The EMBO Journal vol.9 no.4 pp. 1 259 - 1266, 1990 The IS 10 mRNA is destabilized during antisense RNA control

Casey C.Case', Elizabeth L.Simons1 and of double stranded (ds) RNA, and might also induce changes Robert W.Simonsl 2 in target RNA secondary structure in more distal regions. Either or both of these effects could result in altered stability 'Department of Microbiology and the 2Molecular Biology Institute, of the target RNA. The structure and stability of the antisense University of California, Los Angeles, CA 90024, USA RNA, itself, might also change. Communicated by J.H.Miller We have explored these questions in the antisense RNA system that post-transcriptionally regulates transposase (tnp) RNA stability is an important component of gene expression from insertion sequence IS10 (Simons and expression, and antisense have been proposed to Kleckner, 1983, 1988; Case et al., 1989; Kittle et al., 1989). alter target RNA stability. We show here that the ISJO IS10 (Figure 1) has two principal promoters: pIN, which transposase mRNA, RNA-IN, is rendered unstable specifies the IS10 tnp mRNA, RNA-IN; and the stronger, during control by the IS10 antisense RNA, RNA-OUT. opposing , pOUT, which specifies the antisense Destabilization requires RNA-OUT/RNA-1N pairing and RNA, RNA-OUT (Simons et al., 1983; Case et al., 1988). m cleavage. Independent of such cleavage, The 5' ends in RNA-IN and RNA-OUT are complementary RNA-OUT is rendered unstable through disruption of its for 35 bp including the ribosome for the tnp secondary structure. Pairing has no other obvious effects gene (Ma and Simons, 1990). RNA-OUT has a simple on RNA-IN transcription or stability. Nevertheless, RNA- secondary structure consisting of a stable stem domain topped IN destabilization is not required for antisense control by a loosely paired loop domain (Case et al., 1989; Kittle in vivo. In the accompanying paper [Ma,C. and et al., 1989). RNA-IN is complementary to the 5' half of Simons,R.W. (1990) EMBO J., 9, 1267-1274 we show this structure, and RNA-OUT/RNA-IN pairing initiates that pairing blocks ribosome binding to RNA-IN. Were between the RNA-OUT loop domain and the 5' end of RNA- it not for control at this level, destabilization would play IN (Kittle et al., 1989). Mutations in this region alter the a more prominent role. specificity and/or efficiency of pairing in vitro and have Key words: antisense RNA/IS]O/mRNA stability/ribo- corresponding effects on control in vivo (Case et al., 1989; nuclease HI/transposon Kittle et al., 1989). RNA-OUT is an exceptionally stable RNA with a 60 min half-life in vivo, and mutations that increase or decrease its expression or stability have corresponding effects on control (Case et al., 1988, 1989). Introduction We have previously characterized the RNA-OUT and Messenger RNA stability is an important component of gene RNA-IN transcripts expressed in vivo (Case et al., 1988). expression. In , the RNases that process and Here, we show that RNaseIl cleaves the RNA-OUT/RNA- degrade mRNA are often influenced by RNA secondary IN paired species at specific sites in vivo, leading to RNA- structure, and the presence or absence of specific structural IN destabilization. However, such effects play little or no elements frequently provides the basis for regulating the role in antisense control of tnp expression. In the stability of those RNAs (Higgins and Smith, 1986; Belasco accompanying paper (Ma and Simons, 1990), we show that and Higgins, 1988). For example, in 'retro-regulation' of pairing prevents ribosome binding to RNA-IN in vitro, and X int gene expression, the stability of the int propose that this effect accounts for antisense control in vivo. mRNA depends on whether it contains a specific 3' terminal Were it not for control at this level, destabilization would stem-loop structure; when present, this structure is play a more prominent role in regulating tnp expression. processed by ribonuclease III (RNaseIII), initiating int mRNA decay (Guarneros, 1988), RNaseHII also cleaves stem -loop structures found near the ends (usually 5') of Results a number of other mRNAs, in some cases altering their stability (King et al., 1986; Takiff et al., 1989; and Antisense RNA control in multicopy 'cassette' fusions references therein). In order to express RNA-OUT and RNA-IN transcripts over In all of these cases, the relevant secondary structure arises a wide range of detectable levels, we constructed the closely from base pairing between symmetrical sequences present related set of multicopy described in Figure 2 and in cis. Secondary structure can also be influenced by Table I. These plasmids each contain an 'IN-cassette' diffusible antisense RNAs that bind to specific target RNAs. comprised of the outer 340 bp of IS10 (plus flanking his For example, the ColEl antisense RNA alters the sequence) inserted upstream of a suitably engineered lacZ secondary structure of its target RNA, the primer for ColEl gene. In plasmids 1-17, the first 75 codons of the tnp gene replication, preventing primer maturation and DNA synthesis are fused in frame to a truncated lacZ gene to form a (Tomizawa, 1987). More generally, antisense RNA/target tnp'-lacZ' protein fusion; in plasmids 18 and 19 the IN- RNA pairing would be expected to produce a new region cassette is fused to an intact lacZ gene to form a

Oxford University Press 1259 C.C.Case, E.L.Simons and R.W.Simons

hisG AUG tetD

1329 POUT

RNA-IN * cJ1Og R5 s IN primer t 5'-GCGAAAAAUCAAUAAUCAGACAACAAGAUGUGCGAACUCGAUAUUUUACACGACUCUCUUUACCMUUCQICCCC--- 3 '-CCUAUGUGUAGAACAGUAUACUAGUUUACCAAAGCGCUUUUUAGUUAUUAGUCUGUt)GUUCUACACGCU(U)-5S OUT primer I A *t mciSlO RNA-OUT

