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Rev. Biochem. 1991. 60:631-52

Annu. Rev. Biochem. 1991.60:631-652. Downloaded from www.annualreviews.org by NORTH CAROLINA STATE UNIVERSITY on 09/13/12. For personal use only.

ANTISENSE RNA 1 Yutaka EguchF Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Disc!ases, National Institutes of Health, Bethesda, Maryland 20892

Tateo Itoh Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan

lun-ichi Tomizawa National Institute of Genetics, Mishima, Shizuoka-ken KEY WORDS:

411, Japan

RNA-RNA interaction, ColE!, RNA I, RNA II, Rom.

CONTENTS PERSPECTIVES AND SUMMARY . . . . . . . . . . . . . .. . . . .. . . . . . . ..... . . . . . . . . . . . . . . . . . . . .. . . . . . . .. ..

632

BASIC FEATURES OF RNA-RNA INTERACTION.........................................

632

Binding Between Linear RNAs . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . Binding Between Linear RNA and Stem-Loop RNA or Between Two Stem-Loop RNAs . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . RNA Folding. . . . . .. ... .. . . . . . . . . . ..................................................................

632

633

634

BIOLOGICAL REGULATION BY ANTISENSE RNA .. . . . . . . . . . .. . . . . ...... . . . . .. . . ... . . . .

637

Regulation of ColE1 DNA Replication . ..... ... . . . . . . . . . . . . . . . . . . . . . . . ....... . . . . . . . . . . . . . . . . Sequential Steps for Binding of ColE} RNA I to RNA II................................. Early Steps in Binding of RNA I to RNA 11................................................. Binding of Two RNA Stem-Loops at Their Loops . . . . . . . . . . . . .. . . . ... . . . . . . . . . . . . . . . . . . . . .. Early Steps of Binding in the Presence of Rom . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . ... Stabilization by Rom of Complex Formed by RNA Stem-Loops . . . . . . . . . . . . . . . . . . . . . . . .. Regulation by Antisense RNA . . . . . . . . . .. . . . . . . .. . . . .. . . . . . ....... . . . . . . . . . . . . . . . . . . . . . . . . . .. .. .

637

CONCLUSION

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....

638

642 642 643

644 645 648

'The US government has the right to retain a nonexclusive royalty-free license in and to any copyright covering this paper. 2Present Address: The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 1 9 1 04

631

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EGUeHI ET AL

PERSPECTIVES AND SUMMARY RNA-RNA interactions have been shown to play key structural and enzymatic roles in several aspects of cellular processes, including transcription, transla­ tion, RNA processing, and regulation of DNA replication. Recently, it was found that a small RNA can bind to a complementary region of a target RNA and affect its function. An RNA that interferes with the activity of another RNA in this manner is defined as an

antisense

RNA.

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Regulation by antisense RNA was first found during the study of replica­ tion of the

Escherichia coli

plasmid ColEl. Replication of this plasmid

depends on formation of an RNA primer whose precursor RNA is functional only when it assumes a unique structure during its synthesis. Interaction of a small antisense RNA to the primer precursor inhibits formation of the unique structure, and consequently replication of the plasmid. Subsequently, many additional examples of regulation by antisense RNA have been observed, such as those for DNA replication and expression of gene functions. The ability of antisense RNA to inhibit the activity of a specific target mRNA has also led to increasing use of artificial antisense RNAs to study biological function in both prokaryotic and eukaryotic systems (reviewed in 1-3). It has also prompted consideration of antisense RNAs for medical purposes. In this paper, we review regulation by antisense RNAs and examine the more general question of the mechanism of RNA-RNA interactions. An RNA-RNA interaction is biologically significant only when it occurs at a certain time in a biological process, because the effective interaction has to occur during transcription or because the transcript concerned has a certain limited lifetime. Therefore, the rate of RNA-RNA interaction is critically important for effective regulation by antisense RNA. In addition, most RNA transcripts exist in complex folded structures with one or more loops, and the difference in the structure has a profound effect on the kinetics of RNA-RNA

interactions. Accordingly, in this article, the kinetics of interactions between folded RNA molecules are the major subject discussed. Most of the details on the subject are drawn from analysis of the interaction of the precursor RNA of ColEl with its antisense RNA, as outlined above. Although the structures of RNAs and kinetics of their interaction are unique to each system, studies of ColEl RNAs, together with those of complementary oligonucleotides, provide a useful conceptual framework for understanding the mechan­ isms of RNA-RNA interaction and its role in biological regulation.

