylation sites.
1. Introduction The diversity of products of a gene is mostly the consequence of the alternative use of several initiation, splicing and polyadenylation sites (for reviews, see Leff et al., 1986; Breitbart et al., 1987). Usage of the various sites often changes according to the cell type or the stage of development. The early transcription unit 3 (E3$) from adenovirus 2 (Ad2) that we are currently studying produces series of mRNAs with the same initiation site (C) but with different polyadenylation and splice sites (Chow et al., 1979; H&is& et al., 1980; Stllhandske et al., 1983; Cladaras & Wold, 1985). As is t Author to whom all correspondence should be sent. $ Abbreviations used: E3, early transcription unit 3: Ad2, adenovirus 2; DRB, 5,6-dichloro-l-b-nribofuranosyl-benzimidazole; bp, base-pair(s); IVS, intervening sequence; snRNP, small nuclear ribonucleoprotein; hmRNP, heterogeneous nuclear ribonucleoprotein.
frequently the case in viruses, a late transcription unit overlaps early gene 3. In particular, a polyadenylation site (Pl) used for the formation of the 3’ end of a late family of transcripts (Le Moullec et al., 1983; Bhat & Wold, 1986) is found in the promoter-proximal region of E3. We recently detected an early transcript (CPI) with its 5’ end at the E3 capsite C and with its 3’ end at the late polyadenylation site Pl (Gallinaro et aZ., 1986). This finding suggested the existence of a yet-undescribed minor early pathway for the formation of E3 transcripts. The switch to this pathway implies the selection of a polyadenylation site located within an intron of the major early transcripts. A similar situation
occurs in several
other genes (for reviews,
see Leff et al., 1986; Breitbart et aZ., 1987). The mechanism of selection of such sites might be different from that used for polyadenylation sites located, for instance, in the 3’ untranslated region of mRNAs, and our system provides an opportunity to study this process. 1031
0 1988 Academic Press Limited
H. Gallinaro
1032
Our approach to this problem was to search for possible intermediate RNAs that would be related to the formation of CPl. To facilitate such a study, it was of interest to find a way to stimulate the use of the minor, relative to the pathway. We found that 5,6-dichloro-1-/I-n-ribofuranosylbenzimidazole (DRB) stimulated the minor pathway, by enhancing a cleavage event at a specific site of the nascent E3 transcript. The switch to the minor pathway includes this cleavage event; then, the 5’ product of cleavage is used as a precursor for the formation of the polyadenylated RNA CPl. Early work on the effect of DRB on the expression of more or less well-characterized genes suggested that this drug provoked premature transcription termination, pausing or attenuation primarily because small-size nuclear RNA accumulated in the presence of the drug (Sehgal et al., 1979; Laub et al., 1980; Tamm et al., 1980). These results were in contradiction with other in vivo and also with in vitro studies where DRB was shown to inhibit transcription initiation (Sehgal et al., 1976a,b; Winicov & Button, 1982; Egyhazi et al., 1983; Zandomeni et al., 1983; Holst & Egyhazi, 1985). On the other hand, the recent work of Mukherjee & Molloy (1987), who, like us, studied the products of a specific gene demonstrate that DRB inhibits initiation of transcription of the /I-globin gene in vivo and in vitro. In our case which is, as will be discussed, a particular one, we are able to demonstrate a yet-unrecognized effect of DRB; the drug enhances a cleavage event normally used in the cell for the regulation of the adenovirus E3 transcription unit. Therefore, its final effect may be post-transcriptional in particular genes.
2. Materials and Methods (a) Cell culture, viral infection and preparaticm of nuclear poly(A)+ and poly(A)RNA HeLa S3 cells were grown in suspension culture and infected with Ad2 (100 plaque-forming units/cell). Cycloheximide (25 pg/ml) was added to the suspension 2.25 h post-infection and the cells were harvested by lowspeed centrifugation at 3 h post-infection. Cells were resuspended at lo7 cells/ml in fresh medium, 10% (v/v) calf serum, cycloheximide (25 pg/ml) and DRB (25 pg/ml) and then incubated for 20 min at 37°C. For control cells, DRB was omitted. It has been shown that cycloheximide increases the amount of E3 transcripts without affecting the polyadenylation-splicing pattern (Chow et al., 1979; Gallinaro et al., 1986; Sittler et al., 1986). DRB-treated and control cells were labelled for 20 min with 50 PCi [3H]uridine/ml (10 Ci/mmol). For treated cells, the labelling and the drug-treatment were performed simultaneously. They were stopped by transferring the cells on to frozen isotonic buffer. Cell harvesting, purification of nuclei and extraction of RNA were as described (Gallinaro et al., 1983; Sittler et al., 1986). Contaminating DNA was digested by RNase-free DNase and the DNA fragments were removed by chromatography on a Sephadex G-75 column. Poly(A)+ RNA was selected by 3 passages over an oligo(dT)-cellulose column (Collaborative Research Inc.) and the nuclear RNA that did not bind was designated as poly(A)RNA. In order to