Fig. 1. Insertion sequence ISJO and RNA-OUT and RNA-IN transcripts. IS10-Right (as present in the wild type hisG9424::TnJO insertion; Foster et al., 1981) is depicted (not to scale). The element is 1329 bp long and abuts the Salmonella hisG and TnJO tetD (Schollmeier and Hillen, 1984) genes. The tnp coding sequence extends from bp 108 to 1313 (Halling et al., 1982). pIN initiates RNA-IN transcription at bp 81; pOUT initiates RNA-OUT transcription in the opposite direction at bp 115, with a minor start at 116 (Case et al., 1988). A partial restriction map is also shown. The expanded portion shows the first 75 nucleotides of RNA-IN, all of RNA-OUT, and other important features. Three mutations (R5, mciS10 and C(109) are indicated as changes in RNA-OUT or RNA-IN sequence. The AUG start codon and preceding Shine/Dalgarno like sequence for the tnp ribosome binding site (see Ma and Simons, 1990) are underlined (bold). Arrows pointing toward the transcripts are the in vivo RNaseIll cleavage site (*, major sites). Thin lines indicate the IN and OUT primers used in this work.

pIN-lacZ+ fusion. Most plasmids also contain an pIN 'OUT-cassette' located just upstream of the IN-cassette; the OUT-cassettes are isogenic to the IN-cassettes but are not Ti UT4. fused to lacZ. "i: Table I shows the level of fusion expression from each pOUT tnp'-IacZ' fusion plasmid, as well as the relative level of antisense control OUT IN within groups of related plasmids. Plasmids 1-5 contain Cassette Cassette only an IN-cassette and differ by well characterized point mutations that reveal certain important features of ISJO Fig. 2. Structure of multicopy ISIO fusion plasmids. Shown is the antisense control. The R5 mutation severely reduces RNA- basic structure of the multicopy tnp'-lacZ' fusion plasmids used in OUT stability (Case et al., 1989), essentially abolishing these studies (Table I). All plasmids contain an IN-cassette (shaded antisense control and increasing fusion expression 45-fold box) comprised of a -565 bp BglII-AccI fragment obtained from an appropriate wild type or mutant IS10 element inserted (after (cf. plasmids 1 and 2). The G8 mutation increases pOUT appropriate modification of ends) into the protein fusion vector pRS414 transcription - 6-fold (Simons et al., 1983; Case et al., such that the tnp gene is fused in frame to lacZ; or, in the pRS1117 1988) and increases antisense control - 10-fold (plasmid 3). and pRS1141 cases, into the operon fusion vector pRS415 such that This latter effect is largely relieved if the IN-cassette also pIN is fused to an intact lacZ gene. Some plasmids also contain a contains the tandem OUT-cassette (hatched box), which is a similar BglII-AccI R5 mutation (plasmid 4). In contrast, the mciS10 fragment (wild type or mutant) inserted upstream of the IN-cassette. mutation (plasmid 5) provides little relief; mciS10 alters the The solid arrows indicate that RNA-IN and RNA-OUT are the specificity of RNA-OUT/RNA-IN pairing such that it principal transcripts expressed from, respectively, the IN- and OUT- controls its own expression but not that of wild type ISJO cassettes (see text). Individual genotypes are listed in Table I. The and vice versa, as seen below (Case et sources of wild type and mutant Bglll-AccI fragments were: pNK214 al., 1989; C.Ma, (wild-type); pNK225 (R5); pNK234 (G8); pRS819 [G8 R5(=mciS3)]; S.Roels and R.W.Simons, unpublished data). Together, pRS875 (G8 mciS10); pRS1087 (mci30 R5); pRS1088 (HH104 R5); these results show that the outer 340 bp of IS1O are sufficient pCJ291 (mci30 HH104); pCJ292 (mci30 HH104 CJ109). T14, the for efficient and specific antisense control. upstream rrnB operon transcriptional terminators present in pRS414 The IN-cassettes in plasmids 6-19 contain point mutations and pRS415 (Simons et al., 1987). that increase pIN activity (Case et al., 1988; cf. plasmids 2, 6, 9 and 14); most also contain the R5 mutation. antisense control. First, protein fusion expression decreases Therefore, these IN-cassettes can be considered to express with increasing RNA-OUT expression (cf. plasmids 6-8, only RNA-IN. Some ofthese plasmids also contain an OUT- 9-11 and 14-15). Second, when the OUT-cassette contains cassette, which contains either the wild type or G8 pOUT either the R5 or mciS10 mutation, control is reduced promoter, but only the relatively weak wild type pIN pro- substantially (plasmids 12 and 13). The fact that mciS10 has moter. Therefore, these OUT-cassettes can be considered a greater effect in plasmid 13 than in plasmid 5 illustrates to express only RNA-OUT. Consequently, these plasmids the altered specificity phenotype of this mutation. Third, the express high levels of RNA-IN or RNA-OUT from separate pIN - lacZ+ operon fusion experiences much less inhibition cassettes, thereby avoiding any confounding effects that (plasmids 18 and 19) than does the isogenic tnp'-lacZ' might arise from such expression in cis. protein fusion (plasmids 9 and 11), consistent with the post- Plasmids 6-19 also exhibit the essential features of IS1O transcriptional nature of IS1O antisense control (Simons and 1260 mRNA destabilization by antisense RNA