BASIC FEATURES OF RNA-RNA INTERACTION

Binding Between Linear RNAs Formation of a double-stranded RNA by binding of various complementary pairs of short oligoribonucleotides is bimolecular and second-order with the

ANTISENSE RNA

633

association rate constants mostly between 1 x 106 and 3 x 106 M-1 S-I. The initial phase of the binding process was studied by analyzing the effect of temperature on the association rate constants (for review, 4, 5). When the temperature increased, the rate constant of association between As and Ug (6), and that between two molecules of AnUn (n 4, 5, or 6) (7) decreased, giving a negative activation energy. If formation of the first base-pair were rate­ determining, increasing temperature should be accompanied by an increasing association rate. On the other hand, if several base-pairs must form to commit the reactants to binding, the increasing temperature should result in a decreas­ ing association rate, because base-pairing is less stable at a higher tempera­ ture. The activation energy thus obtained agrees with that calculated for involvement of two or three base-pairs in the rate-determining transition state (6). On the other hand, the association rate of AnGC Un (n = 2, 3, or 4) increased by increasing temperature (8). Therefore, in this case the transition state is achieved by a diffusion-controlled reaction, probably formation of a nucleus of just one or two base-pairs. Thereafter, the pairing proceeds through the entire length of the complementary region of the components at the very fast rate of about 106 bases per sec at around 20°C (7). The stabilities of the complexes formed by short oligoribonuc1eotides depend strongly on the chain-length and base-composition of the components (9, 10). In addition, base sequence affects the stability of the complexes (11), indicating that nearest neighbor interactions must affect the contribution of each base··pair to the stability of the helix. The observation that the RNA double-helix is stabilized by the presence of terminal unpaired bases (dangling bases) (12--18) further suggests that stacking of nearest neighbors affects helix stability. Thermodynamic parameters for base-pairing and the effects of the nearest nei.ghbors on the stability of the complex are summarized in Refs. 11, 15, 19-25. These well-characterized thermodynamic parameters allow us to predict the stability of a number of RNA duplexes and the secondary struc­ tures of RNA molecules.

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=

Binding Between Linear RNA and Stem-Loop RNA or Between Two Stem-Loop RNAs Binding of a tRNA to a linear RNA containing a complementary triplet serves as a model for binding of linear RNA and stem-loop RNA. Complexes are formed by a tRNA and a triribonuc1eotide or a larger oligoribonuc1eotide containing a sequence that is complementary to the anticodon (26-30), under the condition where no complex has been detected by a pair of complementary triribonucleotides (31). Two tRNA molecules with complementary anti­ codons also form a complex (32-37)_ The association rate constants obtained for binding between linear RNA and the anticodon loop of tRNA or between two anticodon loops are similar to those for two linear complementary

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634

EGUCHI ET AL

oligonucleotides (32-35, 38--41), even when using the isolated anticodon loop of a tRNA as one of the components (42) in either a linear-to-Ioop or loop-to-Ioop interaction. Because the association rates of these reactions increase by increasing temperature (34,38), the binding is likely to begin with the diffusion-controlled nucleation of pairing of one or two base-pairs prob­ ably facilitated by stacking of bases in the loop. Stability of the complex formed by an anticodon loop and a complementary linear RNA is much higher than expected from the interaction of two linear RNAs. Stability is dependent on the base sequence of the complementary region and increased by the presence of dangling base residues at both ends of the complementary region of the linear component (38, 43--46). Complexes formed by two anticodon loops were 100-1O,000-fold more stable than those of the complexes formed by oligoribonucleotides with tRNA (32, 36, 37). The higher stability is probably due to the loop constraint and to stacking with dangling bases and with modified nucleotides (32). In the model proposed for the complex (32; Figure lA), the three complementary central nucleotides (anticodons) in the seven-nucleotide loops form Watson-Crick base-pairs, and are sandwiched together with two unpaired bases located at the 3' -side of the paired bases between the stems of the components. The overall structure of the resulting complex is similar to a linear A-form double-stranded RNA with two stacked nucleotides looping out from a phosphodiester backbone.

RNA

Folding

Most RNA molecules probably do not exist as random coils, but form secondary structures through intramolecular interactions. Using free-energy parameters obtained for base-pairing and the effects of nearest neighbors and loops on the stability, a possible secondary structure of RNA can be predicted from the nucleotide sequence (19, 48; for recent review, 24). The structures thus predicted for small RNA species frequently match quite well with the biochemical or genetic properties of the molecules. However, agreement is more questionable for large RNA species. For example, the secondary struc­ ture of the wild-type ColEl RNA II (555 nucleotides long) predicted by a computer program (20) is identical to that of a certain mutant RNA II with a single base change (Figure 2B). Nonetheless, the RNase sensitivity and the functional properties of these RNAs are quite different (49). A more plausible wild-type structure can be predicted by adding genetic and biological proper­ ties as constraints to the computer program (Figure 2A). The failure to predict RNA structures accurately by the energy parameters alone probably is due both to formation of tertiary structures and nonconventional structures and to inaccuracies in the values of the basic parameters themselves. The absence of an algorithm that incorporates tertiary interaction is an especially serious problem.

B

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A 3' 0

30

A

5' �

C

3' 5'

5'

Rom

20 10 0

� 1'" ,, ;::;

5'

Figure I

3'

� 5'

3'

(A) Model for complex of two anticodon stem-loops (32). Bases and phosphate backbones are shown by the rods and dotted areas, respectively.

The three complementary central nucleotides (anticodon) in the seven-nucleotide loops pair with those of the other RNA and stack in a helical conform ation together with the neighboring two bases on their 3'-side. The two bases on the 5'-side of the anticodon in the loop are stacked together and looped out from the phosphate backbone. The overall structure is similar to that of a linear A-form double-stranded RNA.

