Developmental regulation of covalent modification of double-stranded RNA during silkmoth oogenesis

J. Mol. Bid. (1991) 218, 517-527

Developmental Regulation of Covalent Modification of Double-stranded RNA during Silkmoth Oogenesis Yasir A. W. Skeiky and Kostas Iatrout University of Calgary, Department 3330 Hospital Drive N. W., Calgary,

of Medical Biochemistry Alberta, Canada T2N 4Nl

(Received 26’ September 1990; accepted 3 December 1990) Follicular cells of the silkmoth Bombyx mori contain an enzymatic activity that modifies RNA duplexes in vitro. The modifying activity converts adenosine residues into inosine in duplex but not single-stranded RNA and mediates the partial unwinding of the complement strands. Because of the modification, the RNA loses its ability to form perfect duplexes with its complement upon reannealing in vitro. The modifying enzyme is localized in the cytoplasm of follicular cells and its activity is modulated in a developmentally regulated manner. In contrast, follicular nuclei contain an activity that inhibits the modification and unwinding of duplex RNA. The modifying activity is also present in the cytoplasm of unfertilized oocytes and its accumulation during oogenesis parallels that of the follicular cells. Examination of an established silkmoth cell line of ovarian origin revealed that, in contrast to the situation with follicular cells, the modifying activity has an exclusive nuclear localization. The cytoplasmic fraction of these cells is not only devoid of modifying activity but, as is the case with the nuclear fraction of follicular cells, contains an activity that inhibits duplex RNA modification and unwinding. We conclude that the modification promoting and inhibiting activities are not restricted to a single cell type and that their compartmentalization is developmentally regulated.

1. Introduction

by an associated activity that, in fact, appears to be responsible for the observed unwinding of the RNA duplexes. The modification was identified as a deamination of adenosine moieties at the 6’ position resulting in their conversion into inosine (Bass & Weintraub, 1988). This paper describes the identification of a similar activity in follicular cells and other ovarian cell types of the silkmoth. The differential distribution and developmental profiles of the modifying activity in the two major cellular compartments, nuclei and cytoplasm, of follicular cells, unfertilized oocytes and an established silkmoth cell line are presented. In addition, we present evidence suggesting that silkmoth cells also contain a counteracting activity that inhibits the deamination of adenosine residues in duplex RNA. The inhibition does not appear to be due to a reversal of t,he modification and the inhibitory activity is localized in a cellular compartment distinct from that of the modifying activity. The differential compartmentalization of the inhibitory activit,y appears to be development,ally regulated.

Choriogenic follicular cells of the silkmoth Bombyx mori contain large quantities of developmentally regulated chorion antisense RNA (Skeiky $ Iatrou, 1990). Chorion antisense RNA was shown to be transcribed from the same loci as its mRNA complement and to accumulate in follicular cytoplasm in parallel with chorion mRNA. Surprisingly, however, antisense RNA was not duplexed with ehorion mRNA, although it could form perfect duplexes with the latter upon incubation in vitro. These observations suggested that follicular cells may contain an enzymatic activity that unwinds RNA duplexes as these are transported from the nucleus into the cytoplasm. The search for such activity was also prompted by the results obtained in other laboratories that suggested the presence of developmentally regulated RNA duplex unwinding activities in Xenopus (Bass & Weintraub, 1987; Rebagliati & Melton, 1987) and in a limited number of other cell types of mammalian origin (Wagner & Nishikura, 1988). Further work in these systems demonstrated not only that RNA-RNA duplexes are unwound by a protein enzymatic activity but also that the unwound RNA is covalently modified

2. Materials

and Methods

(a) Tissue culture cells t Author t,o whom all correspondence should be addressed. otn2-2836/91!070517-1

I $os.oo/o

B. mori tissue culture cells (Bm5; Grace. 1967) were maintained at 28 to 29°C in complete TPL-41 growth

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medium (Weiss et al., 1981) as described (Iatrou et al.. 1985). The cells were subcultured weekly at a density of 0-2x IO6 cells/ml. (b) SlOU e&act preparations (i) Nuclear extracts (a) Follicular

cells

Ovarioles (total or staged) were dissected from 10 to 100 pupae in 1 x RSB (10 mw-Tris.HCI (pH 7.8), 10 mMMgCI,, 1 miv-DTT). The surrounding sheath and other debris were removed by gently squeezing the ovarioles between 2 layers of Whatman paper. Follicles were washed 3 times with RSB and follicular cells lysed with 3 volumes of RSB containing 1 miw-phenyl methane sulphonyl fluoride and 65% (v/v) NP-40 at 0°C for 10 min. The unbroken oocytes were allowed to settle by gravity, and the supernatant centrifuged at 2000g to pellet the follicular nuclei. Nuclear extracts were prepared as described (Skeiky & Iatrou, 1990). (b) Bm5 cells Bm5 cells were harvested by centrifugation at 3OOOg, washed twice in ice-cold RSB and pelleted again. The pellet was resuspended in 3 volumes of RSB containing 1 mhl-phenyl methane sulphonyl fluoride and 95% NP-40 and lysed by homogenization in a Dounce homogenizer (about 15 strokes of a type B pestle). Purification of the nuclei and preparation of nuclear extracts were as described above for follicular cehs. The purity of the nuclei used for extract preparation was assessed by light microscopy and electrophoretic analysis of nucleic acid contents. The latter demonstrated that the nuclei lacked mature size ribosomal RNA species. (ii) Cytoplasmic (a) Follicular