Table 1. Antisense RNA control of tnp'-lacZ' fusion expression in multicopy plasmids Plasmid Plasmid Cassette genotypes tnp'-lacZ' Level of number OUT IN fusion expression control (units (3-Gal) 1 pRS555 -a wt 1 45 2 pRS556 - R5 45 =1 3 pRS492 - G8 <0.1 > 450 4 pRS908 - G8 RS 2 22 5 pRS909 - G8 mciS10 -0.1 -450

6 pRS1112 - mci30 R5 560 =1 7 pRS1142 wt mci3O R5 255 2 8 pRSl140 G8 mci30 RS 10 56

9 pRS1113 - HH104 R5 1550 =1 10 pRS1145 wt HH104 R5 1130 1.5 11 pRS1146 G8 HH104 RS 15 105 12 pRS1147 G8 R5 HH104 RS 260 6 13 pRS1148 G8 mciS10 HH104 RS 130 12

14 pRSIllO - mci3 HH104 14 500 =1 15 pRS1144 G8 mci3O HH104 7250 2 16 pRSIII - mci30 HH104 CJ109 485 =1 17 pRS1143 G8 mci30 HH104 CJ109 240 2

18 pRS1117 - HH104 RS(op)b 13 995 =1 19 pRS1141 G8 HH104 R5(op) 4115 3.4 The indicated plasmids were transformed into DR459 and (3-galactosidase activity determined as described (Simons et al., 1987). 'Level of control' is the level of fusion expression in the absence of control (set at 1 for each group of related plasmids) divided by the level of expression from the plasmid in question. Shown are averages of triplicate experiments repeated three times (standard errors were 20% of the means). The basic structure of these plasmids and their construction are described in Figure 2. aNone present. bOperon fusion. Kleckner, 1983). Finally, the CJ109 mutation, which IN-cassette: 104 104 104 104 104 wt R5 changes the tnp translation initiation codon from AUG to OUT-cassette: -- wt G8 G8 -- C U A G ACG (see Figure 1) and decreases ribosome binding at the tnp translation initiation site in vitro (Ma and Simons, 1990), RI~ ~ ~ ~ ~ R l ,L..... also decreases tnp'- lacZ' protein fusion expression - 30-fold (cf. plasmids 14 and 16). This residual expres- sion remains sensitive to antisense control (plasmid 17). ._. (Control is quite modest in plasmids 14-17 because of the ~'RI _| high RNA-IN:RNA-OUT ratio.) +18R I - 2 - Z 20*am _ ...X _Wr

The transposase mRNA is destabilized durng ..-. antisense control Plasmid: 9 10 11 12 13 1 We used primer extension analysis to examine the 5' region of RNA-IN transcripts expressed from the plasmids listed Fig. 3. Primer extension analysis of RNA-IN during control. Total in Table I. Figure 3 shows that in the absence of antisense cellular RNA was extracted from E.coli strain RS6184 transformed control (plasmid 9) the principal primer extension product with the indicated plasmid and analyzed by primer detected corresponds to the native 5' end of RNA-IN extension as described (Case et al., 1988), using an oligonucleotide et al., 1988). However, complementary to +51 to +69 of RNA-IN (Figure 1). Coordinates (+ lRI), as previously shown (Case are shown relative to the 5' end of RNA-IN. The sequence shown is in the presence of control (plasmids 10, 11) the + 1RI signal that of a transcript spanning the 5' terminus of RNA-IN (bp 44-121 decreases significantly, bands at + 16R, and + 18R, are of ISI0; pRS1 160 transcribed in vitro with T7 RNA ). 104, enhanced (more so at + 16RI), and the amount of total HH104 R5; 30/104, mci3O HH104; S10, mciS1O; -, none present. RNA-IN detected by primer extension decreases. These ef- fects are more pronounced at high levels of control (plasmid Figure 3 also shows that these effects do not depend on 11) and suppressed when the OUT-cassette contains a point high levels of RNA-OUT or RNA-IN expression. When mutation (RS or mciSlO) that decreases control (plasmids RNA-IN transcripts expressed from a wild type IN-cassette 12 and 13). No other significant cleavage events are detected (plasmid 1) are analyzed, little or no + 1RI signal is in the first 265 nucleotides (nt) of RNA-IN, and similar detected, whereas this signal is readily detected if control results are obtained with plasmids 6-8 and 14-17 (not is eliminated by the RS mutation or other point mutations shown). that abolish antisense control (Figure 3, plasmid 2; data not 1261 C.C.Case, E.L.Simons and R.W.Simons