(B) Model for complex Csingle formed by two fully complementary stem-loop RNAs was constructed by a slight modification of that for a complex of two

tRNAs (Al. In this model, all seven nucleotides form base-pairs, and the phosphate backbone of each component kinks at the 5'-end of the loop. The overall

structure is similar to that of a linear A-form double-stranded RNA with a little bend at the center of the complex. Each molecule in (A) and (B) has an axis of dyad symmetry perpendicular to the paper through the bond joining the pairing central bases.

(e) Model for complex Cm'ingle, which consists of a side view of the complex shown in (B) and schematic representation of Rom dimer (47). Open circles in the Rom molecule indicate the position of basic amino acid residues, which are localized on one face of the molecule. The axis of dyad symmetry of the complex (neglecting particular nucleotide sequences) is shown by a horizontal bar.

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636

B

EGUCHI ET

IV

AL

Wild-Type RNA II VI

A

pri 7 RNA II

637

ANTISENSE RNA

RNA 1- bound RNA II

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c

IX s'

3'

Figure 2

RNAI

X

(Parts A and B on facing page) The secondary structures of (A) the wild-type ColEI

RNA II, (B) pri7 mutant RNA 11 (G to A change at 469 nucleotides upstream of the origin), and

(C) the wild,type RNA 11 hybridized with RNA I with the lowest free energy of formation under

certain constraints (48), Sites for preferential cleavage by RNase TI (solid arrowhead), RNase A (open arrowhead), and RNase VI (Y) are indicated, Roman numerals show the names of stem-loops, These figures are not intended to show the structures in detail, for which see references (48, 49),

Intramolecular interaction of a loop region with either another loop region or a linear sequence produces a tertiary structure, A simple structure formed by these interactions is called a pseudoknot. The biological significance of pseudoknotting has been reviewed (51). BIOLOGICAL REGULATION BY ANTISENSE RNA

Regulation of ColE] DNA Replication Studies OIl the replication of plasmid ColEl led to the discovery of biological regulation by antisense RNA. They also showed how folded RNA molecules interact with each other and how the interaction affects the control of ColEl DNA repilicatioll (reviewed in 50, 52, 53), Here we describe the mode of ColE! replication briefly, and then the process of binding of ColEI RNAs in

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638

EGUCHI ET AL

some detail. The sequence of reactions in ColEI RNA primer formation is schematically shown in Figure 3 (left) (54). Replication of ColEI is a multi­ step process, which initiates with synthesis of an RNA primer for DNA synthesis, named RNA II, from a site 555 nucleotides upstream of the site (origin) where DNA replication initiates (55). Initially, the transcript is separated from the template as is normally the case in transcription (Figure 3, steps 1 and 2). However, as the RNA polymerase nears the origin, the transcription switches to a novel mode in which the transcript remains hybri­ dized to the template DNA (Figure 3, steps 3 and 4). Then the hybrid is cleaved by RNase H (Figure 3, step 5), and the 3' -end of the cleaved RNA primes DNA synthesis by DNA polymerase I (Figure 3, steps 6 and 7). What causes the transcript to remain hybridized to the template DNA? Computer analysis of the primer RNA, together with the results of genetic and biochemical analyses, shows that RNA II must fold into a unique structure to form the persistent hybrid with the template DNA (49; Figure 2A). An alteration of the nucleotide sequence of RNA II, even one as small as a single base change, can affect the tertiary structure (Figure 2B), and thereby reduce the ability of RNA II to function as a primer. The region of RNA II about 265 nucleotides upstream of the origin (in structure VII of Figure 2A), which contains six consecutive rG residues, probably interacts with the stretch of six consecutive dC residues in the template DNA strand about 20 nucleotides upstream of the replication origin to promote formation of the persistent hybrid (56). A change in the RNA sequence that prevents this interaction inhibits formation of the persistent hybrid (56). The efficiency of primer formation is determined by a mechanism that regulates the formation of the persistent hybrid of RNA II to the template DNA. A key element of the mechanism is RNA I, a plasmid-specified RNA of about 108 nucleotides (57-59). This RNA is transcribed from the same region of the plasmid as the primer, but in the opposite direction (Figure 3 (right), steps I' and 2'). Binding of RNA I alters the structure of RNA II (Figure 2C) in a way that prevents RNA II from forming a persistent hybrid with the template DNA at the origin (49; Figure 3, steps 3' to 5'). However, to inhibit the formation of the persistent hybrid, RNA I must interact with the nascent RNA II transcript well before the RNA polymerase reaches the replication origin: the elongating RNA II must be between 100 and 360 nucleotides in length to be susceptible to inhibition by RNA I (60). Therefore, a knowledge of the kinetics of the RNA-RNA interaction is critically impor­ tant for understanding the mechanism of regulation of ColEl replication.