et al. remove possible contaminating poly(A)- RNA, poly(A)+ RNA was denatured in 80% (v/v) formamide and rechromatographed on oligo(dT)-cellulose after lowering the formamide concentration to 5%. (b) Preparation
of probes and labelling
The probes used for Northern blotting analysis are shown in Fig. l(b) and were labelled by nick-translation. Probes Da and Db were previously cloned in the SmaI site of pHp34 (Sittler et al., 1986). The probes used for S, mapping analysis as shown in Figs 3 and 4 were all prepared from the EcoRI-Hind111 DNA fragment of 1286 bp (positions -236 to + 1049). For preparation of the TaqI-RaaI probe, it was first digested by RsaI. The largest fragment was purified by centrifugation in a 5% to 20% (w/v) sucrose gradient and then digested by TaqI. End-labelling was performed without further purification. For preparation of the DdeI probe, the 1286 bp fragment was cut by DdeI and the probe was purified by electroelution after electrophoresis on a 5% polyacrylamide gel. For preparation of the AvaI-RsaI probe, the 1286 bp DNA fragment was digested by the 2 enzymes and the probe was purified by sucrose gradient centrifugation. For 3’ end-labelling, the probes were filled in at the 3’ end using the Klenow fra ment of DNA polymerase I in the presence of [a- f2 P]dCTP or [a-32P]dTTP (800 Ci/mmol), respectively, and the other cold dNTPs. 5’ End-labelling was as described by Maxam & Gilbert (1980) in the presence of [Y-~*P]ATP (5000 Ci/mmol; Amersham). The samples were electrophoresed on a strand separation gel and the single-stranded DNA probes were electroeluted. (c) Agarose/formaldehyde
gel electrophoresis and blotting of RNA
RNA was dissolved in 50% formamide, 1.8 Mformaldehyde, 25 mlcl-Mops, denatured by heating for 10 min at 65°C and separated by electrophoresis through horizontal 1.5% (w/v) agarose gels cast in 25 m&r-Mops, 5 mhr-sodium acetate, 1 mM-EDTA (final pH 7.0), and 2.2 M-formaldehyde. The migration was at 7.5 V/cm for 4 h. The gel was placed in 20 x SSC (SSC is 0.15 M-NaCl, 0.015 M-trisodium citrate, pH 7) for 0.75 h and the RNAs were transferred to nitrocellulose sheets (BA 85 Schleicher and Schiill) by blotting overnight. The prehybridization and hybridization with nick-translated probes were as described (Gallinaro et al., 1983). (d) 8, nuclease mapping S, nuclease analysis of RNA was carried out as described by Weaver & Weissmann (1979). An excess of the end-labelled DNA probes (8000 to 11,000 cts/min) and the RNA to be analyzed were coprecipitated by ethanol. They were dissolved in 10 ~1 of FNPE buffer (80% formamide, 0.4 M-NaCl, 40 mnr-Pipes (pH 6*4), 1 m&r-EDTA), denatured by heating for 10 min at 85°C and hybridized at 53°C for 1.5 h, then at 50°C for another 1.5 h and, finally, at 48°C for 0.5 h. The samples were diluted in 0.4 ml of cold digestion buffer and treated with 160 units of S, nuclease (Appligene) at 20°C for 1 h. Digestion with different concentrations of enzyme or for longer periods of time did not change the pattern of protected bands significantly. Digestion was stopped and the hybrids were precipitated with ethanol. After denaturation of the hybrids by heating in 80% formamide, 5 mw-Tris-borate (pH 8.3), 0.1 mM-EDTA for
10 min at 85”C, the protected DNA fragments were analysed by electrophoresis on an So,0 polyacrylamide/ 8 M-urea sequencing gel. To determine precisely the end of the DNA fragments, the homologous 5’ end-labelled single-stranded probes were cleaved according to Maxam & Gilbert (1980) and the reaction products were run in parallel. (e) Primer extension analysis The RNA sample was coprecipitated with an excess of the 5’ end-labelled primers p26 or p57 (Sittler et al., 1986). The pellet was dissolved in 10 ~1 of 250 mM-NaC1, 40 mMPipes (pH 6.4), 5 mM-EDTA, 0.2% (w/v) SDS, heated at 65°C for 5 min and hybridized by slow cooling to 30°C overnight. The hybrids were precipitated with ethanol and dissolved in 10 ~1 of 31 mw-Tris. HCl (pH 8.3), 25 mM-KCl, 3.75 mM-MgCl,, 5 mM-dithiothreitol, 0.2 mM of each dNTP and 2 units of RNasin (Promega Biotec). cDNA was synthesized by addition of 4 units of AMV reverse transcriptase and incubation at 41°C for 30 min. For sequence control, we used the same 5’ end-labelled primer and, as template. a single-stranded non-coding DNA fragment that overlaps the RNA extremities (positions 603 to 1189); cDNA synthesis was performed as above but in the presence of 1 of the 4 ddNTPs (50 PM for ddATP. 60 PM for ddCTP and ddGTP, 80 PM for ddTTP). The elongation products were analyzed on an 8% polyacrylamide/8 M-urea sequencing gel.