IN-cassette: -- 30 104 30 104 control in vivo, and then further destabilized. We indicate 104 the major (*) and minor RNA-IN cleavage sites in Figure 1, OUT-cassette: GB GB GB GB SboGB where they are seen to lie within the ribosome binding site for the tnp gene. 0 + 1 _10 _ I. RNA-OUT is also destabilized during antisense control We also examined RNA-OUT under these same conditions (Figure 4). In the absence of significant RNA-IN expression (plasmid 3), a strong primer extension signal corresponding ,*.~~~~~~~~~~~~~~~~~~~~00, to the 5' end of RNA-OUT (+ IRO) is observed, as well as a number of minor products. These minor bands are seen with RNA-OUT transcripts prepared in vitro (not shown) +21 RO +23_ - _ and presumably result from pausing or premature termination :B. ~t by reverse transcriptase as it traverses through the RNA- OUT secondary structure. In the presence of increasing RNA-IN expression (plasmids 8, 11 and 15), the +1RO Plasmid: 3 8 11 15 13 signal decreases, the bands at +21RO and +23RO are enhanced (more so at +23RO), and there is a Fig. 4. Primer extension analysis of RNA-OUT during control. Total general cellular RNA was extracted from E.coli strain RS6184 transformed decrease in the amount of RNA-OUT detected. These effects with the indicated plasmid and analyzed as in Figure 3, except an are suppressed when RNA-OUT contains the mciS10 oligonucleotide complementary to +51 to +69 of RNA-OUT mutation (plasmid 13), and are not seen with RNA-OUT (Figure 1) was used. Coordinates are shown relative to the 5' end of transcripts synthesized in vitro, whether or not they are RNA-OUT. For this experiment we consider plasmid 3 to contain an to RNA-IN OUT-cassette. 104, HH104 RS; 30/104, mci30 HH104; S10, mciSJO; paired (not shown). -, non present. These observations are exactly analogous to those obtained with RNA-IN, and strongly suggest that RNA-OUT and A B RNA-IN are cleaved in a concerted fashion once they pair to one another in vivo. We indicate these sites of concerted RNA-IN RNA-OUT cleavage in Figure 1; at both the major (*) and minor sites, IN-cassette: 104 104 104 104 30 30 104 104 cleavage appears to occur in a 2 bp staggered fashion. OUT-cassette: -- G8 -- G8 G8 G8 G8 G8 rnc (host): + + - - + + Ribonuclease 111 cleaves the RNA-OUT/RNA-IN paired species in vivo and in vitro +1RI--- _ m_ _ -- 1RO RNaseIll cleaves many of its substrates in a 2 bp staggered fashion (Daniels et al., 1988). To explore the possibility that RNaseHI also cleaves the RNA-OUT/RNA-IN duplex, we examined the fate of RNA-IN and RNA-OUT in isogenic +16RI - m +18RI RNaseIII+ and RNaselI- hosts (Figure 5). In the absence - +21R0 of high levels of RNA-OUT (Figure 5A, plasmid 9), no - +23RO substantial differences are seen between RNA-IN transcripts expressed in RNaseHIl+ and RNaseIIH cells, except for suppression of the weak +16R, and +18R, signals in the RNaseIII- host. However, when both transcripts are in the same under conditions where Plasmid: 9 11 9 11 3 15 3 15 expressed cell efficient pairing is expected to occur (plasmid 11), the severe RNA- Fig. 5. Effect of the mc- host mutation on RNA-IN and RNA-OUT IN cleavage and destabilization observed in the RNaseIII+ in vivo. Total cellular RNA was extractedi from E.coli strains RS6184 host (i.e. decreased + 1RI and increased + 16R, and + 18RI (+, mc+) or RS6181 (-, rnclO5) transfiormed with the indicated signals) are completely suppressed in the RNaseIII- host plasmid and (A) RNA-IN or (B) RNA-O1UT transcripts analyzed by (plasmid 11). Other results (not shown) reveal no significant reverse transcriptase primer extension as (described in Figures 3 and 4 respectively. 104, HH104 RS; 30/104, mc cleavage anywhere in the first 265 nt of RNA-IN expressed present. in the presence or absence of RNA-OUT in the RNaseIII- host. Additionally, the failure of high RNA-OUT levels to shown). We estimate that RNA-I]N is destabilized at least decrease the + Iu signal in RNasell- cells shows that 5-fold in the wild type case. RNA-OUT does not induce termination of RNA-IN These effects do not result from decreased translation of transcription (this is also true when a primer complementary the tnp gene, per se; the CJ109 mutation, which decreases to the adjoining lac sequences is used; not shown). tnp translation initiation, does not give rise to these signals The situation for RNA-OUT is similar but somewhat more in the absence of antisense control ('not shown). Nor do these complicated (Figure SB). In the absence of RNA-IN effects result from pausing or prremature termination by expression (plasmid 3), very little difference is seen between reverse transcriptase; they are not observed when RNA-IN RNA-OUT expressed in the two hosts, consistent with other transcripts prepared in vitro are a nalyzed, whether or not studies showing that RNaseIII plays little or no direct role such transcripts are paired to RNAi-OUT (Ma and Simons, in RNA-OUT stability (Case et al., 1989). However, when 1990). The simplest explanation ffor these observations is both transcripts are expressed in the same cell (plasmid 15), that RNA-IN is cleaved at specifiic sites during antisense RNA-OUT cleavage at +21Ro and +23Ro observed in the 1262 mRNA destabilization by antisense RNA

RNaseIH+ host is completely suppressed in the RNaselll- A B host. Nevertheless, the + IRO signal still decreases in the RNA- I N RNA-OUT RNaseHI- host when RNA-IN is present. These results strongly suggest that RNaseIH cleaves the RNA-OUT/RNA- t-ali r'ea: yes yes no

.. IN duplex, and that pairing renders RNA-OUT unstable by RNoseIIl: - + t + - a mechanism independent of such cleavage.