Sequential Steps for Binding of ColE] RNA I to RNA II Binding of RNA I and RNA II was first detected by formation of a structure that is sensitive to RNase III that specifically cleaves double-stranded RNA

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INHIBITION OF PRIMER FORMATION BY RNA I

FORMATION OF PRIMER AND ITS REMOVAL 1,

Transcription initiation by RNA polymerase

2,

3, RNA/DNA

4,

5,

6,

H

Addition of dNMP by

Elongation of DNA removal of primer

Origin

�

6.

Jtr

�

&

�\� 6.

�� �;: 6.

..

l' , Transcrlptlon . . . .. . Imtlatlon by RNA polymerase

Origin

..

�

�)

6.

..

vw

�

Elongation of hybrid

DNA polymerase I

7,

_ RNA I

�

Absence of coupling

Cleavage by RNase

...,;;

6.

�

hybrid

formation, coupling

(

-445

11

-555 11

Elongation of RNA

RNA

..

2',

Completion of RNA

I

synthesis

3',

Interaction of RNA I and RNA

4',

11

RNA/RNA hybrid formation

5',

Inhibition of RNA/DNA hybrid fromation, uncoupling

Figure 3

The processes of ColEI primer formation and of inhibition of

primer formation by RNA I are illustrated schematically (54). Straight lines represent double-strands of DNA; wavy lines, RNA transcripts; small circles,

RNA polymerase. The DNA template forms an eye structure from the origin of DNA replication (filled triangle)

One alternative of step 3 causes primer . hybridization, and the other (shown in parentheses) does not. Arrows in the

eye structures in steps 5 and 6 indicate cleavage of hybridized RNA by RNase

H. The thick lines in the eye structures are newly synthesized DNA strands.

Binding of RNA I to RNA II in step 3' inhibits formation of RNA/DNA hybrid and consequently primer formation.

�

tn

� tn m

� 0"1 W \0

640

EGUCHI ET AL

Process of Binding

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RNA I

RNA II

Figure 4

The stepwise process of binding of ColEI RNA I to RNA II is illustrated schematical­

Iy. RNA I and RNA II interact at the loops of their folded structures to form C**. This interaction facilitates pairing that starts at the 5' -end of RNA I. The pairing propagates progressively as the stem-loop structures unfold, and finally. RNA I hybridizes along the entire length to RNA II.

(54). Subsequently, binding of RNA I and RNA II was studied by quantitating the product by polyacrylamide gel electrophoresis (61). While RNA I has a unique structure consisting of three stem-loops and a short tail at its 5' -end (see Figure 4), an RNA II transcript changes both its folded structure and its propensity to bind RNA I during elongation (60, 62, 63). The use of a 241-nucleotide RNA II transcript in the binding studies as a representative of

ANTISENSE RNA

641

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species whose binding to RNA I inhibits subsequent primer formation is considered to be reasonable (60). Binding of RNA I to RNA II results in the formation of a complex, designated CS ( " stands for "stable"), which has a distinct mobility in poly­ acrylamide gel electrophoresis. The rate constant of formation of CS between the wild-type RNA I and RNA II is about 1 x 106 M-1 S-I at 25°C (60, 64). However, the rate constant is altered by even a single base change in any loop of the folded structures common to RNA I and RNA II. In particular, if the sequences at any corresponding pair of loops are not complementary, the binding rate is very low. These results suggest the importance of loop-to-Ioop

interactions in the binding. The loop sequences in free RNA I can be cleaved with a low concentration (0.1 unit/ml) of RNase TI. If the two RNA com­ ponents are mixed and then treated with the enzyme, the loop regions of RNA I become resistant to the enzyme quite rapidly (64). However, most com­ plexes that are resistant to the enzyme at a low concentration are still sensitive to the enzyme at a high concentration (2000 units/mI). These complexes are converted very slowly to highly resistant forms: about half in 30 min (61). These results indicate the presence of a reversible loop-to-Ioop interaction followed by its slow conversion to a much more stable pairing. The complex thus formed by reversible loop-to-Ioop interactions is designated C**. The rate constant of association of the wild-type RNA I and RNA II to form C** is 3 x 106 M - 1 S- 1 (64). The conversion of the three loop structures to a more stable one is a sequential process that initiates at the loop and the tail region located near the 5 I -end of RNA I, showing unidirectional progression of complete hybridization. The process of binding of the wild-type RNA I and RNA II, deduced from the properties of the binding described above, is schematically shown in Figure 4. On the polyacrylamide gel, the three types of elongating complexes have the same mobility as the completely hybridized complex. They are considered to be in the same class (C') in the kinetic analysis described below. When five or nine nucleotides are removed from the 5'-end of the wild-type RNA I, CS is not made with RNA II (61, 65). However, the removal does not prevent formation of C** (64). These results suggest that C** converts to CS through a process that requires pairing of the tail region of RNA I. The presence of a tail structure for at least one of the components and of a complementary single-stranded region in another com­ ponent is generally required for stable binding of folded RNAs (60), even for an RNA with a single stem-loop (Y. Eguchi, J. Tomizawa, unpublished observation). The wild-type bacteria can eliminate five nucleotides from the 5 I -end of RN A I. Loss of this cleavage activity by a bacterial mutation results in a decrease in plasmid copy number, indicating that the cleaved molecule is inactive in inhibition of in vivo ColEl replication (66). Formation of the complete double-strand is a very slow process, because simultaneous melting

642

EGUCHI ET AL

of complementary stems is required for progression of hybridization. Exten­ sion of hybridization into the first stem-loops (top of the three CS diagrams in Figure 4) is sufficient to inhibit primer formation (61).