exkructs cells

Total choriogenic or staged follicles were treated as described above. The post-nuclear supernatant (cytoplasmic fraction) was brought to 10% (v/v) glycerol and dialyzed exhaustively against TGKED buffer (50 IIIMTris. HCI (pH 7+3), 25% glycerol, 50 miv-KC], 91 mMEDTA: 65 mM-DTT; Bass & Weintraub, 1988). (b) Oocyten and Bm5 cells Following lysis of the follicular cells, the unbroken oocytes were allowed to settle by gravity and washed 3 times with RSB. Cytoplasmic extracts were prepared from oocytes (devoid of follicular cells) and Bm5 cells by homogenization in a Dounce homogenizer with 3 volumes of TGKED buffer using 15 strokes of a type B loose-fit pestle followed by centrifugation at 100,000 g for 60 min. In the case of the oocyte extracts, following centrifugation the tubes were frozen in liquid nitrogen and the top portion (containing lipid components) was cut off and discarded. The absence of detectable nuclear contaminants from the follicular and Bm5 cell cytoplasmic preparations was established by 2 independent criteria: electrophoretic analyses of nucleic acid contents to document the absence of genomic DNA and gel retardation assays to document the lack of chorion promoter DNA-binding activities known to exist in follicular and Bm5 cell nuclei (Skeiky & Iatrou. 1991). All extracts were frozen in liquid nitrogen and stored at -76°C. The protein concentration of the SlOO extracts was det,ermined by the Bradford (1976) protein assay using the BioRad protein assay reagent and bovine serum albumin as a standard.

and K. Iatrou

(c) Preparation

of duplex

RNA

Tabelled sense and antisense RNAs were transcribed from the linearized plasmid pgHcB(O.5) using SP6 and T7 RNA polymerase, respectively, in the presence of [a-32P]UTP as described (Skeiky & Iatrou, 1990). The labelled sense and antisense transcripts were annealed at a 1 : 1 molar ratio in formamide annealing buffer (80% (v/v) formamide, 40 mM-Pipes (pH 67), 400 mM-NaCl, 1 mMEDTA) at a final concentration of 2 pmol RNA per 20 ~1. The solution was heated at 95°C for 5 min and then rapidly transferred to a water bath pre-set at 55°C. After annealing for 6 to 9 h, the reaction volume was brought to 100 ~1 with diethyl pyrocarbonate-treated water and digested with RNase A by adding 600 ~1 of ice-cold 0.3 MNaCl, 10 mw-Tris.HCl (pH 7.5), 5 mM-EDTA and 40 pg RNase A/ml for 1 h at 35°C (Melton et al., 1984). This treatment was done to ensure that the single-stranded non-complementary overhangs (28 and 25 nucleotides of the sense and antisense transcripts, respectively), derived from sequences comprising the polylinker of the transcription vector pGEM-1, were hydrolyzed. Following digestion. the RNase A-resistant duplexes were purified as described (Bass & Weintraub. 1987). (d) RNA unujinding reactions Unwinding reactions were performed essentially as described by Bass & Weintraub (1987). Duplex RNA (05 to 1 fmol) was mixed with 10 to 20 pg of the appropriate extract in a final volume of 20 ~1 containing 1 x TGKED and 5 mM-MgCI, and incubated for 3 h (unless otherwise indicated) at 25°C. Following deproteinization and ethanol precipitation. the reactions were analysed on a native 496 polyacrylamide gel followed by autoradiography at -70°C. (e) .~uclsus~ PI digestion and thin-layer chromatography Purified RNA from unwinding reactions was digested t,o completion with nuclease Pl by resuspension of the RNA in a nuclease digestion reaction containing 10 mMTris. HCI (pH 7.4). 1 mM-EDTA and 500 units of nucleasr Pi/ml. Tncubation was at 50°C for 1 h (Bass & Wcintraub, 1988). The digested products were purified by extractions with phenol. chloroform and ether. The samples were lyophilized and resuspended in 2 to 20~1 of diethyl pyrocarbonate-water. Equal samples of nucleasedigested reactions were spotted at the bottom of a 20 cm x 20 cm PEI-cellulose plate with fluorescent indicator (Cel 300 PEI/IJV254; Brinkman Instruments Inc.). Mixtures of 10 mM of each unlabelled 5’ ribonucleoside monophosphate (5’AMP, 5’CMP, 5’UMP, 5’ GMP and 5’ TMP) were spotted alongside the experimental samples to serve as standards. Chromatography was in 1 dimension using as a solvent a mixture of saturated (NH,)zS04. 61 M-NaOAc (pH 6.0) isopropanol (79 : 19 : 2). At the end of the run. the plate was air dried and the positions of the cold standards located with an ultraviolet lamp (254 nm). The plate was t,hen autoradiographed at -70°C.

3. Results (a)

putative

Detection

RNA

of RNA modifying

duplex

activity call types

unwinding in different

and a ovarian

Nuclear and cytoplasmic extracts prepared from follicular cells of choriogenic follicles, their oocyt’es (cytoplasmic extracts only) and Rm5 tissue culture cells (ovarian origin) were tested for unwinding activity using as substrate duplex RNA generated