To directly examine RNaseIH cleavage of the RNA- + I T go" _w-*?4w +1RO OUT/RNA-IN paired species, we incubated paired and unpaired transcripts with purified RNaseIH and analyzed the 0 products by primer extension. Figure 6 shows that both strands of RNA-OUT/RNA-IN are cleaved in vitro at the sites of primary cleavage in vivo (+ 16RI and +23RO; lanes 2 and 6). Under these same in vitro conditions, unpaired +23RO RNA-IN is not detectably cleaved (Figure 6, lane 4), whereas unpaired RNA-OUT is (lane 8), albeit to a somewhat lesser extent than when it is paired to RNA-IN (lane 6). Together, these results confirm that RNaselI is responsible for specific 1 ? 5 6 7 8 RNA-OUT/RNA-IN cleavage and necessary for further RNA-IN destabilization during antisense control in vivo. Fig. 6. RNaseIII cleavage of paired and unpaired transcripts in vitro. Total cellular RNA extracts containing uncleaved RNA-IN, uncleaved RNA-OUT, or neither of these transcripts (control RNA) were RNA-IN destabilization is not required for antisense obtained from Ecoli strain RS6181 (rnclO5) transformed with control in vivo pRS1 110 (mci30 HH104), pRS492 (G8) or no plasmid respectively. The observations described above prompted us to determine These extracted RNAs were then combined (-22 yg total RNA/50 ILI whether, and to what extent, RNaselI dependent destabiliza- final volume) as follows: lanes 1 and 2, RNA-IN+RNA-OUT (1:10); lanes 3 and 4, RNA-IN+control RNA (1:10); lanes 5 and 6, RNA- tion of RNA-IN is required for antisense control in vivo. OUT+RNA-IN (1: 10); lanes 7 and 8, RNA-OUT+control RNA To do this, we compared tnp'- lacZ' fusion expression from (1:10). They were then incubated for 2 h at 37°C in pairing buffer various cassette plasmids in isogenic RNaseIII+ and (see Ma and Simons, 1990) to allow RNA-OUT/RNA-IN pairing to RNaseII- hosts. Table II shows that fusion expression in occur, brought to 2 mM dithiothreitol and 20 ug/ml acetylated BSA the absence of control decreases generally -2-fold in the (Sigma), and portions incubated at 37°C with and without purified RNaseIII (- 15 sAg/ml) for 35 min. The RNA was then purified RNaseHl- host. More importantly, Table II also shows that (essentially according to the procedure for total cellular RNA the level of antisense control of fusion expression decreases extraction) and analyzed as described in Figure 5. no more than 2-fold in the RNaseIII- host, over a broad range Moreover, point of RNA-OUT and RNA-IN levels. Table II. Effect of the mncl05 host mutation on antisense control mutations in RNA-OUT that alter control (R5 or mciSJO) have similar effects in both strains. We also examined the Plasmid Cassette genotypes RS6184 (mc+) RS6181 (mncl05) inhibition of single copy tnp'-lacZ' fusion expression or OUT IN Fusion Level Fusion Level single copy TnJO transposition by RNA-OUT expressed in exp'na controlb exp'na controlb trans from a multicopy plasmid and saw no more than a 2-fold decrease in the RNaseII- host (not shown). These 1 -c wt 1.3 29 1 16 2 - RS 38 =1 15.8 =1 results strongly suggest that RNaseIII dependent destabiliza- 3 - G8 <0.1 >380 <0.1 > 160 tion of RNA-IN plays little or no role in ISJO antisense 4 - G8 R5 2.4 16 0.9 18 control under the conditions examined here. 5 - G8 mciS10 - 0. 1 -380 -0.1 - 160

Identification of a dsRNA remnant of the RNA- 6 - mci3O RS 670 =1 365 =1 OUTIRNA-IN paired species in vivo 7 wt mci3O RS 340 2 160 2.3 8 mci3O R5 16 42 11 33 The results shown in Figure SB suggest that RNA-OUT is G8 rendered unstable during control in vivo, whether or not 9 - HH104 R5 2020 =1 1070 =1 RNaselII is present. To explore this possibility, we used 10 wt HH104 RS 1690 1.2 815 1.3 Northern blot hybridization to further characterize RNA- 11 G8 HH104 RS 50 40 60 19 OUT transcripts expressed in RNaseIII+ and RNaseIII- 12 G8 RS HH104 RS 290 7 325 3 hosts. Figure 7A shows that RNA-OUT levels decrease in 13 G8 mciS10 HH104 RS 200 10 270 4 both hosts when RNA-IN is also expressed (plasmid 11), confirming that pairing alone destabilizes the - 70 nt RNA- The indicated plasmids were transformed into RS6181 (nclO5) or RS6184 (rnc+) and ,B-galactosidase activities determined as described OUT transcript. However, Figure 7A also shows that a in Table I. shorter RNA-OUT transcript persists in the presence of high aUnits ,B-galactosidase activity. levels of RNA-IN, but only in the RNaselIl- host. This bAs defined in Table I. 35 nt species corresponds to the 5' 'half of RNA-OUT, cNone present. and it has a remarkably long half-life (>2 h; see Materials and methods). The properties of this transcript and the conditions of its under these conditions. Figure 7B shows this to be the case: appearance suggests that it is one strand of a stable, dsRNA a - 35 nt RNA-IN transcript is detected, but only in remnant of the RNA-OUT/RNA-IN paired species. If so, RNaseIII- cells expressing both RNA-OUT and RNA-IN. an equally stable RNA-IN remnant should also be present Like its RNA-OUT counterpart, this species corresponds to 1263 C.C.Case, E.L.Simons and R.W.Simons