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Early Steps in Binding of RNA I to RNA II The presence of a precursor of C** in the binding reaction is suggested by the following inhibition kinetics. RSF1030 is a close relative of CoIEI. Its replication is regulated by RSF RNA I, which is very similar to ColEl RNA I except for the loop III region (67, 68). RSF RNA I inhibits C' formation between ColEl RNA I and RNA II (64, 69). The inhibition is presumably due to formation of a very unstable complex, named C* for the heterologous pair, whose rate constant of formation has been measured to be about 6 X 106 M-1 s-I (64). If a corresponding complex is made between the homologous ColEl pair, one would expect an even larger value for its association rate constant. The faster rate of formation of C* than of C**, together with other evidence, suggests that C* is a precursor of C** (64). Formation of C* by a heterologous pair requires homology at both loop I and loop II and probably in the tail region as well. Because binding of RNA I and RNA II is likely to begin with an interaction at only one of these three regions, it is unlikely that C*, which involves multiple regions of interaction, is the very first product of interaction. The very first product is likely to be an as-yet-undetected complex, named CX, formed by a homologous pair by nucleation at one of the interacting loops. Its formation is probably the target of inhibition by formation of C* by the heterologous pair (64, 70). Thus, the binding of RNA I to RNA II consists of the followirig sequence of reactions, producing a series of progressively more stable intermediates leading to the final product (64): RNA I + RNA II � CX � C* � C**

�

C'

Binding of Two RNA Stem-Loops at Their Loops The binding of two RNA stem-loops with fully complementary loop se­ quences was analyzed in detail using pairs of complementary single stem­ loops bearing partial sequences of RNA I and RNA II and their derivatives (71, 72; Y. Eguchi, J. Tomizawa, in preparation). Each RNA has seven nucleotides in the loop and 5 or 20 base-pairs in the stem, so the structure may be similar to that of an anticodon loop. Binding of these stem-loops was examined by comparing the cleavage patterns by RNases. The complex formed by the binding of two single stem-loops, named complex Csingle (the subscript distinguishes it from complexes formed by RNA with multiple stem-loops), has a different cleavage pattern at the loops of the components from that of a duplex formed by annealing the components, and is much less stable than the duplex. Therefore, the complex is not an ordinary duplex but a

ANTISENSE RNA

643

hetero-dimeric molecule with interacting loops. The association rate constants

for the interaction of two stem-loops with fully complementary sequences range from I X 105 to 3 X 106 M I S I, depending on the sequence. These -

-

values are similar to those obtained for the binding of the other types of RNAs described above. The observations of rapid and similar rates of binding of complementary RNAs with different structures suggest that their binding

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shares a common basic mech anism

for the rate-determining steps, most likely the nucleation of base-pairing. Replacement of a base at different positions in the loop of a component by a noncomplementary base reduces the stability of the resultant complex. The

complexes formed by pairs in which only the central three or five bases are complementary are much less stable. These results suggest that all seven

complementary nucleotides in the loops of the wild-type pair form base-pairs. The stability of the complex is highly dependent on the loop sequence as well, showing that base-stacking as well as base-pairing determines stability. Analysis of the wild-type complex by native gel electrophoresis suggests that the complex is bending a little at the interacting region. Based on these

fully com­ has been proposed as illustrated in Figure IB and Ie (72; Y. Eguchi, 1. Tomizawa, in preparation). In the model, all seven nucleotides in the loops form base-pairs, and the phosphate backbone of each component kinks at the 5' -end of the loop. The overall structure is similar to a observations, a structural model for the complex formed by

plementary stem-loops

linear A-fiJrm double-stranded RNA with a small bend at the center of the complex. Bending assists in the pairing of all seven complementary nucleo­

tides in the loop.

Early St,eps of Binding in the Presence of Rom The presence of a certain region downstream from the origin of replication of ColEi reduces the plasmid copy number (67, 73). The speculation that the region specifies a hypothetical repressor protein named Rop that inhibits synthesis of RNA II (74) was not supported experimentally (75, 76). Instead, the protein isolated affects the mode of binding of RNA I to RNA II and consequently, affects the inhibitory activ ity of RNA I (60, 70, 75). This protein of 63 amino acids was named Rom to emphasize its function as the "BNA-.Qnt� lDodulator." The Rom protein acts at an early step in the binding process (60, 70). The following scheme has been proposed for formation of CS in the presence of Rom (70):

RNA

I + RNA II � ex � C* � C** '" + Rom it CS 7' cm* � Cm**

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644

EGUCHI ET AL

Binding of a single dimer molecule of Rom to C* forms cm*, whose presence is shown by the effect of Rom on inhibition of binding by RSF RNA I. The complex is then converted to Cm**, whose RNase sensitivity differs from that of free RNA or C**. Rom does not convert C** to Cm**, but instead creates a second pathway for stable binding of ColEI RNA I to RNA II by converting an unstable early intermediate to a more stable complex. The apparent rate constant of formation of es from wild-type ColEl RNA I and RNA II in the presence of Rom is 3 X 106 M-I S-I and that of cm** is 5 X 106 M- I S - I . Rom does not always enhance the formation of C'; it is actually inhibitory for certain lengths of RNA II (for example, 89-nucleotide long RNA II; see 60). Thus, the rate of conversion of cm* to CS is not always higher than the rate of C* to C'.