RNA Duplex Modi$cation from two complementary riboprobes synthesized in vitro from clone pgHcB(O.5) (Fig. 1 (a)). As shown in Figure l(b), incubation of the duplexes (lane I)) with the rytoplasmic follicular (lane F’) or oocyte (lane 0’) ext,ract, caused a considerable change in t,heir electrophoretic behaviour: the purified labelled species migrated towards the region where singlestranded RNA migrates (lane M). The apparent unwinding effect was much more pronounced with the oocyte cytoplasmic extract. which caused all of the starting duplex RNA to shift towards the position of single-stranded RNA. The lesser unwinding effort observed with the follicular extract was shown not to have resulted from the presence of NP-40, which was included in the extraction buffer; addition of an equivalent amount of NP-40 to the oocyte extract did not alter the magnitude of the oocyte unwinding effect, (not shown). Judging from

its polydisperse electrophoretic mobility, the incubated double-stranded RNA was not completely dissociated into the two monomers. The diffuse of the treated duplexes remained mobility unchanged even when longer incubat,ion times (5 h) were employed or fresh extracts were added t,o the partially unwound duplexes (not shown). Incubation of the RNA duplexes with the follicular nuclear extract (lane F”) caused only a minimal change in t)he electrophoretic mobilit,y of the duplexes; no species reaching the region of singlestranded RNA were detected. Therefore. follicular nuclei appear to be largely devoid of t,hr putative unwinding activity. In fact, t’he low level of unwinding activity of the nuclear extracts may be due to some cytoplasmic contamination. Finally, in contrast to the situat,ion observed with the follicular and oocpte +oplasmic fractions.

(a) -

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0’

8”

C

6’

FC

Be

0’

MW

64s i S *

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220 ‘200 l 175 l

D- 115 l

100

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Figure 1. Duplex RI\;A unwinding activities in silkmoth cell extracts. (a) Preparation of duplex RPI;A. transcripts were synthesized in vitro from plasmid pgHcB(05). The radioactive sense (S) and antisense wer? annealrd at a 1: I molar ratio, treated with RP;ase A to remove the single-stranded overhangs rlertrophoresis in a native polyacrylamide gel (lane C in (b) below). The length of the R&se A-resistant

Complementary (AS) transcripts and purified by

duplex RNA is 475 base-pairs. (b) and (c) Unwinding/modifying activities of follicular, oocyte and Bm5 extracts. Duplex RKA (1 fmol) was incubated with IO pg of extract in a final reaction volume of 20 ~1. Following deproteinization and precipitation with ethanol. each reaction was analysed in 2 different ways: half was loaded directly on a native 4% (w/v) polyacrylamide gel (b). The remaining half was treated with RNase ,4 prior to analysis on the same gel system (c). Lanes C contain the RiVase A-resistant S-AS duplexes prior to purification of the full-length duplex (D). The low molecular weight fragments (asterisks) are due to RNase A cleavage in A/U-rich regions of the duplex. Lanes M and D contain the native monomeric single-stranded species (S and AS) and the RNase A-resistant duplexes (S-AS), respectively. Lanes F”. F’. O’, H” and Kc contain duplexes derived from incubations with follicular nuclear, follicular cytoplasmic, oocyte cyt,oplasmir. Bm5 nuclear and Bm5 cytoplasmic extracts, respectively. Lane MW contains molecular weight markers (numbers represent lengths in base-pairs).

Y. A. W. Skeiky and K. Iatrou

520

analysis of the nuclear and cytoplasmic fractions of Bm5 cells revealed a reciprocal distribution of the unwinding activity; while considerable unwinding was obtained with the nuclear fraction (lane B”), incubation with the cytoplasmic extract (lane B”) resulted in no unwinding of the duplexes. The presumed partial single-stranded nature of extract-treated duplexes was confirmed by digesting the unwound RNA with RNase A. As shown in Figure l(c), in contrast to the duplexes that were incubated without extract (lane C), which were largely resistant to the RNase treatment, those incubated with the cytoplasmic follicular (lane F”) or oocyte (lane 0’) extract were hydrolysed by RNase A. The degree of duplex sensitivity to RNase A correlated with the level of the presumed unwinding activity (compare lanes F” to 0” in Fig. l(b) and (c); under the same conditions, control single-stranded RNA (not shown) was hydrolysed to mononucleotide size). The most prominent RNase A-resistant species obtained following incubation with the oocyte or follicular cytoplasmic extract had a size of about 100 bpt, indicative of the presence of regions of persisting sequence complementarity in the unwound duplexes. The residual susceptibility of the duplexes (even of those that had not been incubated with any extracts; see also Fig. l(b), lane C), is probably due to RNase cleavage occurring at A/U-rich regions. The sizes of these fragments are consistent with those predicted to arise from the digestion of two A/U-rich blocks distributed asymmetrically within the duplex (see Tsitilou & Iatrou, 1983, for sequence details). Finally, RNase A treatment of duplexes that were incubated with the Bm5 cytoplasmic extract that is devoid of unwinding activity (lanes B’) resulted in a pattern of RNase A-resistant fragments indistinguishable from that of the control, thus confirming the absence of unwinding activity in this extract. (b) Specificity of the unwinding

activity

A quantitative evaluation of the unwinding activity present in the oocyte extract was carried out through the time course analysis presented in Figure 2(a) (upper). Using as substrate 1 fmol of double-stranded RNA and 10 pg of oocyte extract, duplex unwinding increased in a linear fashion with time and complete disappearance of the RNA duplex occurred within a period of three hours. Electrophoretic analysis of replica samples on a native polyacrylamide gel following heat denaturation (Fig. 2(a), lower), revealed that both monomer strands were intact. However, the mobility of the monomers appeared to be increasingly affected with progressive unwinding (gradual convergence of the 2 monomer bands). This effect was reminiscent of the result of RNA base modification reported by Bass &

7 Abbreviations polyethyleneimine.

used: bp, base-pair(s);

