Editing of plant mi to chon drial trans fer RNAs

Transcription

1 Vol. 48 No. 2/ QUARTERLY Minireview Editing of plant mi to chon drial trans fer RNAs Julien Fey 1, Jacques H. Weil 1, K. Tomita 1, Anne Cos set 1, André Dietrich 1, Ian Small 2 and Laurence Maréchal-Drouard 1 1 Institut de Bi o logie Moléculaire des Plantes, CNRS, Strasbourg, France and 2 Sta tion de Génétique et d Amelioration des Plantes, INRA, Versailles, France Received: 14 February, 2001; accepted: 15 April, 2001 Key words: plant mi to chon dria, RNA ed it ing, trna, pro cess ing, pseudouridine Editing in plant mi to chon dria con sists in C to U changes and mainly af fects mes sen - ger RNAs, thus pro vid ing the cor rect ge netic in for ma tion for the biosynthesis of mi to - chon drial (mt) pro teins. But ed it ing can also af fect some of the plant mt trnas en - coded by the mt ge nome. In di cots, a C to U ed it ing event cor rects a C:A mis match into a U:A base-pair in the ac cep tor stem of mt trna Phe (GAA). In larch mi to chon dria, three C to U ed it ing events re store U:A base-pairs in the ac cep tor stem, D stem and anticodon stem, re spec tively, of mt trna His (GUG). For both these mt trnas ed it ing of the pre cur sors is a pre req ui site for their pro cess ing into ma ture trnas. In po tato mt trna Cys (GCA), ed it ing con verts a C28:U42 mis match in the anticodon stem into a U28:U42 non-canonical base-pair, and re verse tran scrip tase minisequencing has shown that the ma ture mt trna Cys is fully ed ited. In the bryophyte Marchantia polymorpha this U res i due is en coded in the mt ge nome and evo lu tion ary stud ies sug gest that res to ra tion of the U28 res i due is nec es sary when it is not en coded in the gene. How ever, in vi tro stud ies have shown that nei ther pro cess - ing of the pre cur sor nor amino acylation of trna Cys re quires C to U ed it ing at this po - si tion. But se quenc ing of the pu ri fied mt trna Cys has shown that Ψ is pres ent at po si - tion 28, in di cat ing that C to U ed it ing is a pre req ui site for the sub se quent isomerization of U into Ψ at po si tion 28. Pres ented at the In ter na tional Con fer ence on Mo lec u lar Ar chi tec ture of Evo lu tion, Pri mary and Sec - ondary Determinants Poznañ, Poland, Oc to ber 29 31, To whom cor re spon dence should be ad dressed: Institut de Biologie Moléculaire des Plantes, CNRS, 12 rue du Gen eral Zimmer, Strasbourg, France; tel: (33 3) ; fax: (33 3) Laurence.drouard@ibmp.u-strasbg.fr Abbreviation: mt, mitochondrial.