3. Results (a) The steady-state profoundly
E3 transcript
pattern
is
in DRB-treated cells
modi$ed
The structure and nucleotide sequence of the early region 3 as well as t,he major hallmarks for (0)
.I
1033
Polyadenylation-splicing
Alternative
synthesis and processing of transcripts have been described (Chow et al., 1979; H&is& et al., 1980; H&is& & Galibert, 1981; St&lhandske et al., 1983; Cladaras & ,Wold, 1985) and are summarized in Figure l(b). Of the four polyadenylation sites, only P2 and P4 are actively used at the early phase of infection, generat’ing the two major primary transcripts pre-c and pre-a (Fig. l(a)). Pl is mostly used at the late phase of infection for the formation of the 3’ end of RNAs from the L4 family (Le Moullec et al., 1983; Bhat & Wold, 1986). However, at the early phase of infection we found a small amount of an RNA (CPl) starting at the E3 capsite and terminating at Pl (Gallinaro et al., 1986). As for P3 (not shown in Fig. l), its existence was suggested from electron microscopic studies of transcripts (Chow et al., 1979) but it has never been detected by other methods. To study the effect of DRB on the production of the E3 transcripts ending at PI, P2 and P4. we first analysed the nuclear poly(A)+ RNA from untreated and DRB-treated cells by Northern blotting. The optimal conditions for DRB treatment were found to be 25 pg/ml for 20 minutes. The effect of the drug did not change upon doubling its concentration. The maximum effect was reached between 10 and 15 minutes, and persisted up to 30 minutes (data not shown). In poly(A)+ RNA from untreated cells, probe Db, specific to IVSl sequences (Fig. l(b)), displays the two precursors pre-a and pre-c, and the two related intermediates TVSl-exon 2-poly(A) (Fig. 2, lanes 1 and 3). These RNAs have been fully
.
h
Late
L4
pre-maNA
I
CPl
IVSl
EXl (b)
,----C I 1
Da
Ex2
,------,-------Dl I 372
PlA2 ‘L’ 620 768
02 951
A3 1740
A4 2157
L
P2
A6
P4
2190
2830
3255
.
Db
Figure 1. (a) The Ad-2 genome and the E3 and L4 primary transcripts. The r strand of the genome is divided into 100 units. Above is shown the late L4 pre-mRNA that terminates at Pl. The black squares represent the 3 leaders common to all major late families of RNA. Below is shown the early transcription unit 3 with its 3 polyadenylation sites. (b) The landmarks of the E3 transcription unit (from Stllhandske et al., 1983; Cladaras t Wold, 1985). C is the capsite, Ds are 5’ splice sites, As are 3’ splice sites and Ps are polyadenylation signals. The probes Da and Db used for Northern blot analysis are shown below (for isolation, see Materials and Methods).
1034
H. Gallinaro et al.
F
1234
5
6
7
8
Figure 2. Northern blot analysis of nuclear E3 RNA. The probes used to display the RNAs are indicated at the top and shown in Fig. l(b). Lanes marked + indicate the DRB-treated cells and lanes marked - indicate the control cells: 1.8% of either the poly(A)+ RNA fraction or the poly(A)- fraction was analysed. This represented: for poly(A)+ RNA (lanes 1 to 4), 1.35 pg (lanes - ) and 1.40 pg (lanes +); for poly(A)- RNA (lanes 5 to 8), 18 pg (lanes -) and 16pg (lanes +). Lanes 3 and 4 show overexposure of lanes 1 and 2. The E3 RNAs were previously characterized by other methods (Gallinaro et al., 1986; Sittler et al., 1986, 1987). characterized (Sittler et al., 1986, 1987). An RNA of about 800 nucleotides (indicated as CPl) is also detected and is more clearly visualized after overexposure of the gel (lane 3). This RNA is a very minor species when compared to the transcripts terminating at P2 and P4. In DRB-treated cells, we observe an almost complete disappearance of pre-c, a considerable decrease of pre-a and the disappearance of the two IVSl-exon 2-poly(A) RNAs (lanes 2 and 4). In contrast, the amount of the small RNA CPl increases, as well as its apparent complexity (lane 4). This increased complexity is not due to an increased heterogeneity of the 3’ ends of the core of CPl. This was shown by an S, nuclease protection experiment (Fig. 4(a)). The number of protected bands is not modified by treatment with DRB; only their intensity increases. Therefore, we assume that the heterogeneity observed by Northern blotting is due to a heterogeneity of the length of the poly(A) tail and we conclude that DRB provokes a marked increase of the proportion of WI relative to that of pre-a and pre-c. By using an exon l-specific probe, we verified that the amount of nuclear E3 mRNAs did not change much during the 20 minute treatment with DRB (data not shown). This apparent stability of mRNA is due to its long half-life and has been described (Harlow & Molloy, 1980; Nilsen et al., 1983; Edwards et al., 1985). Nuclear non-polyadenylated RNA was studied. It consists primarily of growing transcripts and of
non-polyadenylated splicing intermediates or products. Probes Da and Db specific to exon 1 and IVSI sequences, respectively (see Fig. l(b)), display a background signal due to the heterogeneous growing transcripts (Fig. 2, lanes 5 and 7). Over this background, we systematically detect a novel RNA or F-RNA of about 750 nucleotides. In addition, free IVSl is detected (lane 7). In DRBtreated cells, the general pattern of poly(A)- RNA is strikingly modified. The amount of large transcripts and of free IVSl decreases, while that of the small transcripts, and in particular of F-RNA, increases (lanes 6 and 8). Thus, while minor in untreated cells, F-RNA becomes a relatively abundant component in DRB-treated ceils. These results show that DRB considerably modifies the pattern of the E3 nuclear transcripts. It affects the selection of the polyadenylation sites, as shown by the changes of distribution of the primary transcripts terminating at Pl, P2 and P4 (Fig. 2). The decrease of the precursors terminating at P2 and P4 leads to a decrease of the related splicing intermediates and products (IVSI-exon 2 and free IVSI). On the other hand, the amount of the small promoter-proximal RNAs termed F-RNA and CPI increases. As can be seen in Figure I, the use of Pl and the excision of the first E3 TVS (DlA2) are mutally exclusive. In the following sections, we shall try to establish whether, and how, the increase of CPl and F-RNA is related to the mechanism of selection of Pl or excision of TVSI.