Stabilization by Rom of Complex Formed by RNA Stem-Loops The molecular mechanism of the action of Rom has been studied in detail using the system of binding of two complementary single stem-loops (71, 72; Y. Eguchi, 1. Tomizawa, in preparation). A single dimer molecule of Rom binds to the complex Csingle, forming the complex cmsingle' Formation of Cmsingle was shown by resistance to cleavage by RNase V at the loop region I and on the 5' -side of the stem of the RNA components. Cffisingle is separable from Csingle, single stem-loops, or Rom by gel filtration. While Rom binds neither to each component alone nor to A-form double-stranded RNA, it binds with a similar affinity to complexes formed by complementary stem-loops having various sequences. The affinity to Csingle decreases greatly when one or two noncomplementary bases are present at various positions of the loop sequence. Therefore, Rom recognizes the structure of a target rather than its exact nucleotide sequences. The binding of Rom decreases the rates of dissociation of the complexes about one hundred fold. The gross structure of complex Csingle, formed by pairing of all the bases in the loops, has a convex surface (Figure Ie). While all the phosphate groups of Csingle can be alkylated by ethylnitrosourea, binding of Rom suppresses alkylation of the groups on the convex surface (72). On the other hand, the Rom dimer has a concave surface on which are located several basic amino acid residues that are important for the function of Rom (77; see Figure Ie). These two surfaces fit to each other quite well, and their interaction must be responsible for formation of complex emsingle (Figure I e). Analysis of the interactions of pairs of single stem-loops to form Csingle and cmsingle suggests a mechanism for complex formation between RNA I and RNA II. The apparent rate constant for formation of C** from RNA I and RNA II is about 2.5 x 106 M-I S-I, which is close to that for the formation of Csingle' This suggests that the rate-determining step in formation of C** is interaction at either one of the loop regions, and this interaction induces

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ANTISENSE RNA

645

interaction at other stem-loop regions to produce C** that is more stable than Csingle' Because binding of only one dimer molecule of Rom is sufficient for the formation of Cm**, stabilization of a single pair of interacting stem-loops should be responsible for the direct effect of Rom on binding of RNA I and RNA II. Complex C* is slightly more stable than Csingle, yet C* is sensitive to RNase A while Csingle is resistant. The rate constant of formation of C* is also higher than that of Csingle' It is possible that the rapid and weak interaction that produces C* does not involve simultaneously all the three loops or all the seven basl�-pairs of each loop.

Regulation by Antisense RNA A mutation in the RNA I region of the ColEl plasmid alters both RNA I and RNA II. A comparison of the copy numbers of the mutant plasmids and the binding rates of their RNA I and RNA II shows an inverse correlation (61, 75). The characteristics of binding among homologous and heterologous pairs of RNA I and RNA II also explain very well the property of incompatibility, in which one member of a group of plasmids is excluded from a bacterial cell by another member. The inhibitory activity of ColE1 RNA I is supprcssed by RSF RNA I and vice versa (69). This mutual suppression explains the increase in copy numbers of both ColE1 and RSF1030 by coexistence. These observations indicate that the binding of RNA I to RNA II is responsible for in vivo regulation of plasmid replication. In several groups of ColEl-type plasmids, in vitro formation of the primer for DNA synthesis is inhibited by the group-specific RNA I (57). Regulation of copy number and incompatibility of these plasmids can bc explained by the group-specific interaction of RNA I and RNA II. These RNA I species are very similar in size and structure; they have three similar-sized stem-loops and a similar short tail. A total of 12 stem-loops of four different groups of plasmids, ColE1, RSF1030, p15A, and CloDFI3, consist of only four types of stem-loops with different nucleotide sequences in their loops (68). The specific regulatory system for each group of plasmids may have evolved by duplication and recombination of the DNA sequences for these stem-loops. The efficiency of replication of ColEI in vivo fits reasonably well with expectations based on the concentrations and rates of synthesis of RNA I, RNA II, and Rom, as well as the rate constant of formation of CS (78). Several theoretical models for binding of RNA I to RNA II and for replication of ColE I-type plasmid have been proposed (79-82). Functional regulation of specific target RNAs by antisense RNA has been shown or proposed to operate in various prokaryotic systems besides the ColEl system described above (reviewed in 83-85). In most of the systems listed in Table 1, antisense RNAs and their target RNAs are transcribed from

646

EGUCHI ET AL

Table 1

Naturally occurring antisense RNAs Antisense

Target

Function

Level of

Type of

Selected

RNA

RNA

controlled

control"

mechanism

refs.