PEI,

Weintraub (1988) and Wagner et al. (1989), and was less pronounced in the case of the follicular cytoplasmic extract which contains a lower level of unwinding activity (data not shown). Electrophoresis of the unwound samples on a denaturing gel (Fig. 2(b)) resulted in the appearance of a single (monomeric) band, indistinguishable in migration from that of the unincubated mixture of the two monomer strands, thus ruling out the possibility that the altered mobilities observed in the native gel have resulted from changes in the molecular weight of the RNA. Electrophoretic analysis of one of the monomer RNAs following incubation with increasing amounts of the oocyte extract, revealed no alteration of the native electrophoretic mobility of the treated monomer (not shown). Thus, assuming that the altered mobility of the partially unwound monomers on the native gel is, indeed, due to a modifying activity, this activity should be specific for double-stranded RNA. This was confirmed through competitive inhibition assays (Fig. 2(c)), which revealed that although duplex unwinding was completely inhibited by the presence of an excess of unlabelled duplex, it was not affected by the presence of an excess of double-stranded DNA or single-stranded RNA containing intramolecular double-stranded regions (tRNA). Moreover, an excess of either of the two monomer strands that are devoid of regions of self-complementarity (Skeiky & Iatrou, 1990) was unable to inhibit the unwinding of duplex RNA (not shown). Finally, preincubation of follicular cytoplasmic extracts with proteinase K or EDTA prior to the addition of the duplex RNA resulted in the complete elimination of the unwinding effect (Fig. 2(c)), suggesting that the unwinding activity has a protein component. (c) Unwinding of duplex RNA is correlated with stable aderwsine to inosine modi&ation of the monomer strands The abnormal electrophoretic mobility of the monomer strands of the partially unwound duplexes in native gels following heat denaturation suggests the presence of a structural heterogeneity in the two monomer populations. To determine whether the RNA strands of the unwound duplexes were covalently modified, purified duplex RNA that had been subjected to incubations with follicular (F’), oocyte (0’) or Bm5 (BC) cytoplasmic extracts (Fig. 3(a)) was heat-denatured and reannealed in vitro, and the resultant duplexes analysed on a native polyacrylamide gel (Fig. 3(b)). The electrophoretic analysis demonstrated that most of the reannealed monomers derived from the follicular and oocyte cytoplasmic incubations failed to migrate as duplexes. Their behaviour was similar to that of the extract-treated duplexes (Fig. 3(a)) except that the retarded species migrated closer to the origin of the gel. This is probably due to the creation of complex concatameric forms resulting from the reannealing of partially complementary molecules. In contrast,

RNA Duplex Modi$cation (a 1 5

521

(b)

15

30

60

120

180

(min)

F”

S/AS

Fc

Oc

D

-

AS-

Figure 2. Time course and substrate specificity of the unwinding activity. (a) Time-dependent unwinding of doublestranded RNA by the oocyte extract. Duplex RNA (S/AS) (@5 fmol) was incubated for the indicated times with 10 pg of oocgte extract. Following deproteinization, half of each time point was analysed directly on a native 49’, polyacrylamide gel (upper), while the remaining half was heat-denatured at 90°C for 3 min in formamide prior to analysis on a native 4% polyacrylamide gel (lower). Bands identified as S/AS, S and AS contain the duplex and the monomeric single-stranded species. respectivelv. (b) Following incubation of the duplex RKA (S/AS) with 10 pg of follicular nuclear (lane F”). follicular cytoplasnk (lane F’) or oocyte cytoplasmic (lane 0’) extract for 3 h, the deproteinized samples were analysed on a 6y0 denaturing polyacrvlamide gel. Lane D is the control duplex RPjA standard. (c) Left: unwinding reactions employing IO pg of oocyte extract were carried out in the presence of proteinase K or 100 mM-EDTA, as indicated. Lane - contains the standard control reaction without any additions. Right: competition analysis using 05 fmol of radiolabelled duplex RKA and IO pg oocyte extract in the presence of a 50 M excess of cold competitor nucleic acid as indicated

the RNA monomers recovered from the incubation with the Bm5 cytoplasmic extract (lane B’), which is devoid of unwinding activity, were able to form

perfect duplexes under the same conditions. Therefore, the monomer strands that have been subjected t.o unwinding by the follicular and oocyte extracts have lost their ability to form perfect duplexes, presumably because of some type of stable modification that cannot be reversed by the usual methods of nucleic acid purification. Previous work on other experimental systems has established that RNA duplex unwinding may be the consequence of adenosine deamination occurring during the incubation of RNA duplexes and resulting in introduction of base-pair the mismatches that prevent stable duplex formation (Bass & Weintraub, 1988; Wagner et al., 1989). To determine whether the unwinding effect of the follicular cell and oocyte cytoplasm is due to the presence of an analogous enzymatic activity, a radioactive sense strand synthesized in the presence of [c+‘~P]ATP was annealed to an excess of unlabelled antisense RNA, and the resultant labelled duplexes subjected to incubation with oocyte, fol-

licular cytoplasmic or Bm5 cytoplasmic extracts. Following purification and heat denaturation of the unwound duplexes, the RNA was digested to completion with nuclease Pl and the resultant mononucleotides fractionated chromatographically (Fig. 3(c)). Tn contrast to the mock-incubated control that yielded only a single radioactive nucleotide migrating at the adenosine monophosphate position (lane C), two radioactive spots were obtained in the hydrolysates of the RNA that had been unwound by the oocyte and follicular cytoplasmic extracts (lanes 0’ and F”). The migration rate of the additional spot was indistinguishable from that of the inosine monophosphate standard. As expected, the sample that was incubated with the Bm5 cytoplasmic extract (lane B”) contained a single radioactive spot identical to that of the mock incubated control. It is, therefore, concluded that unwound RNA contains modified adenosine residues and that the modification involves deamination of adenosine into inosine. Densitometric quantification of the radioactive spots revealed that 15% of the adenosine residues of the labelled sense strand were converted into inosine.