(b) Possible mechanism of selection of the polyadenylation site Pl The Northern blot analyses in Figure 2 suggest that F-RNA is a promoter-proximal RNA with a 3 end close to the 3’ end of TVSl. S, nuclease mapping and primer extension experiments were performed in order to determine its extremities more precisely. For the determination of its 5’ end, F-RNA from untreated cells was first hybridselected with the IVSl-specific probe Db. The selected RNAs were then electrophoresed in polyacrylamide gels. RNA 2, which has the size of FRNA and which can be revealed by hybridization with the exon l-specific probe Da (data not shown), was eluted (Fig. 3(a)). A primer, ~57 (Fig. 3(b), bottom) was then annealed to RNA 2 and extended. A 627 nucleotide cDNA was obtained (Fig. 3(b), lane 2), clearly different from the 255 nucleotide product synthesized from free lariat IVSl (lane l), which is the major hybrid-selected RNA species (Fig. 3(a)). The results indicate that the 5’ end of F-RNA is at the E3 capsite. This conclusion was confirmed by an S, mapping experiment using the 5’ end-labelled probe pr756 overlapping the capsite (Fig. 3(c)). The 3’ end of F-RNA was determined on the hybrid-selected RNA 2, by S, nuclease protection experiments, and was shown to be heterogeneous and located slightly upstream from the A2 3’ splice
Alternative (.a 1
1035
Polyadenylation-splicing (c) M
(b) Ml23
627
527-
:
.255
242238217-
* -p57 180-
AvuI C
Le2
Lel Dl
I-W
Le2
Lel
RSUI
I
721
-34
,+
Fok I
I 372
571 627
768
--Y
P57
-
pr756 721nt
255nt 627nf
Figure 3. Determination of the 5’ end of the F-RNA. (a) Hybrid selection. The method was as described (Sittler et al., 1986). The in viwo ‘H-labelled nuclear poly(A)- RNAs of 399 to 1996 nucleotides from untreated cells were hybridized to probe Db covalently linked to diazobenzyloxymethyl (DBM) paper. The hybrid-selected RNAs were fractionated on 5% polyacrylamide/&3 ~-urea gels and displayed by fluorography. RNA 2 was electroeluted and used for the experiments in (b) and (c). RNAs 1, 3 and 4 are different IVSl forms (Sittler et al., 1987). (b) Primer extension. Primer p57 was hybridized to eluted RNA 1 (lane 1) and RNA 2 (lane 2) and extended. Lane 3 shows a control experiment (probe treated in the absence of RNA). (c) S1 nuclease protection experiment. The 5’ end-labelled single-stranded AvaIRsaI probe was hybridized to RNA 2. The protected DNA fragment (721 nucleotides) and the residual probe (756 M-urea sequencing gel. Size markers: pBR322 digested by Mspl nucleotides) were analysed on a 5% polyacrylamide/8 (lanes M) or by FokI (lane Ml). nt, nucleotides.
site (results not shown). This heterogeneity, as well as the precise location of the 3’ ends, is illustrated in Figure 4(b), which shows that the same pattern of
bands is obtained in untreated and DRB-treated cells. The data indicate that DRB enhances the formation of F-RNA but that it is not responsible for its heterogeneity. By comparing the mobility of the protected DNA bands with that of the products of sequencing reactions and by combining the results of three we determined that the separate experiments, predominant 3’ termini of F-RNA were between positions 746 and 753 with minor termini up to positions 743 upstream and 759 downstream. As
shown in Figure 6, the sequence overlapping
the 3’
end region of F-RNA
a low
stability
is A + U-rich,
suggesting
of the hybrids and a possible trimming
S1 nuclease.