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Plasmid ColE I

RNA I

RNA II

Replication

Primer formation

IncF

copA RNA

repA mR NA

Replication

mRNA stability

II

86-91

IncI

inc RNA

repZ mRNA

Replication

Translation

CoIE2

copRNA

rep mRNA

Replication

uncertain

II II

97, b

Rl l 62

ct RNA

rep) mRNA

Replication

(Translation)

R6K

silencer

activator

Replication

uncertain

pT l81

RNA I

repC mRNA

Replication

Transcription

II 92-96 98 uncertain

99

II

100-102

II

105

termination IncF

finP RNAc

traJ mRNA

Conjugation

(Translation)

IncAI

sok RNA

hok mRNA

Host killing

(Translation)

103, 104

Phage lambda

aQ RNA

Q mRNA

Lysis/lysogeny

uncertain

lambda

oop RNA

cII mRNA

Lysis/lysogeny

mRNA stability

109

P22

sar RNA

ant mRNA

Lysis/lysogeny

(Translation)

110

c4 repressort

ant mRNA

Lysis/lysogeny

(Translation)

I II

RNA-OUT

tnp mRNA

Transposition

Translation

112-114

E. coli

micF RNA"

ompF mRNA OmpF synthesis Translation

E. coli

tic RNN

crp mRNA

P I, P7

uncertain

106-108

Transposon IS10 Bacterial Crp synthesis

115-117

Transcription

118

termination a

Speculated level of control is given in parentheses.

bH. Yasueda, S. Takechi, T. Itoh, in preparation C

The antisense control of conjugation by finP RNA requires the finO gene product, which might be RNA (119) or a

protein (120). dThe

c4

repressor is an antisense RNA containing two short sequence elements that are complementary to target

sequences in the e

ant

mRNA encompassing the ribosome-binding site involved in

ant

expression.

The micF RNA is 70% complementary to the 5' -region of the ompF mRNA containing the ribosome-binding site and

the initiation codon. 'The tic RNA is complementary to 10 out of the first II nucleotides of the crp mRNA.

the complementary strands of the same region of DNA. However, for the two

Escherichia coli chromosomal genes, each antisense RNA is transcribed from a region away from the target gene (115-118). On the other hand, the ant mRNA of phage Pi (and P7) and its antisense RNA (c4 repressor) are transcribed from the same promoter (111). The shorter antisense RNA prob­ ably interacts with a distal region of the target RNA. Binding of an antisense RNA may disrupt the function of its target RNA either directly by blocking function of the region that is complementary to the antiscnse RNA (Type I), or indirectly by altering the structure of the target RNA (Type 11). Inhibition of translation by blocking the ribosomal binding site and/or the initiation codon as seen for regulation of IS 10 transposition (114) and Escherichia coli OmpF synthesis (116, 117) are examples of the Type I mechanism. Regulation by the Type II mechanism can take multiple

ANTISENSE RNA

647

forms. Regulation of ColE1 primer formation as described above results from the confOImational change of RNA II that prevents it from making a structure necessary for primer formation. Binding of antisense RNA can also affect translation by altering the secondary structure of the target transcript in such a way that the ribosomes-binding site and/or the initiation codon is inaccessible to ribosome, as is proposed for regulation of ·replication of IncFII plasmid (121) and of Incla ( IncIl) plasmid ColIb-P9 (96), and host killing of the IncFII plasmid (105). In addition, binding of an antisense RNA could induce premature termination of transcription of the target RNA by forming a transcription terminator or terminator-like structure, as is the case for repC transcription of plasmid pT181 (102) or for crp transcription of Escherichia coli (118). Antisense RNA binding can also render a target RNA susceptible to digestion by RNase III or other RNases as seen for rep mRNA of IncFII plasmid RI (91) and lambda clI mRNA (109).

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=

All known antisense RNAs except the tic RNA are predicted to form very

stable secondary structures. The copA RNA of IncFII plasmid RI (122), sar RNA of bacteriophage P22 (123), and RNA-OUT of IS10 (124), in fact form stable stern-loop structures. Genetic analyses using mutants of IncFII plasmid (125, 126), ColE2 plasmid (S. Takechi, H. Yasueda, T. Itoh, in preparation), ColIb-P9JPlasmid (96), bacteriophage P22 (cited in 85), bacteriophage P I and P7 (Il l ), or IS10 transposon (127), and phylogenetic comparisons of the finP RNAs of IncF plasmids (128), have suggested that the specificity of initial recognition between the antisense RNA and target RNA is determined by the nucleotide: sequence of the loop portion of one major stem-loop structure of each antisense RNA. The antisense RNAs named cop RNAs of closely related but mutually compatible plasmids of the IncF family (IncFI and IncFII) (86, 89, 90, 129) and those of the IncI and IncB families (92-94) share an identical sequence (5'-CGCCAA-3') in the loop portions of the major stem-loop structures. On the other hand, the cop RNAs of three closely related but mutually compatible plasmids of the CoIE2 family share another identical sequence (5'-UUGGCG3') in the loop portions of their major stem-loop structures (S . Hiraga, T. Itoh, unpublished). Because these shared sequences are complementary, the initial interaction of the antisense RNAs and the target RNAs of all these plasmids probably occurs between the identical combination of complemen­ tary nucleotide sequences, suggesting an evolutionary significance. Binding between purified antisense RNA and the target RNA was demon­ strated for the systems of IncFII plasmid RI (copA RNAlrepA mRNA) (130--132), of ColE2 plasmid (T. Sugiyama, T. Itoh, in preparation), of bacteriophage P22 (133), and of transposon IS10 (127). The second order rate constants of stable binding in these systems are 3 x 105 to 3 X 106 M-I S- I , comparable to that between RNA I and RNA II of CoIEI. The effects of mutations in the loop portions of the antisense RNAs of RI, Co1E2 , and ISIO