A B but significant role in ISJO antisense control under the RNIA OUT RNA- I N conditions examined here and, perhaps, a more important [N- cdssette: 104 104 104 role under conditions we have not examined. OUT cassette: Gd G8 G8 rnc (host): t 1 e + Comparisons with other antisense RNA systems In addition to ISJO, there are several other documented cases where an antisense RNA is complementary to the translation RNAA 1OU - :,.* initiation region of its target mRNA (Mizuno et al., 1984; Finlay et al., 1986; Kim and Meyer, 1986; Dempsey, 1987; :35 nt Liao et al., 1987; Wu et al., 1987; Simons, 1988; Simons and Kleckner, 1988). In all such cases, we believe it likely that control occurs by inhibition of ribosome binding, without Plasmid: "I 11 9 11 the need for cleavage and/or destabilization of the target mRNA, even though it may occur. However, when the Fig. 7. Identification of a remnant of the paired species. (A) Total antisense RNA is to some cellular RNA was extracted from E.coli strain RS6184 (+, rnc+) or complementary other region of RS6181 (-, rnclOS) transformed with the indicated plasmids and the target mRNA, such effects may play important or even analyzed by Northern blot hybridization as described (Case et al., essential roles. Indeed, a requirement for RNaseIH cleavage 1988), using a probe synthesized containing IS10 sequence has been implicated in the inhibition of bacteriophage X cII complementary to RNA-OUT (bp 44-121 of S110; pRS1 160 gene expression by the X oopRNA, which is complementary transcribed in vitro with T7 RNA polymerase). The - 35 nt RNA- OUT band hybridized only to probes containing sequences to the 3' region of the cIl mRNA (Krinke and Wulff, 1987). complementary to the 5' half of RNA-OUT (not shown). (B) A similar When an antisense RNA binds to a region upstream or Northern blot hybridization using a probe containing IS10 sequence downstream of its target gene, control is likely to occur complementary to the 5' terminal 265 nucleotides of RNA-IN (pRS999 through some alteration of target RNA secondary structure transcribed with T7 RNA polymerase in vitro). The -35 nt RNA-IN that leads to at a distal band hybridized only to probes containing sequences complementary to cleavage position, sequestration of the first -40 nt of RNA-IN (not shown). Sizes were estimated with the ribosome binding site, or premature termination of end-labelled, glyoxylated DNA fragments from a HaelI digestion of transcription. This latter mechanism operates to control repC pBR322 (not shown). 104, HH104 RS; -, none present. expression in plasmid pTl81 (Novick et al., 1989), and a sequestration model has been proposed for control of repA the 5'-end of RNA-IN and has a very long half-life (22 h; expression in IncFll plasmids (Womble et al., 1987). see Materials and methods). The nature of the RNase/l sites RNaseIII cleaves the RNA-OUT/RNA-IN paired species at Discussion specific sites. The primary site is the same in vivo and in vitro and accounts for 80-90% of cleavage in both cases. RNA-IN is destabilized during antisense control A secondary site is detected in vivo and, with less certainty, The most significant finding reported here is that both RNA- several secondary sites are observed in vitro. At both the IN and RNA-OUT are destabilized during antisense control. primary and secondary in vivo sites, RNaseIII appears to RNA-IN destabilization requires RNaseIII cleavage. Three cleave RNA-OUT/RNA-IN in a 2 bp staggered fashion. This lines of evidence show that these effects occur only when type of cleavage and its degree of specificity (one or two RNA-OUT/RNA-IN pairing occurs: (i) destabilization is cuts in 35 bp of dsRNA) is characteristic of other RNaseIll readily detected when both transcripts are expressed in the substrates (Robertson, 1982; Daniels et al., 1988) and same cell but not when they are expressed separately; (ii) consistent with the notion that RNasellI possesses rather mutations that prevent pairing in vitro also prevent specific substrate requirements. However, the exact nature destabilization in vivo; and (iii) the paired species is of these requirements remains unresolved. Daniels et al. efficiently and specifically cleaved by RNaseIfl in vitro and (1988) recently proposed a consensus sequence for RNaseIH in vivo. These results establish that an antisense RNA can recognition. Although the primary cleavage site in RNA- alter the stability of its target. OUT/RNA-IN matches this sequence at four out of six In the particular case of IS10, destabilization is not positions, there are eight other four out of six matches, as required for antisense control. We believe this is because well as a perfect match, where no RNA-OUT/RNA-IN pairing alone is sufficient to manifest control. In the accom- cleavage is observed. While the proposed consensus panying paper (Ma and Simons, 1990) we show that sequence may play some role in site selection, it is either ribosomes are unable to bind to the tnp translation initiation insufficient or can be overridden by other substrate features. site in vitro when RNA-IN is paired to RNA-OUT. Were Interestingly, unpaired RNA-OUT is efficiently cleaved it not for efficient control at this level, destabilization would by RNaseIII in vitro even though no such cleavage of play a more prominent role. In support ofthis overall model, unpaired RNA-OUT is observed in vivo (see above), nor other results described here show that pairing has no effects is RNA-OUT more stable in an RNaseIII- host (Case on RNA-IN transcription or stability (aside from RNaseIII- et al., 1989). Moreover, the cleavage site in unpaired RNA- dependent destabilization) that might account for control. OUT detected in vitro is identical to the primary site in RNA- Although RNaseIlH cleavage does not play a crucial role OUT cleavage in the RNA-OUT/RNA-IN paired species in IS10 antisense control, a small but reproducible decrease (+23Ro). This difference between the in vitro and in vivo in control is seen in the RNasellF host. This may be due cases may reflect differences in RNaseIII specificity and/or to differences in the physiology of RNaseIII+ and a special property of the RNA-OUT transcript (or something RNaseIII- cells. Alternatively, cleavage might play a small associated with it) that confers RNaseIII-resistance in vivo. 1264 mRNA destabilization by antisense RNA