However,
the pattern
by
of bands did not
change when the nuclease concentration was modified (Fig. 4(d)), suggesting that nuclease trimming may be responsible for minor quantitative changes but it is not the major cause of the heterogeneity, which is likely to reflect the situation in vivo. Thus, F-RNA is an RNA starting at the E3 capsite and extending up to the region between the IVSl branch point and 3’ splice site. It contains the entire sequence of the non-polyadenylated core of
(b)
(a)
(c)
MM+
-I
M
premRNA 242 238 217 201 _, ws"QO 160
CPl4 A2
Q Bi
RNA c 14’.
G-RNAC
C
01
Pl
El
A2
C
Bl
A2
Ode1 pr Ode
Bl
A2
740
768
DC?*1
OdeL * 912
pr Ode 593
+-----r-
323nt
/VSl
17snt
F-RNA
372
Ode I
s-2
Tr CPl
Dl
C I I
A2
TI G-RNA
----cI=c32Ool \\~147/140nl
159/,71”,
-1 Cd) -
31 RNA
2
1 3 1.5 III /
F-RNA
1234GA -IQ
Figure 4. S1 nuclease mapping experiments. The 5’ or 3’ end-labelled (*) probes are shown at the bottom of each panel as well as the positions of the major protected fragments; - and + refer to the absence or presence of DRB. Lanes M, 5’ end-labelled Ms$ cleavage products of pBR322. Lane 0, probe treated in the absence of RNA. Lanes G, A + G, T + C and C, products of sequencing reactions. The sites of interest (A2, Bl) are indicated on these gels. So as to precisely localize the extremity of the fragments protected by the probe, we took into account the difference of migration of 2 nucleotides between the sequencing products and S, nuclease-treated fragments (Contreras et al., 1982; Gallinaro et al., 1986). (a) The 3’ extremity of CPl; 1.54pg (lane -) and 15Opg (lane +) of poly(A)+ RNA, representing 2% of the total poly(A)+ fraction, were analysed. (b) The 3’ extremities of F-RNA: (c) The 5’ extremities of G-RNA. In both (b) and (c), 18.8 pg (lanes - ) and 20.0 pg (lanes + ) of poly(A) - RNA representing 2% of the total fraction were analysed. Tr, transcripts that protect the full-length probe. (d) S, nuclease control for F-RNA. RNA was analysed as in (b), except that the ratio of S1 nuclease to RNA was changed as indicated at the top. Lanes 2 and 4 contain twice as much RNA as lanes 1 and 3. Note that each band is detected in all lanes and that only its intensity changes from one lane to another. nt, nucleotides.
1037
Alternative Polyadenylation-splicing CPl, as previously characterized (Gallinaro et al., 1986), plus about 130 nucleotides at the 3’ end. In fact, it has all the characteristics required for a substrate of the cleavage-polyadenylation reactions leading to the formation of CPl (for a review, see Birnstiel et al., 1985). Unfortunately, the precursorproduct relationship is difficult to demonstrate by pulse-chase experiments in vivo, since equilibration of the nucleotide pool takes about one minute, while CPl is already fully labelled two minutes after addition of a labelled nucleoside (Gallinaro et al., 1986). This makes interpretation of chase experiments rather hazardous. Nevertheless, the parallel stimulation of the formation of F-RNA and CPl in DRB-treated cells (Figs 2 and 4) supports the idea of a precursor-product relationship. In addition, recent experiments in vitro demonstrate that an RNA with its 3’ end close to that of F-RNA is faithfully cleaved and polyadenylated at Pl (Gallinaro, Meyer & Jacob, unpublished results). Note that the formation of F-RNA prevents the formation of pre-a and pre-c, while allowing that of CPl. Therefore, the formation or non-formation of F-RNA might be a crucial step in the selection of the E3 polyadenylation sites.
G-RNA were found between positions 764 and 770, whereas minor termini were found up to positions 752 and 775 (Fig. 6). As for F-RNA (see above), we were able to exclude the possibility that the major termini were artifactually produced by S, nuclease trimming. This conclusion was further confirmed by a primer extension analysis. An oligonucleotide p26 complementary to an exon 2 sequence was labelled at its 5’ end, annealed to nuclear poly(A)RNA of untreated and DRB-treated cells and extended (Fig. 5). A group of extension products ending in the region of the 3’ splice site between positions 763 and 768 was predominant and more intense in DRB-treated cells than in normal cells. This 763768 sequence is very close to the 764-770 sequence determined by S, nuclease mapping (Fig. 6). Thus, the two methods give comparable results, as far as the major 5’ ends are concerned, but not for the M
AGC
T
(c) Formation of F-RNA by cleavage of the primary transcript If F-RNA is involved in the selection of the E3 polyadenylation sites, then it is of importance to understand how it is formed. F-RNA might be either a product of transcription termination or a product of cleavage of the primary transcript. Both possibilities may be envisaged. The active use of Pl at the late phase of infection (Fig. l(a)) indicates that transcription termination may occur at some site downstream from Pl; at the early phase, the same type of event may provoke the formation of F-RNA. On the other hand, endonucleolytic cleavages might be important events in the mechanisms regulating pre-RNA processing as shown, for instance for the Ll ribosomal protein gene of Xenopus laevis (Caffarelli et al., 1987). To decide between these alternatives, we considered that an RNA with its 5’ end in the branch point-3’ splice site region should obligatorily exist in the case of post-transcriptional cleavage but not in the case of transcription termination. In addition, if this RNA is generated by cleavage, its amount should vary in parallel with that of F-RNA in the presence of DRB. We have looked for such an RNA in the nuclear non-polyadenylated RNAs. For this purpose, S, nuclease protection experiments were performed using the same probe as for the determination of the 3’ end of F-RNA but labelled at the 5’ end instead of the 3’ end. Figure 4(c) shows that an RNA with a heterogeneous 5’ end around the A2 3’ splice site is detected. This RNA is designated as G-RNA. Its amount increases in DRB-treated cells, as expected if it is formed simultaneously with F-RNA by a cleavage of the primary transcript. The predominant 5’ termini of
Bl
G-RNA
A2
Lel
871 -I+ e
G-RNA Tr
Figure
p
P26 104/109nl 871nl
5. Primer extension analysis of G-RNA. RNA from control (lane -) and DRB-treated
Poly(A)cells (lane +) were used as templates. The localization of primer p26 and of its extension products is indicated at the bottom. Lane M, size markers as in Fig. 4. Lanes A, G, C and T, extension products made with p26 using a single-stranded non-coding DNA fragment as template and 1 of the 4 ddNTPs. The positions of A2 and Bl are indicated. nt, nucleotides.