2 384 J. Fey and oth ers 2001 RNA ed it ing RNA ed it ing is usu ally de fined as a mod i fi ca - tion of the RNA pri mary se quence, thus caus - ing a divergence from the cod ing DNA se - quence. Rather than an un usual phe nom e non re stricted to a few ge netic sys tems, RNA ed it - ing ap pears more and more to be an es sen tial step in gene ex pres sion in a wide va ri ety of or - gan isms such as protists, an i mals and plants, and in dif fer ent compartments such as mito - chon dria and chloroplasts (re viewed by Smith et al., 1997). This was again re cently illus - trated when ed it ing of an RNA from HIV was de scribed by Bourara et al. (2000). For each type of RNA ed it ing the same ques - tions have to be con sid ered: How did RNA ed - iting appear and evolve? How do nu cle o tide de le tions and in ser tions or nu cle o tide con ver - sions oc cur? What is the func tion of RNA ed it - ing? RNA ed it ing in plant mi to chon dria In plants, a sim i lar kind of RNA ed it ing, con - sist ing in C to U con ver sions, has been iden ti - fied in both chloroplasts and mi to chon dria (reviewed by Maier et al., 1996). One of the striking differences, however, is the number of ed it ing sites per genome. In a chloroplast ge nome only a few ed it ing sites can be iden ti - fied. In plant mitochondria, studies based on sin gle tran script led to the pre dic tion that sev - eral hun dreds of ed it ing sites could be pres ent in a genome. This was re cently confirmed by Giegé & Brennicke (1999) who found 441 ed it - ing sites in tran scripts cov er ing the whole mi - tochondrial genome of Arabidopsis thaliana. Editing has been observed in all the ma jor groups of land plants, but not in al gae (Hiesel et al., 1994; Malek et al., 1996). A hy poth e sis to ex plain how RNA ed it ing ap - peared and was main tained in plant orga - nelles is to con sider that the fac tor(s) re spon - sible for this nu cle o tide conversion first de - rived from another RNA pro cess ing activity. Following this event, mutations were con - served in the genome while be ing compen - sated at the RNA level. This idea orig i nates es - sen tially from the fact that editing often makes it possible to main tain sequence con - servation at the protein level. The ques tion of how RNA ed it ing oc curs has only partially been an swered. Yu & Schuster (1995) were able to dem on strate that editing results from the deamination of a C residue into U, rather than nu cle o tide re place ment, but un til now at tempts to identify a nu clear gene coding for the cytidine deaminase in - volved have been un suc cess ful (Faivre- Nitschke et al., 2000). An other ma jor as pect of the ed it ing mechanism remains com pletely un known in plant mi to chon dria: the deamination reaction has to be restricted to spe cific C res i dues and the cis- and/or trans-act ing factors in volved in the selection of these ed it ing sites still have to be iden ti fied. The func tion of ed it ing can vary. Most of the ed it ing sites are in the cod ing se quences of messenger RNAs, so that ed it ing re sults in a mod i fi ca tion at the pro tein level and of ten in - creases the con ser va tion of pro tein se quences be tween dif fer ent spe cies. How ever, the ques - tion of their ne ces sity should be ad dressed for each pro tein. The role of ed it ing in non-coding se quences is even more dif fi cult to iden tify, as the im por tance of 5 and 3 un trans lated re - gions of mitochondrial mrnas in mech a - nisms such as RNA sta bil ity, pro cess ing or deg ra da tion and trans la tion ini ti a tion re - mains poorly documented. However, a study of an ed it ing event in an intron showed that it was a pre req ui site to splic ing (Börner et al., 1995). Al though ed it ing mainly af fects mrnas, ed - iting sites have also been identified in three different plant mi to chon drial trnas. In this re port, we sum ma rize the re sults ob tained on the ed it ing of these three trnas.