RNA-OUT degradation during antisense control regions of dsRNA 50 bp or more in length are likely to Several unexpected and interesting complexities were contain at least one RNaselII site (Robertson, 1982) and that revealed in the analysis of RNA-OUT and RNA-IN analogous to RNaselI may be present in other transcripts during antisense control. The normally stable organisms (Shapiro et al., 1987). RNA-OUT transcript is always degraded during control, but We also show that in the absence of RNaselH, RNA-OUT the degradation products observed by Northern blot analysis dramatically stabilizes the 5' -35 nt of RNA-IN. Under depend on whether the host is RNaseIl+ or RNaseJI-: no certain circumstances, an entire RNA might be rendered stable degradation products are seen in RNaseIII+ cells, more stable by antisense RNA binding. For example, an whereas a very stable species corresponding to the first antisense RNA directed to the 3' end of an mRNA might -35 nt of RNA-OUT is detected in the RNaseIH- host. protect that transcript against 3' exonuclease attack. In this With respect to RNA-IN, a very stable species corresponding way, antisense RNAs, artificially constructed or naturally to its first - 35 nt is also detected during control in occurring, could 'activate' gene expression. Other RNaseIL- cells, but no stable RNA-IN species is observed considerations for the design of efficient and stable antisense in the RNaselH+ host, whether or not control is occurring. RNAs are discussed elsewhere (Simons, 1988; Simons and We interpret these observations in the following model. Kleckner, 1988; Case et al., 1989; Kittle et al., 1989; Ma RNA-OUT has a simple stem-and-loop secondary structure, and Simons, 1990). and RNA-OUT's unusual stability in vivo (t½/2 -60 min) depends primarily upon base pairing within the stem domain (Case et al., 1989). RNA-IN, on the other hand, is very Materials and methods see Materials and methods). unstable in vivo (t½/2 -30 s; Media, and chemicals As the RNA-OUT/RNA-IN duplex forms, base pairing in Media, growth conditions and transformation procedures were as described the RNA-OUT stem domain gives way, leaving the 3' end (Simons et al., 1987). AMV reverse transcriptase and T7 RNA polymerase of RNA-OUT unprotected. At this point the 3' ends of both were purchased from Promega. T4 Polynucleotide , restriction RNA-OUT and RNA-IN are more or less equally sensitive endonucleases and other DNA modifying enzymes were purchased from which New England Biolabs. Oligonucleotides were obtained from New England to single strand specific 3' exonucleolytic degradation, Biolabs or the UCLA DNA Synthesis Facility. [a-32P]CTP and proceeds along both transcripts until the RNA-OUT/RNA- [Fy-32P]ATP were purchased from Amersham and ICN respectively. Kodak IN duplex region is encountered, leaving a - 35 bp dsRNA XAR-5 film was used for all autoradiography. Other chemicals were obtained remnant. In the absence of RNaseHl, this remnant is very from 1BI, Sigma or Calbiochem. in the stable, by virtue of its dsRNA character. However, Bacterial strains and plasmids presence of RNaseHI it is cleaved to products that are either Escherichia coli strains were: DR459, AlacX74 galOP308 rpsL A(tonB- further degraded in the cell or too small for detection in trpA)905 trpR (constructed by D.Roberts); RS6184, A(lac-pro) ara thi Northern blots. glyA::Tn5; RS6181, A(lac-pro) ara thi glyA::Tn5 rnc105. Plasmids were These observations have several important implications. as follows. pRS999 is pGEM-3Z (obtained from Promega) with a - 300 bp A12-AccI ISJO fragment inserted into the polylinker such that the T7 First, the 35 bp remnant represents the first physical evidence promoter (of pGEM-3Z) transcribes ISJO in the same direction as pOUT. that RNA-OUT/RNA-IN pairing actually occurs in vivo. pRS1160 is pGEM-3Z with a - 75 bp AJ2- TaqI ISJO fragment inserted Furthermore, the extraordinary stability of this species (in into the polylinker such that the T7 promoter transcribes ISJO in the same the absence of RNaseIfl cleavage) suggests that pairing is direction as pIN. pNK214, pNK225, pNK234, pRS819 and pRS875 have both complete and essentially irreversible under biological been described (Case et al., 1989), as have pRS414 and pRS415 (Simons et al., 1987). pRS1087 [mci3O(=pc2)RS] and pRS1088 [(HHJ04(=pc3) conditions. Second, whenever RNA-IN is expressed at a R5] are derivatives of pRS967, which contains an RS tnp'-kan' protein higher than normal rate, RNA-OUT levels will tend to fall. fusion; they were isolated on the basis of increased kanamycin resistance We have observed such an effect under two different after mutD-mutagenesis and will be described in detail elsewhere (J.Sussman, experimental conditions: with ISJO point mutations that C.Masada-Pepe, E.L.Simons and R.W.Simons, in preparation). pCJ291 and in E. coli strains (mci30 HH104) and pCJ292 (mci3O HH104 CJ109) were obtained from increase pIN activity (Case et al., 1989) C.Jain. Other plasmids are described in Figure 2 and Table I. Complete deficient for DNA -adenine methylation (dam), where pIN details of all constructions will be made available upon request to activity increases 10- to 20-fold (Roberts et al., 1985; Case R.W.Simons. et al., 1988). We do not know whether dam methylation a role in modulating RNA-OUT levels in DNA and RNA methods plays significant All DNA manipulations were performed essentially as described (Maniatis wild type cells. However, this or some similar effect could, et al., 1982). RNA extraction, probe synthesis, fragment end labelling, RNA if it persisted over many cell generations, decrease RNA- sequencing and Northern blot hybridizations were as described (Case et al., OUT levels and thereby lessen antisense control. Finally, 1988). In vitro transcription with T7 RNA polymerase was carried out the need to account according to the supplier (Promega). Where possible, all procedures involved these observations underscore carefully autoclaved for cleavage or other complexities that might arise whenever DEPC treated and reagents. RNA/RNA pairing occurs in vivo. The half-lives of the paired species remnant and the 5' end of RNA-IN Implications for the design of efficient artificial The half-life of the 35 bp remnant was determined by extracting RNA at appropriate time intervals from RS6181 transformed with pRSl 146 and antisense systems comparing relative levels by quantitative Northern blot hybridization as Our studies show that antisense RNA/target RNA pairing described (Case et al., 1989). The half-life of the paired species remnant can decrease RNA stability in at least two ways: cleavage was found to be >2 h at 37'C, equal to or exceeding that of the ribosomal of the species by an endonuclease and perturbation RNA used to standardize the experiment. The half-life of the 5' terminal paired same method of RNA secondary structure (RNA-OUT in this case) leading region of RNA-IN was determined by essentially the except that samples were analyzed in triplicate by primer extension and to exonucleolytic degradation (presumably). Both of these autoradiography, and the relative intensity of the band corresponding to possibilities should be considered in the design of artificial the 5' end of RNA-IN quantitated by densitometry. The half-life of this antisense RNA control. In this regard, we point out that band was found to be -30 s at 370C. 1265 C.C.Case, E.L.Simons and R.W.Simons