1038
H. Gallinaro Branch point
%Cyw
3’ Spliie
IVS 1
4
734
et al.
750
740
760
~~UUUA~CG~~~UCUUC~UUUGUAAUU~ACAA~AGUU~~AG~G~ F-RNA G-RNA G-RNA
l
junction I
Ex 2
770
777
.000~*~~~*e~000000~~~
* *
SI lluctoaeo
. . . . . ..*.00000000000oeo*~mo*ooooo~ q . . . . ..o...*......~...clo.....
ooovTlllll~uvtl~vv
6
lz *I3
wmnebn
vPvllTv~vv~~v~oo
Figure 6. The branch point-3’ splice site region. The sequence of the region (H&s& et al., 1980) is presented. The extremities of the fragments as determined by S, nuclease mapping (average of 3 experiments) is shown below the sequence. (e) Strong bands; (0) weak bands; (0) faint bands. The 5’ extremities of G-RNA as determined by primer extension are also indicated. (a) Strong bands; (0) weak bands; (0) faint bands. The intensity of all these bands increases in DRB-treated cells. Others, whose intensitv decreases, are marked by a star. A and B indicate the 2 possible interpretations of the results (see the text).
minor bands. It is not possible to decide whether these minor bands are artifactually generated by S,
nuclease trimming or whether they are not, displayed in the primer extension experiments for some reason(s) to be determined. On the other hand, the DRB-induced increase was apparently lower when studied by primer extension than by S1 nuclease mapping or Northern blotting. We have no explanation for this discrepancy. Nevertheless, we would like to emphasize that a DRB-induced increase was systematically observed in all experiments with all methods. In conclusion, we have characterized a family of G-RNAs with a heterogeneous 5’ end around the 3’ splice site of the first IVS. It is likely to be generated simultaneously with F-RNA by a cleavage of the primary transcript and the possible modalities of such a cleavage will be examined in the next section. Note that the production of both F-RNA and G-RNA is similarly enhanced in the presence of DRB (Fig. 4), suggesting that the drug is involved in cleavage stimulation. (d) Generation
of F-RNA
and
G-RNA
The demonstration of two families of RNAs, F-RNA with a heterogeneous 3’ end in the branch point-3’ splice site region of IVSl and G-RNA with a heterogeneous 5’ end around the 3’ splice site indicates that a nuclease cleavage has occurred in this region. The inspection of the precise localization of the major 3’ termini of F-RNA and of the major 5’ termini of G-RNA (Fig. 6) shows that these ends do not coincide, as would b;e’ expected if single internucleotide bonds were cleaved. Rather, there is a gap of about ten nucleotides between the major extremities, suggesting that whole segments of the sequence were removed. Due to the fact that minor bands may be indicative either of high efficiency of cleavage (little material left) or of low efficiency of cleavage (most material uncleaved), it is not
possible to decide as to the exact primary cleavage sites (A or B, Fig. 6). Nevertheless, the accessibility of a short sequence (A) or sequences (B) to intranuclear nucleases suggest that one or several of the factors that normally bind the branch point-3’ splicing region do not bind a fraction of the nascent RNA molecules. This fraction is small in untreated cells and much larger in DRB-treated cells. It is well-known that the branch point-3’ splice site region is crucial for the assembly of the splicing complex (Green, 1986; Padgett et al., 1986; Aebi & Weissmann, 1987; Maniatis & Reed, 1987) and, therefore, we may assume that assembly is inhibited for a fraction of the molecules and that DRB accentuates this inhibition. 4. Discussion The demonstration of novel E3 nuclear RNAs in adenovirus 2-infected HeLa cells allows us to describe a novel early pathway for the formation of E3 transcripts and to propose a mechanism for the switch from the major pathways to the minor one (Fig. 7). It should be emphasized that the minor pathway is functional in infected HeLa cells in the absence of both DRB and cycloheximide. This was shown by the detection of CPl (Gallinaro et al., 1986) and F-RNA (Sittler, 1986). Cycloheximide, which allows a relative increase of all E3 RNA, and DRB, which enhances the minor pathway, and has helped us to specify the transcripts involved in the regulation process. The major event in the switch seems to be a cleavage of the growing transcript. If we examine the alternative possibility of generation of F-RNA by transcription termination, we observe that it implies the formation of G-RNA by re-initiation immediately downstream from the termination site. The DRB
data allow this possibility
to be excluded;
indeed, the drug that enhances the formation of both RNAs would thus enhance both termination and initiation of transcription, which is unlikely
Alternative
Polyadenylation-splicing
Naacsnt C t
4
Dl I 372
II
Transcript
pxiiiiq I 507
p1 I, 620
j7iiFiq
Bl 1 740
CI.w8go ,,
F-RNA
/I
1039
A2 I 768
l
I Hlll G-RNA + 1111
CIw48g8 polyrdonylrllon
I
CPl I,
r,
1 “n
Figure 7. Proposed mechanism for Pl selection at the early phase of infection.