522

Y. A. W. Skeiky

C

Cc

Fc

6’

C

Oc

Fc

and K. Iatrou

8’

C

Oc

C-

SC

Fc

D-

(a)

(b)

! Origin

-e

(c) Figure 3. The modification of the duplex RNA is stable and results into adenosine conversion into inosine. 32P-labelled ATP duplex RNA (@5 fmol) was incubated for 3 h with 40 pg of follicular cytoplasmic (F”), oocyte cytoplasmic (0’) or Bm5 cytoplasmic (B’) extract. Lane C contains the mock incubated (no extract) duplex RNA (D). Following deproteinization, the recovered RNA from each reaction was analysed in 3 ways: (a) one third was loaded directly on a native 4% polyacrylamide gel; (b) a second third was first denatured in formamide and then allowed to renature prior to analysis on a native 4% polyacrylamide gel; (c) the remaining third was subjected to complete digestion with nuclease Pl and then analysed by PEI-cellulose thin-layer chromatography. C and C- are control duplex RNA (no extract) with or without nuclease Pl digestion, respectively. Circles indicate the position of migration of the unlabelled standard 5’ ribonucleoside monophosphates (a, AMP; g, GMP; i, IMP; c, CMP and u, UMP) used for determining the nature of the

modification.

(d) The modifying

activity is developmentally regulated

The occurrence

of quantitative changes in the level of the modifying/unwinding activity during incubations of choriogenesis was assessed through the duplex RNA with follicular cell and oocyte cytoplasmic extracts prepared from staged follicles

(Fig. 4(a)), panels F and 0). The two types of extracts showed parallel unwinding behaviour during choriogenesis. The unwinding effect was very prominent with extracts derived from early choriogenie follicles (lanes E), maintained at similar levels in follicles of middle developmental specificity (lanes M) but was considerably weaker with extracts prepared from late choriogenic stages (lanes L). It is worth noting that in this experiment the follicular extracts were more active than the oocyte ones, thus excluding the possibility that the modification activity present in follicular cells is due to contamination by oocyte constituents that leak out during fractionation. To determine whether the low levels of unwinding effect in the late follicles may be due to proteolysis, duplex RNA was incubated with equal quantities of the early and late extracts in a single reaction. The subsequent electrophoretic analysis (lanes E/L) revealed an additive unwinding effect, thus demonstrating that

the low levels of unwinding obtained with the late extracts simply reflect reduced levels of unwinding

activity in the cytoplasmic compartment. The developmental staging of the extracts was confirmed through an electrophoretic analysis of the yolk proteins of the corresponding oocyte fractions (Fig. 4(b)). Th e oocyte extracts were found to contain

the

normal

complements

of

proteins

previously described by Indrasith et al. (1988) and their staging was correlated with the changing accumulation profiles of identifiable polypeptides during oogenesis, particularly ESP-H, ESP-L, 30KP and the two minor proteins in the 95 to 98 kDa region (Indrasith et al., 1988). In addition, the oocyte protein analysis confirmed the notion that the quantitative changes in the levels of oocyte modifying activity during choriogenesis were not due to proteolysis that occurred during the preparation of the extracts. Therefore, it appears that the decrease in the modifying/unwinding activity observed in the late extracts is the result of genuine down-regulation. (e) Bm5 cytoplasmic and follicular nuclear extracts contain an activity that inhibits duplex RNA modi$cation and unwinding

The possibility that the lack of an unwinding effect of the Bm5 cytoplasmic extracts on duplex

RNA Duplex Modzlfication

523 -

D

E

m

L

E/L

0

kDo

m

E

m

66-

L

mat

(

Vtn-H

<

ESP-H

*

ESP-L

30 KP

(b)

Figure 4. The unwinding activity is developmentally regulated. (a) Choriogenic follicles were dissected from developing ovarioles as groups representing the early, middle and late stages of oogenesis. Cytoplasmio extracts were subsequently prepared from follicular cells (F) and oocytes (0) of the pooled follicles. Lanes D contain the unt,reated duplex RK’A control. Lanes E. M and L contain unwinding reactions using @5 fmol of duplex RNA and 10 pg of respectively. Lanes E/L contain duplexes from cytoplasmic extracts prepared from early, middle and late follicles, unwinding reactions employing mixtures of 10 pg each of early and late extracts in a single incubation reaction. (b) Developmental changes in the polypeptide composition of oocytes during oogenesis. Samples (5 pg) of early (E). middle (M) or late (L) choriogenic oocyte extract used in the unwinding analysis shown in (a) were analysed on a lO?h SDS/polyacrylamide gel. Lane Mat contains an equivalent amount of oocytr extract prepared from mature (ovulated) oocytes. The proteins were visualized by Coomassie blue staining. The major yolk proteins were identified by csomparisons to previously published work (Indrasith et al., 1988). Vtn-H. heavy subunit of vitellin (178 klla); ESP-I,. light subunit of egg-specific protein (64 kDa); Vtn-L, light subunit of vitellin (43 kDa): 30KP. 30 kl)a proteins. Lane RI contains molecular weight standards