3 Vol. 48 Editing of plant mi to chon drial RNAs 385 trna EDITING IN PLANT MITOCHONDRIA The three ed ited trnas In dicot mi to chon dria a C to U ed it ing event cor rects a C:A mis match into a U:A base-pair in the ac cep tor stem of trna Phe (GAA) (Binder et al., 1994; Maréchal-Drouard et al., 1993). In the mitochondria of the gym no - sperm Larix leptoeuropaea, three C to U con - ver sions re store U:A base-pairs in the accep - tor stem, D stem and anticodon stem, re spec - tively, of trna His (GUG) (Maréchal-Drouard et al., 1996b). The third ex am ple is the na tive trna Cys (GCA) expressed in dicot mi to chon - dria where a C28:U42 mis match is con verted into a U28:U42 non ca non i cal base-pair (Binder et al., 1994; Fey et al., 2000). These ed - iting sites are in di cated in Fig. 1. ed it ing event could be ob served (Schock et al., 1998) Thus, a mis match alone in a trna arm is not suf fi cient to lead to a specific editing event. Func tion of trna ed it ing In the case of both trna Phe and trna His, ed it ing of pre cur sor tran scripts was shown to be a prerequisite for 5 and 3 pro cess ing to generate mature trna (Maréchal-Drouard et al., 1996a, 1996b; Marchfelder et al., 1996, Kunzmann et al., 1998). These experiments were con ducted by in cu bat ing in vi tro syn the - sized RNA (cor re spond ing to a trna pre cur - sor tran script) in the pres ence of par tially pu - ri fied mi to chon drial pro teins con tain ing both RNase P and RNase Z pro cess ing activities (Fig. 2). In the case of mt trna Phe, since ed it - ing of the C4-A69 mispairing into the nor mal Fig ure 1. Editing sites in plant mi to chon drial trnas and their func tions. About the mechanism con fer ring spec i fic ity As ed it ing oc curs in dou ble-stranded re gions of these trnas and re stores base-pairing, it could be hy poth e sized that trna ed it ing is di - rected by the pres ence of a mis match in a trna arm. How ever, sim i lar mis matches are pres ent in trna Gly (GCC) and trna Ser (GCA) of pea and po tato mi to chon dria, but no U4-A69 base pair ap peared to pro mote ef fi - cient pro cess ing of the cor re spond ing pre cur - sor RNA in vi tro, we wondered whether the same ef fect would be ob tained when this mis - match is changed into an other base pair C4-G69. Although less efficiently, a ma ture trna-size prod uct was also ob tained when the C4-G69 in vi tro transcript of this mu tated gene was in cu bated in the pres ence of the mi -

4 386 J. Fey and oth ers 2001 to chon drial lysate, sug gest ing that proper fold ing of the trna pre cur sor is prob a bly re - quired for recognition by RNase P and/or other pro cess ing enzymes. More re cently we have stud ied the ed it ing of po tato mitochondrial trna Cys (Fey et al., 2000). In that case, in vi tro pro cess ing ex per i - ments showed no ev i dence that ed it ing was re - polymorpha, we first se quenced this gene from several plant spe cies. While the an ces tral U cod ing se quence was also found in a fern and in the Prespermaphyte Gingko biloba, two Cy - cads (also Prespermaphytes) and Magnolia grandiflora (belonging to the earliest dicot sub-class) ex hib ited a C in the same gene at po - sition 28. If this trna was not ed ited in the Fig ure 2. Editing is a pre req ui site for the pro cess ing of trna His and trna Phe pre cur sors. The up per part of the fig ure shows sche matic rep re sen ta tions of the gene con structs used as tem plates for the syn - the sis of A) trna His and B) trna Phe pre cur sors. Lower part: in vi tro-syn the sized la beled pre cur sor RNAs cor re - spond ing to the nonedited (NE) or ed ited (E) forms of A) trna His and B) trna Phe were in cu bated in the pres ence (+) or ab sence ( ) of a po tato mi to chon drial