(Mukherjee & Molloy, 1987). The particular cleavage of the E3 pre-RNA occurs in the branch point-3’ splice site region of IVSl. This region is of the utmost importance for the formation of the splicing complex in vitro (for reviews, see Green, 1986; Padgett et al., 1986; Aebi & Weissmann, 1987; Maniatis & Reed, 1987) and probably also in vivo. Our data suggest that, for a fraction of the transcripts, one or several constituents of the splicing complex (snRNPs or other factors) do not bind their specific sites on the RNA, leaving the way open to intranuclear nucleases. We observed that the average size of hnRNP decreased upon treatment with DRB and that it became more sensitive to degradation (data not shown). This is compatible with perturbations of assembly but obviously does not prove it. In vitro investigations are in progress to solve this problem. Once the primary transcript has been cleaved,
of polyadenylation and splice sites (Bovenberg et al., 1986; Leff et al., 1986, 1987; Peterson & Perry, 1986; Breitbart et al., 1987; Galli et al., 1987; Tsurushita et al., 1987). That DRB does not cleave any intervening sequence is proven by the absence in other E3 IVS of detectable cleavages similar to those found in IVSl. On the other hand, the fact that DRB may act post-transcriptionally at the level of premRNA processing underlies some earlier observations. For instance, it has been shown that DRB severely inhibits only RNAs that require extensive processing for their formation (Harlow & Molloy, 1980). Moreover, the synthesis of unspliced RNAs such as Ad-2 polypeptide IX mRNA mRNA (Vennstr6m et al., 1979) or interferon (Sehgal & Tamm, 1979) is insensitive to the drug. Studies of the effect of DRB on the expression of other selected genes might be helpful for our
the major early precursors longer be formed, whereas
understanding of the molecular mechanism underlying the events observed in DRB-treated cells.
pre-a and pre-c can no the promoter-proximal
cleavage product F-RNA may generate CPl (Fig. 7). Thus, a defect in the assembly of the splicing complex at the level of IVSl might trigger the use of an upstream polyadenylation signal and thus
interfere
with
polyadenylation
site selection.
An alternative to this mechanism is that DRB may affect the recognition of the polyadenylation site Pl, and that this event may provoke a cleavage at the far 3’ end of IVSl. Though this alternative cannot be excluded, we rather favour the first possibility, which follows the canonical rules of 3’ end formation of mRNA (Birnstiel et al., 1985).
Our results give some indications
as to the mode
those whose regulation
& Molloy,
1987) in agreement
concern a large number particular
cleavage
probably
restricted
event
of genes, if not all, the that
we
describe
is
to a few genes; for instance,
use
References
with
earlier studies (Sehgal et al., 1976a, b; Winicov & Button, 1982; Egyhazi et al., 1983; Zandomeni et al., 1983; Holst & Egyhazi, 1985). Using another selected gene, we show that DRB enhances a posttranscriptional cleavage event. In contrast to the inhibition of transcription initiation, which may
the alternative
We are grateful to Mrs L. Kister for her invaluable technical assistance throughout this work, and to Dr G. Richards for a critical reading of the manuscript. We thank J. L. Weickert and M. Gilbert for growing HeLa cells, C. Kutschis, C. Werl& and B. Boulay for assistance in the preparation of the manuscript. This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la San6 et de la Recherche MBdicale, the Fondation pour la Recherche MBdicale Franpaise, the Association pour la Recherche sur le Cancer and the Ligue Nationale Franpaise contre le Cancer.
of action of DRB. Investigations on the transcripts of the /?-globin gene demonstrate that DRB inhibits initiation in vivo and transcription in vitro
(Mukherjee
implies
Aebi, M. & Weissmann, C. (1987). Trends Genet. 3, 102107. Bhat, B. M. 6 Wold, W. S. M. (1986). J. Viral. 60, 54-63. Birnstiel, M. L., Busslinger, M. & &rub, K. (1985). Cell, 41, 349-359.