RNA (Fig. 1) is simply due to the absence of the modifying activity was examined through mixing experiments in which double-stranded RNA was simultaneously incubated with active and inactive extracts (Fig. 5). Incubation of the duplex RNA with a mixture of follicular and Bm5 cytoplasmic extra&s (Fig. 5(a), lane 4) resulted in a severe reduction of the unwinding effect relative to that observed with the follicular cytoplasmic extract alone (lane 2), suggesting that the Bm5 cytoplasmic extract contains an inhibitor of the modifying/ unwinding activity. This was directly confirmed through a chromatographic separation of the corre-

nucleotide mixtures sponding resulting from nuclease Pl digestion of the breated duplexes (Fig. 5(b)). This analysis revealed that,, while inosine was present in the hydrolysate of the RNA that had been incubated with the follicular cytoplasmic extract, no inosine was present in the sample derived from the mixed incubation. It is, therefore, concluded that the cytoplasm of Bm5 cells contains an activity that inhibits the deamination of adenosine residues in duplex RNA (see Discussion). To determine whether the inhibition of modification may be due to the presence of endogenous duplex RNA in the Bm5 cytoplasmic rxtract’ that

Y. A. W. Skeiky and K. Iatrou

524 I

2

3

4

5

6

7

I

2

3

Figure 5. Bm5 cytoplasmic and follicular nuclear extracts inhibit duplex RNA modification and unwinding. (a) Duplex RNA (0.5 fmol) was incubated with 10 pg of follicular cytoplasmic or Bm5 cytoplasmic extract either in separate reactions or in a single reaction and analysed directly on a native 4% polyacrylamide gel. Lane 1, contains the intact RNA duplex; lanes 2 and 3, contain duplexes recovered from incubations with follicular cytoplasmic (PC) and Bm5 cytoplasmic (B’) extract, respectively; lane 4, contains duplexes recovered from incubations with a mixture of 10 pg each of F’ and B’; lane 5, is as lane 4 except that the Bm5 extract was heat-denatured at 75°C for 20 min prior to the incubation; lane 6, contains duplexes recovered from incubation with F” in the presence of nucleic acid contained in 40 pg of B’ ; lane 7, is as lane 2 except that the purified duplexes were further incubated with 10 pg of Bm5 cytoplasmic extract prior to analysis on the gel. (b) PEI-cellulose thin-layer chromatography of nuclease Pl digested duplexes. Lane 1. contains control duplex RNA (no extract); lane 2, contains the nuclease Pl digested duplexes recovered from the incubation with a mixture of follicular and Bm5 cytoplasmic extracts; lane 3, contains duplexes recovered from the incubation with follicular cytoplasmic extract alone. The arrowhead indicates the origin of the chromatogram and spots a and i are as described in Fig. 3. (c) RNA duplexes recovered from incubations with different extracts were analysed as described for (a). Lanes 1, 2 and 3, contain duplexes recovered from incubations with follicular nuclear (F”), oocyte cytoplasmic (0’) and a mixture of the 2 extracts, respectively. Lanes 4 and 5, contain duplexes recovered from incubations with Bm5 nuclear extract (Bm) and a mixture of Bm5 nuclear and oocyte cytoplasmic extracts (B”/O’), respectively.

may act as competitor in the unwinding reactions, purified nucleic acid from 40 pg of Bm5 cytoplasmic extract was mixed with labelled duplex RNA and the mixture incubated with 10 pg of follicular cytoplasmic extract. This experiment (Fig. 5(a)), lane 6) demonstrated that the labelled duplexes were unwound in a manner indistinguishable from that of the direct incubation with the follicular extract (lane 2). Therefore, the inhibitory activity of the Bm5 cytoplasmic extract cannot be attributed solely to the presence of naked double-stranded RNA. Furthermore, heat treatment of the Bm5 cytoplasmic fraction resulted in the inactivation of the inhibitory activity (Fig. 5(a)), lane 5). Taken together, these experiments suggest that the inhibitory activity of the Bm5 cytoplasmic extracts may be due to a protein that may or may not be associated with RNA into a ribonucleoprotein particle.

Because it is possible that the inhibition may be exerted through reversal of the modification, unwound duplex RNA purified from a reaction with follicular cytoplasmic extract (Fig. 5(a), lane 2) was subjected to a further incubation with an excess of Bm5 cytoplasmic extract. Following deproteinization, the labelled RNA was analysed electrophoretitally (lane 7). No difference was observed in the electrophoretic migration of the RNA relative to that of the control duplex RNA that was subjected to the single incubation reaction with follicular cytoplasmic extract. It can be concluded, therefore, that

the Bm5 inhibitory

activity

does not involve

reversal of the modification of duplex RNA. This conclusion is based on the fact that the unwound duplex RNA derived from the follicular cytoplasmic regions contains double-stranded incubation (Fig. l(c)). Should the Bm5 inhibitory activity be

RNA Duplex Modi&ation due to a reversal of the modification, these regions would have served as nuclei mediating the instantaneous reannealing of the partially unwound molecules. A similar type of analysis was carried out using t’he follicular nuclear extract that was also shown to be unable to mediate duplex RNA unwinding Mixed incubations involving the (Fig. l(b)). unwinding oocyte extract and the follicular nuclear extract (Fig. 5(c)) revealed that, as was the case with the Bm5 cytoplasmic extract, follicular nuclei contain an activity that inhibits duplex unwinding (Fig. 5(c)), lane 3). Finally, because extracts derived from Bm5 nuclei were found to contain relatively low levels of duplex unwinding activity (Fig. l(b)), they were also examined for the possible presence of an inhibitor. Incubation of RNA duplexes with a mixture of Bm5 nuclear and oocyte extracts resulted in an increased unwinding of the duplexes (Fig. 5(c), lane 5), suggesting that, unlike the case of the follicular nuclear extracts, the lower level of Bm5 nuclear unwinding (modifying) activity is not due to the presence of an inhibitor. Furthermore, chromatographic analysis of nuclease Pl digestion of RNA purified from the reactions with the oocyte ext,ract and the mixture of the oocyte and Bm5 nuclear extracts revealed similar ratios of modified to unmodified residues (not shown). These experiments demonstrate that the modifying and inhibitory activities present in follicular and Bm5 (reciprocal) to differential cells are subject compartmentalization.