pro tein ex tract. Pro cessing prod ucts were an a lyzed on 15% polyacrylamide gels. quired for trna mat u ra tion. Other in vi tro as - says were de vel oped, but again ed it ing seemed to affect neither the aminoacylation of this trna, nor the addition of the 3 -CCA se quence. We then de cided to study the evo lu - tion of this trna sequence. Hav ing no ticed that the C to U ed it ing at po si tion 28 re stores the an ces tral se quence found in the mi to chon - drial trnc gene of the Bryophyte Marchantia two Cy cads, it would have sug gested that this ed it ing site is not es sen tial. On the con trary, we found that trna Cys is also edited in the two Cycads. As this study clearly in di cated that ed it ing has al lowed the con ser va tion of a U res i due at po si tion 28 dur ing evo lu tion, we re con sid ered the im por tance of this trna ed - it ing. The iden ti fi ca tion of ed it ing sites is usu - ally based on reverse tran scrip tion of trna

5 Vol. 48 Editing of plant mi to chon drial RNAs 387 and se quenc ing of the resulting cdna. To check the in vivo se quence of trna Cys, we pu - rified it from po tato mitochondria and found that the residue at position 28 was not a uridine but rather a pseudouridine (Ψ). This re sult raises the ques tion whether the mech a - nism al low ing the con ver sion of a C residue into Ψ could still be considered as RNA ed it - ing. Only one sim i lar case has been pre vi ously de scribed in Esch e richia coli, where trna Ser (GGA) un der goes a con ver sion of the C20 res i - due into dihydrouridine (Motorin et al., 1996), prob a bly due to a two-step mech a nism in volv - ing first a C to U deamination (personal com - munication of H. Grosjean in Price & Gray, 1998). As C to U deamination and U to Ψ isomerization (by a Ψ synthase) af fect dif fer - ent at oms in the py rim i dine ring, we pro pose a two step model to explain how Ψ28 can be gen er ated in po tato mt trna Cys (Fig. 3). The 1999). More generally, these res i dues could rep re sent fa vor able sites for trna hydration, where a wa ter mol e cule can be in volved in hy - dro gen bonds with the phosphate back bone and with the Nl atom in the Ψ py rim i dine ring (Arnez & Steitz, 1994; Westhof & Moras, 1988). CONCLUSION We have come to a point where a more de - tailed un der stand ing of the func tion of ed it ing in these three trnas is re lated to other bi o log - ical pro cesses. In the case of trna Phe and trna His, the iden ti fi ca tion of the sequence and struc tural fea tures required for a trna precursor to be recognized as a substrate by RNase P and/or RNase Z will probably ex - plain the importance of these ed it ing events. Fig ure 3. A two-step model pro posed to gen er ate Ψ at po si tion 28 in po tato mt trna Cys. necessity for this editing event could be re - lated to the function of this Ψ28. It is gen er - ally assumed that Ψ res i dues pro vide a fine improvement of the trna struc ture. For in - stance, it has been shown that Ψ res i dues can stabilize RNA by improving RNA stacking (Davis, 1995). Stabilization of trna structure by a Ψ res i - due at po si tion 39 was also shown in the case of E. coli trna Phe (Davis & Poulter, 1991) and hu man trna Lys-3 (Durant & Da vis, In the case of trna Cys, more ex per i men tal ev - idence for the im por tance of Ψ res i dues in trnas will be required before one can con - clude on the necessity of this ed it ing site to stabilize the structure of this trna in vivo. Other editing sites in mt trnas might be identified in the fu ture, but our studies have already pro vided evidence that the im por - tance of RNA ed it ing is not re stricted to mes - senger RNAs in plant mitochondrial gene ex - pression, but also to struc tural RNAs.