Bovenberg, R. A. L., van de Meerendonk, W. P. M., Baas, P. D., Steenbergh, P. H., Lips, C. J. M. & Jansz, H. S. (1986). Nucl. Acids Res. 14, 8785-8803. Breitbart, R. E., Andreadis, A. & Nadal-Ginard. B. (1987). Annu. Rev. Biochem. 56, 467495.
1040
H. Gallinaro
Caffarelli, E., Fragapane, P., Gehring, C. & Bozzoni, I. (1987). EMBO J. 6, 3493-3498. Chow, L. T., Broker, T. R. & Lewis, J. B. (1979). J. Mol. Biol. 134, 265-303. Cladaras, C. & Wold, W. S. M. (1985). Virology, 140, 2% 43. J., van de Contreras, R., Gheysen, D., Knowland, Voorde, A. & Fiers, W. (1982). Nature (London), 390, X%505. Edwards, D. R., Parfett, C. L. & Denhardt, D. T. (1985). Mol. Cell. Biol. 5, 32863288. Egyhazi, E., Holst, M. & Tayip, U. (1983). Eur. J. Biochem. 130, 223-226. Galli, G., Guise, J. W., McDevitt, M. A., Tucker, P. W. & Nevins, J. R. (1987). Genes Develop. 1, 471481. Gallinaro, H., Puvion, E., Kister, L. Jacob, M. (1983). EMBO J. 2, 953-960. Gallinaro, H., Sittler, A., Kister, L. & Jacob, M. (1986). Biochimie, 68, 1009-1017. Green, M. R. (1986). Annu. Rev. Genet. 20, 671-708. Harlow, P. & Molloy, G. (1980). Arch. Biochem. Biophys. 203, 764-773. H&is&, J. & Galibert, F. (1981). Nucl. Acids Res. 9, 12291240. H&is&, J., Courtois, G. & Galibert, F. (1980). Nucl. Acids Res. 8, 2173-2192. Holst, M. & Egyhazi, E. (1985). J. Cell. Biochem. 29, 115126. Laub, O., Jakobovits, E. B. & Aloni, Y. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 3297-3301. Leff, S. E., Rosenfeld, M. G. & Evans, R. M. (1986). Annu. Rev. B&hem. 55, 1091-1117. Leff, S. E., Evans, R. M. & Rosenfeld, M. G. (1987). CeZE, 48, 517-524. Le Moullec, J. M., Akusjiirvi, G., Stalhandske, P., Pettersson, U., Chambraud, B., Gilardi, P., Nasri, M. & Perricaudet, M. (1983). J. Viral. 48, 127-134. Maniatis, T. & Reed, R. (1987). Nature (London), 325, 673-678.
Edited
et al. Maxam, A. M. & Gilbert, W. (1980). Methods Enzymol. 65, 499-560. Mukherjee, R. & Molloy, G. R. (1987). J. BioE. Chem. 262, 13697-13705. Nilsen, T. W., Maroney, P. A. & Baglioni, C. (1983). Mol. Cell. Biol. 3, 64-69. Padgett. R. A., Grabowski, 1’. J., Konarska, M. M., Seiler, S. & Sharp, P .A. (1986). Annu. Rev. Biochcwb. 55, 1119-1150. Peterson, M. L. & Perry, R. P. (1986). Proc. Nat. Acad. Sci., U.S.A. 83, 8883-8887. Sehgal, P. B. & Tamm, I. (1979). Virology, 92, 240-244. Sehgal, P. B., Darnell, J. E., Jr & Tamm, I. (1976u). Cell. 9, 473480. Sehgal, P. B., Derman, E., Molloy, 0. R., Tamm, I. & Darnell, J. E. (19763). Science, 194, 431433. Sehgal, P. B., Fraser, N. W. & Darnell, J. E.. Jr (1979). Virology, 94, 185-191. Sittler, A. (1986). Ph.D. thesis. University Louis Pasteur, Strasbourg. Sittler, A., Gallinaro, H. & Jacob, M. (1986). Nucl. Acids Res. 14, 1187-1207. Sittler, A., Gallinaro, H., Kister, L. & Jacob, M. (1987). J. Mol. Biol. 197, 737-742. Stblhandske, P., Persson, H., Perricaudet, M., Philipson, L. & Pettersson, U. (1983). Gene, 22, 157-165. Tamm, I., Kikuchi, T.. Darnell, J. E., Jr & SaldittGeorgieff, M. (1980). Biochemistry, 19, 2743-2748. Tsurushita, N., Avdalovic, N. M. & Korn, L. J. (1987). Nucl. Acids Res. 15, 46034615. Vennstriim, B., Persson, H., Pettersson, U. & Philipson, L. (1979). Nucl. Acids Res. 7, 1405-1418. Weaver, R. F. & Weissmann, C. (1979). Nucl. Acids Res. 7, 117551193. Winicov, I. & Button, D. (1982). Eur. J. Biochem. 124, 239244. Zandomeni, R., Bunick, D., Ackerman, S., Mittleman, B. & Weinmann, R. (1983). J. Mol. Biol. 167, 561-574.
by B. Mach