648

EGUCHI ET AL

on the binding rates correlate very well with their in vivo phenotypes, such as

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increased copy numbers, reduced incompatibility, and reduced multicopy

inhibition ofIS lO transposition. By analogy with the ColEI RNAs, binding of RNAs in systems other than IS 10 is thought to be initiated by interaction between loops followed by stable binding that could start from the single­ stranded tail extending from the stem-loop structure. However, for ISIO RNAs, pairing of RNA-OUT and tnp mRNA initiates between the free single-stranded 5'-end of tnp mRNA and the loop of RNA-OUT and then proceeds down one side of the RNA-OUT stem by strand displacement (127). In all cases examined, the final products of RNA-RNA interaction are com­ plete duplex RNAs. However, it is not known whether formation of a complete hybrid is required for inhibition by an antisense RNA, except for ColEl for which complete hybridization has been shown to be unnecessary (64). So far no natu rally occurring antisense RNA regulation has been proven in eukaryotic systems. However, there are numerous cases known in eukaryotic systems in which both strands of a DNA segment are transcribed, or (at least partially) complementary RNAs are detected (83, 134, 135). Some of these could be involved in an ti se ns e RNA regulation. For example, an RNA species

found in neurons latently infected with herpes simplex virus is complementary to the mRNA for an immediate early viral protein required for the lytic process. Suppression of the lytic process by this RNA could be responsible for maintaining the latent state (136, 137). Small RNAs, called translational control (tc) RNAs, capable of specifically inhibiting in vitro translation for the chicken myosin heavy chain (MHC), are partially complementary to the 5' -end region and the 3' -part of MHC mRNA and might regulate expression of the mRNA (138). Synthesis of an RNA species complementary to the barley alpha-amyla se mRNA is developmentally regulated. The antisense RNA might play a regulatory role in preventing expression of the alpha­ amylase and thereby protecting the storage contents of the seeds (139). An RNA complementary to a part of the mRNA for the basic fibroblast growth factor in Xenopus oocyte directs the double-strand specific covalent modifica­ tion (adenosine to inosine) (140) of the complementary region of the mRNA, and this modification might be the cause of the abrupt degradation of the mRNA during maturation of the oocyte (141).

CONCLUSION Nucleation by pairing of a very small number of nucleotides initiates binding of complementary oligonucleotides of various structures. The rate constant of association of the components is relatively independent of their nucleotide

sequences and structures. On the other hand, the rate constant of dissociation depends very much on the nucleotide sequences and structures in the region of

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ANTISENSE RNA

649

several nucleotides around the site of nucleation. Formation of a seven­ nucleotide loop in the anticodon loop of tRNA or in the ColEl RNA provides very favorable conditions for the binding through structural constraint by a particular base-stacking. The process of binding complex molecules consists of a sequence of reactions producing a series of progressively more stable intermediates, leading to the final stably bound product. Binding may express the regulatory function without completing the sequence. Many functional RNA molecules form specific folded structures: for ex­ ample, all kinds of tRNA molecules form L-shaped structures and all types of RNA I of ColEl analogues are composed of three stem-loops. Interaction of folded structures has various advantages. If RNA molecules interact only in extended linear structures, the specificity of the interaction is determined solely by the nucleotide sequences. On the other hand, when one or both components of interaction is folded, the structure itself dictates an additional specificity of interaction and thus increases the functional and genetic versatil­ ity of the interaction. For example, a very small change in the primary sequence of folded structure can profoundly affect the rate and specificity of interaction of folded RNAs. In addition, formation of a group-specific struc­ ture allows a member to interact with a group-specific protein. This further increases the specificity and strength of the interaction. Furthermore, an analogous but genetically distinct regulatory system could evolve by geneti­ cally exchanging the stem-loop sequences. All these advantages of regulatory systems based on interactions between folded RNAs are clearly seen in regulation of the replication of plasmid ColEI and its relatives. RNA-RNA interaction through recognition of the complementarity in the nucleotide sequences is also found in systems other than those for antisense regulation. Recognition of the codon by the anticodon, determination of the sites of initiation of translation by rRNA, and selection of the site of splicing in RNA processing are just a few examples. These subjects were not dis­ cussed here as they have been reviewed previously (142-144). ACKNOWLEDGMENT

We are thankful for valuable advice on the manuscript by Drs. Nobuo Shimamoto, Kiyoshi Mizobuchi, and Michael Brenner.

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