6 388 J. Fey and oth ers 2001 REF ER ENCES Arnez, J. & Steitz, T. (1994) Crys tal struc ture of unmodified trna Gln complexed with gluta - minyl-trna synthetase and ATP suggests a possible role for pseudouridines in sta bi li za - tion of RNA struc ture. Bio chem is try 33, Binder, S., Marchfelder, A. & Brennicke, A. (1994) RNA ed it ing of trna Phe and trna Cys in mi to - chon dria of Oenothera berteriana is ini ti ated in precursor molecules. Mol. Gen. Genet. 244, Börner, G., Morl, M., Wissinger, B., Brennicke, A. & Schmelzer, C. (1995) RNA ed it ing of a group II intron in Oenothera as a prerequisite for splicing. Mol. Gen. Genet. 246, Bourara, K., Litvak, S. & Araya, A. (2000) Gen er a - tion of G-to-A and C-to-U changes in HIV-1 tran scripts by RNA editing. Sci ence 289, Da vis, D. (1995) Sta bi li za tion of RNA stack ing by pseudouridine. Nu cleic Acids Res. 23, Da vis, D. & Poulter, C.D. (1991) 1 H- 15 N NMR stud - ies of Esch e richia coli trna Phe from hist mu - tants: A struc tural role for pseudouridine. Bio - chem is try 30, Durant, P.C. & Da vis, D.R. (1999) Sta bi li za tion of the anticodon stem-loop of trna Lys,3 by an A + -C base-pair and by pseudouridine. J. Mol. Biol. 285, Faivre-Nitschke, S.-E., Grienenberger, J.-M. & Gualberto, J. (2000) A prokaryotic-type cyti - dine deaminase from Arabidopsis thaliana Gene ex pres sion and func tional char ac ter iza - tion. Eur. J. Biochem. 263, Fey, J., Tomita, K., Bergdoll, M. & Maréchal- Drouard, L. (2000) Evolutionary and func - tional as pects of C-to-U ed it ing at po si tion 28 of trna Cys (GCA) in plant mi to chon dria. RNA 6, Giegé, P. & Brennicke, A. (1999) RNA editing in Arabidopsis mitochondria effects 441 C to U changes in ORFs. Proc. Natl. Acad. Sci. U.S.A. 96, Hiesel, R., Combettes, B. & Brennicke, A. (1994) Evidence of RNA editing in mitochondria of all major groups of land plants except the bryophyta. Proc. Natl. Acad. Sci. U.S.A. 91, Kunzmann, A., Brennicke, A. & Marchfelder, A. (1998) 5 End maturation and RNA editing have to precede trna 3 processing in plant mi to chon dria. Proc. Natl. Acad. Sci. U.S.A. 95, Maier, R., Zeitz, P., Kossel, H., Bonnard, G., Gualberto, J. & Grienenberger, J.-M. (1996) RNA ed it ing in plant mi to chon dria and chloroplasts. Plant Mol. Biol. 32, Malek, O., Lättig, K., Hiesel, R., Brennicke, A. & Knoop, V. (1996) RNA editing in bryophytes and a molecular phytogeny of land plants. EMBO J. 15, Marchfelder, A., Brennicke, A. & Binder, S. (1996) RNA editing is re quired for ef fi cient ex ci sion of trna(phe) from precursors in plant mi to - chon dria. J. Biol. Chem. 271, Maréchal-Drouard, L., Cosset, A., Remade, C., Ramamonjisoa, D. & Dietrich, A. (1996a) A single editing event is a prerequisite for ef fi - cient pro cess ing of po tato mi to chon drial phenylalanine trna. Mol. Cell. Biol. 16, Maréchal-Drouard, L., Kumar, R., Remade, C. & Small, I. (1996b) RNA editing of larch mi to - chon drial trna(his) pre cur sors is a pre req ui - site for processing. Nucleic Acids Res. 24, Motorin, Y.A., Auxilien, S. & Grosjean, H. (1996) Potential Editing of Cytidine to Dihy dro - uridine in E. coli trna Ser (GGA). Proc. EMBO Work shop on RNA Editing, p. 75. Price, D. & Gray, M. (1998) Editing of trna; in Modification and Editing of RNA (Grosjean, H., ed.) pp , ASM Press, Washington. Schock, I., Maréchal-Drouard, L., Marchfelder, A. & Binder, S. (1998) Pro cessing of plant mi to -

7 Vol. 48 Editing of plant mi to chon drial RNAs 389 chon drial trna Gly and trna Ser (GCU) is in de - pend ent of RNA ed it ing. Mol. Gen. Genet. 257, Smith, H., Gott, J. & Hanson, M. (1997) A guide to RNA editing. RNA 3, Westhof, E. & Moras, D. (1988) Hydration of transfer RNA molecules: A crystallographic study. Biochimie 70, Yu, W. & Schuster, W. (1995) Evidence for a site-specific cytidine deamination re ac tion in - volved in C to U RNA editing of plant mi to - chon dria. J. Biol. Chem. 270,