4. Discussion The results described above demonstrate that follicular cells and oocytes contain a cytoplasmic activitv that modifies RNA duplexes in vitro. The modifi\;ing activity converts adenosine into inosine residues in duplex but not single-stranded RNA. The modification results in the partial unwinding of RNA duplexes, and the monomer strands lose their ability to form perfect duplexes with their complements when reannealed in vitro. The modifying activity appears to be similar to that found to exist in Xenopus (Bass & Weintraub, 1988) and a number of mammalian cell types (Wagner et al., 1989) and is developmentally regulated. However, the developmental regulation of the Bombyx modifying activity in follicular cells and oocytes is different from that of the Xenopus enzyme: unwinding of RNA duplexes does not occur in the cytoplasm of stage VI Xenopus oocytes (meiotic metaphase I stage) but does take place in the cytoplasmic fractions of mature eggs (arrested at metaphase II) and early embryos (Rebagliati & Melton, 1987; Bass & Weintraub, 1987, 1988); in contrast, the silkmoth activity is present at high levels in the cytoplasm of choriogenic follicles but immature, early/middle decreases during maturation (late choriogenesis), both in the oocyte itself and in its accessory folli-

525

cular cells. It should be noted that the oocyte of all choriogenic follicles is equivalent to stage VI frog oocytes (chromosomes at metaphase I, disrupted nuclear envelope) and that mature eggs are arrested at the same stage until fertilization (Yamauchi & Yoshitake, 1984). On the other hand, no mitotic divisions occur in the cells of the follicular epithelium during choriogenesis. Therefore, the observed fluctuations in enzyme activity are due to changes in the differentiation state of these cells rather than to cell cycle variations. Although the modifying activity does not appear to be specific for any particular double-stranded RNA, at least as shown by the ability of the oocyte extracts to unwind chorion RNA sequences that are only present in follicular cells, the choice of the specific adenosine residues that are subject to modification appears to be influenced b,7: the nature of their flanking sequences. The probability of A modification was shown previously to be higher if the 5’ neighbour is an A or a U, whereas a G and. to a lesser extent, a C on the 5’ side decreases the probability of a modification (Kimelman & Kirschner, 1989). Wagner et al. (1989) also reported that the modification has a 3’ neighbour preference of G = C > U > A. Therefore, the lower level of A modification (15%) described for the follicular extracts compared to the higher conversion (25 to 4016) reported for other systems (Bass & Weintraub. 1988; Wagner et al., 1989) may simply reflect differences in the primary structure of the subst,rates used in our studies. The most attractive physiological role of the modifying activity appears to be its involvement in the controlled turnover of cytoplasmit mRNA in vivo. This hypothesis was recentlv strengthened by the demonstration that an exteniive in viva modification (adenosine to inosine) of the Xenopus bFGF mRNA exclusive to the region of overlap with a complementary mRNA, parallels the degradation of bFGF during meiosis (Kimelman & Kirschner, 1989). In addition, these authors reported that the modification of bFGF mRNA was observed only after germinal vesicle breakdown but not in fullgrown or in younger oocytes. This is consistent with earlier reports that showed that the modifying activity is present only in the oocyte nucleus before the onset of the second meiotic division and in the cytoplasm at and after the second meiotic metaphase (Bass & Weintraub, 1987: Rebagliati & Melton, 1987). Although the unwinding activity present in silkmoth follicles may play a similar regulatory role, its natural substrates have yet to- be identified. Recently, silkmoth follicular cells have been shown to contain significant quantities of chorion antisense RNA that accumulates in parallel to rhorion mRNA in the cytoplasmic fraction (Skeiky & Iatrou, 1990). However, despite the fact that chorion mRNA is five to ten times more abundant than t,he corresponding antisense RNA, the latter is apparently neither duplexed to the mRNA nor modified. Considering the in vitro properties of the silkmoth

Y. A. W. Skeiky and K. Iatrou

526

RNA modifying activity, and the implicit suggestion that the mammalian and amphibian modifying activities may be responsible for the observed heterogeneity in the primary structure of a number of viral and cellular RNA sequences (Cattaneo et al.. 1988; Bass et al., 1989; Kimelman & Kirschner, 1989), the detection of unmodified single-stranded chorion antisense RNA in follicular cytoplasm is intriguing. Although it is formally possible that chorion antisense RNA is never duplexed to its mRNA complement, the parallel accumulation of the two complementary types of RNA in follicular cytoplasm renders this possibility unlikely. Our attempts to detect chorion pre-mRNA and antisense RNA transcripts in follicular nuclei by methodologies have conventional hybridization been unsuccessful, probably because newly synthesized transcripts are rapidly processed and transported into the cytoplasm. Should, however, the presence of chorion pre-mRNA-antisense RNA duplexes be demonstrated through the employment of more sensitive techniques, such as reverse polymerase chain reaction amplification of RNA duplexes surviving RNase digestion of hnRNA, an mRNA stabilization function could be assigned to antisense RNA. In such a case, differential cytoplasmic sub-compartmentalization of the modifying activity and the chorion mRNA-antisense RNA duplexes would have to be postulated to explain the fact that the latter are not, subject to modification. This could be achieved by the association of the chorion RNA duplexes with the translational machinery of the rough endoplasmic reticulum. In fact, in such a scenario, the translating ribosome would be responsible for the dissociation of the chorion mRNA from its complementary antisense RNA, since the latter does not extend into the region of ribosome binding and translation initiation (Skeiky & Iatrou, 1990). Chorion mRNA and antisense RNA dissociated through t.he movement. of the translating ribosomes would, of course, not be modified. In contrast to the situation with follicular cells and oocytes, the modifying activity in a silkmoth cell line, Hm5, was found to be localized in the nuclear fraction. Although this cell line was derived from silkmoth ovaries, the exact cell lineage is unknown. It is, however, likely that Hm5 cells have originated from the same mesodermal progenitors that give rise to terminally differentiated follicular cells. This notion is based on our finding that the nuclei of Bm5 cells contain a ahorion promoterspecific DNA binding protein that is identicai to one of the follicular nuclear factors that has been implicated in the tissue-specific expression of chorion genes (Skeiky & Iatrou, 1991). It is, therefore, possible that in the absence of normal environmental conditions (e.g. hormonal stimulation) and in viva selection, the progenitors of Rm5 cells have diff’erentiated towards a new path that can be stably propagated in vitro. By analogy, the RNA duplex modifying activity is subject to differential cellular