Acknowledgements We gratefully acknowledge the participation of all members of our laboratory in discussions of the ideas presented here. We especially thank Su-Min Chen and Don Court for the generous gift of purified RNaseIII, Chaitanya Jain and Nancy Kleckner for strains and the communication of unpublished results, and Chuck Ma for determining the pairing specificity of the mciS1O mutation. C.C.C. was supported in part by a postdoctoral fellowship from the American Cancer Society. R.W.S. was supported by a Junior Faculty Research Award from the American Cancer Society (JFRA-130). This work was funded by a research grant to R.W.S. from the National Institutes of Health (GM35322). References Belasco,J.G. and Higgins,C.F. (1988) Gene, 72, 15-24. Case,C.C., Roels,S.M., Simons,E.L. and Simons,R.W. (1988) Gene, 72, 219-236. Case,C.C., Roels,S.R., Jensen,P., Lee,J., Kleckner,N. and Simons,R.W. (1989) EMBO J., 8, 4297-4305. Daniels,D.L., Subbarao,M.N., Blattner,F.R. and Lozeron,H.A. (1988) Virology, 167, 568-577. Dempsey,W.B. (1987) Mol. Gen. Genet., 209, 533-544. Finlay,B.B., Frost,L.S., Paranchych,W. and Willets,N.S. (1986) J. Bacteriol., 167, 754-757. Foster,T., Davis,M.A., Takeshita,K., Roberts,D.E. and Kleckner,N. (1981) Cell, 23, 215-227. Guameros,G. (1988) Curr. Top. Microbiol. Immunol., 136, 1-19. Halling,S.M., Simons,R.W., Walsh,R.B. and Kleckner,N. (1982) Proc. Natl. Acad. Sci. USA, 79, 2608-2612. Higgins,C.F. and Smith,N.H. (1986) In Booth,I.R. and Higgins,C.F. (eds), Regulation ofGene Expression, 25 Years on. Cambridge University Press, Cambridge, pp. 179-198. Kim,K. and Meyer,R.J. (1986) Nucleic Acids Res., 14, 8027-8046. King,T.C., Sirdeskmukh,R. and Schlessinger,D. (1986) Microbiol. Rev., 50, 428-451. Kittle,J.D., Simons,R.W., Lee,J. and Kleckner,N. (1989) J. Mol. Biol., 210, 561-572. Krinke,L. and Wulff,D.L. (1987) Genes Dev., 1, 1005-1013. Liao,S., Wu,T., Chiang,C.H., Susskind,M.M. and McClure,W.R. (1987) Genes Dev., 1, 197-203. Ma,C. and Simons,R.W. (1990) EMBO J., 9, 1267-1274. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) In Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Mizuno,T., Chou,M. and Inouye,M. (1984) Proc. Natl. Acad. Sci. USA, 81, 1966-1970. Novick,R.P., Iordanescu,S., Projan,S.J., Komblum,J. and Edelman,I. (1989) Cell, 59, 395-404. Roberts,D., Hoopes,B.C., McClure,W.R. and Kleckner,N. (1985) Cell, 43, 117-130. Robertson,H.D. (1982) Cell, 30, 669-672. Schollmeier,K. and Hillen,W. (1984) J. Bacteriol., 160, 499-503. Shapiro,D.J., Blume,J.E. and Nielsen,D.A. (1987) Bioessays, 6, 221-226. Simons,R. (1988) Gene, 72, 35-44. Simons,R.W. and Kleckner,N. (1983) Cell, 34, 683-691. Simons,R. and Kleckner,N. (1988) Annu. Rev. Genet., 22, 567-600. Simons,R.W., Hoopes,B.C., McClure,W.R. and Kleckner,N. (1983) Cell, 34, 673-682. Simons,R.W., Houman,F. and Kleckner,N. (1987) Gene, 53, 85-96. Takiff,H.E., Chen,S.-M. and Court,D.L. (1989) J. Bacteriol., 171, 2581 -2590. Tomizawa,J. (1987) In Inouye,M. and Dudock,B.S. (eds), Molecular Biology ofRNA: New Perspectives. Academic Press, New York, pp. 249-259. Womble,D.D., Dong,X. and Rownd,R.H. (1987) In Inouye,M. and Dudock,B.S. (eds), Molecular Biology of RNA: New Perspectives. Academic Press, New York, pp. 225-247. Wu,T., Liao,S., McClure,W.R. and Susskind,M.M. (1987) Genes Dev., 1, 204-212. Received on November 27, 1989

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