compartmentalization.

This

conclusion

is

consistent with the demonstration

that the Xenopus

modifying

from

activity

is relocalized

the unferti-

lized egg nucleus into the cytoplasm following fertilization and back into the nucleus after the midblastula transition of the embryo (Bass & Weintraub, 1988). Finally,

follicular

cell nuclei

and Hm5 cell cyto-

plasm were shown to contain an activity that inhibits the modification of double-stranded RNA. Two independent lines of evidence suggest that the inhibition is specific for the duplex RNA modifying enzyme rather than the result of the presence of a non-specific protease. First, both cytoplasmic and nuclear fractions of follicular and Bm.5 cells have been used as sources for the isolation of .specific DNA binding activities that are implicated in the transcriptional control of chorion genes (Skeiky & Iatrou. 1991; Y.A.W.S. and K.T., unpublished results) and no structural differences have been observed in the identified polypeptides. Secondly, addition of an excess of Rm5 cytoplasmic extract (containing the inhibitory activity) to a chloramphenicol acetyl transferase reaction caused no measurable reduction in the degree of chloramphenicol acetylation (not shown). The mechanistic details of the inhibition cannot be deduced from our experiments but at least two possibilities are envisaged: (I) the inhibitory activity could aet directly on the modifying enzyme and degrade it (specific proteolysis) or prevent its interaction with the substrate (modification): or (2) the inhibitory activity could interact with the duplex RNA substrate. thus prevent~ing access of the modifying enzyme. Although the modifying and inhibitory activities appear t,o have mutually exclusive cellulat compartmentalizations, it is possible t,hat the apparent reciprocal distribution in the nuclei and cytoplasm of follicular and Bm5 cells is simply a reflection of reciprocal quantitative changes occurring during the terminal differentiation of these cells. That is, the control of gene expression at. the level of duplex RNA modification may be exerted through the controlled modulation of the level of the two counteracting activities. Irrespective of the details of such modulations, the differential and reciprocal distributions of the two apparent opposing activities in follicular and Bm5 cells and oocytes suggest that duplex RNA modification in Hombyx mori represents a developmentally regulated activity rather than a general housekeeping function. We thank B. Kalisch and Dr R. Pon for advice and materials used for the chromatographic analyses; Dr R. Johnston for his continuous interest in the progress of this work and useful suggestions; C. Berglind for expert secretarial support. This work has been supported by the Medical Research Council of Canada. References Bass, B. I,. & Weintraub, Bass. B. L. & Weintraub,

H. (1987). CeZl, 48, 607-613. H. (1988). GeZZ,55, 1089-1098.

RNA Duplex Modification Bass, 1%L.. Weintraub, H.. Cattanes, R. & Billeter, M. A. ( 1989). Cell, 56, 331. Bradford, M. M. (1976). Anal. Rio&em. 72, 248-254. (‘attaneo, R., Schmid, A., Eschle. D., Baczko, K., Meuten, V.-T. B Biltrter. M. A. (1988). Cell, 55, 255-265. Grace. T. 1). C. (1967). Nature (London), 216, 613. tatrou. K.. Ito. K. & Witkiewicz. H. (1985). J. ViroZ. 54. 436-445. tndrasit,h. I,. S., Sasaki, T.. Yaginuma, T. & Yamashita. 0. (198X). J. Ponq. Physiol. 158, l-7. 1). B Kirchner. M. W. (1989). Cell, 59, Kimelman. 687-696. Melt,on, I>. A.. Krieg, P. A., Rebagliati, M. R., Maniatis, T.. Zinn, K. & Green. M. R. (1984). Nucl. Acids Res. 12. 7035--7056. l+ttagliat,i. M. R. & Melton, T). A. (1987). (‘ell, 48, 599--605. Edited

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Bkeiky, Y. A. W. & Iatrou, K. (1990). .I. &fol. Hiol. 213. 53-66. Skeiky, Y. A. W. & Iatrou. K. (1991). Mol. (‘~11. Biol. In the press. Tsitilou, S. G. & Tatrou, K. (1983). /*:,VHO .J. 2. IKItt 440. Wagner, R. W. & Nishikura, ti. (l!%X). .Vol. (‘rll. Hiol. 8. 770-777. Wagner, R. W., Smith, J. E.. (looperman, 1s. S. & Nshikura, K. (1989). t’mc. Mnf. L4~nd. Rci.. /‘.!.A. 86. 6647-2651. Weiss, S. A., Smith, U. C.. Kalter. S. S. & Vaughn. .t. L. (1981). In vitro, 17. 495-502. Yamauchi. H. Br Yoshitakr, N. (1983). ./. 1Uorph~ol. 179. 21-31.

by R. Schleif