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RNA EDITING IN TRYPANOSOMATID Annu. Rev. Microbiol. 1991.45:327-344. Downloaded from www.annualreviews.org by York University on 08/12/14. For personal use only.

MITOCHONDRIA K. Stuart Seattle Biomedical Research Institute, Seattle, Washington 98109-1651 KEY WORDS:

RNA processing, mitochondrion, Kinetoplastida, gene regulation

CONTENTS INTRODUCTION.....................................................................................

327

kDNA.................................................................................................... Ma.ti circle s ......................................................................................... Mi nicircle s . . ..... . . . . . . . ......... . . .. . . . . . . . . . . . .. . .. . . ... . . . . . . . ............. .. . . . . . . . ...... . . . . . . kDNA Mutati o ns . .... ... . .... . . .. . .. . .. . . .. . . . . .. . .. ... . . . . ... ..... . .. .. . . . . . . . . . . . . .. .. . . . . . . . .

328

RNA EDITING IS TRANSCRIPT AND TRANSCRIPT-REGION SPECIFIC ........... E diti ng of R e stricte d Reg i o ns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... Exte nsive E diti ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONSEQUENCES OF EDITING..................................................................

332 332 332 334

GUIDE RNA........................................................................................... g RNA Characteri stics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g RNA C o di ng Seque nce s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

335 335 335

329 329 331

PA RTIALL Y EDITED mRNAs.................................................................... Junctio ns. . . . . . . ..... .. . . . ... . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . ... . . . . . ... Mo dels of g RNA Usag e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The E diting Machine ry .......................................................................... DEVELOPMENTAL REGULATION.............................................................

340 340

PERSPECTIVE. . . . . ..... .... .. . . . . . . . . .... ...... .............. ................................... .....

341

337 337 339

INTRODUCTION Studies of mitochondrial gene organization and expression in kinetoplastid protozoans led to the discovery of a form of RNA processing called RNA 327

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STUART

editing. However, RNA editing in kinetoplastids is distinct from other types of RNA processing, some of which are also termed RNA editing (13, 43), and perhaps should have a distinctive designation such as K-RNA editing. K­ RNA editing is characterized by the addition and removal of uridines, prob­ ably employs a different mechanism, and has been the subject of several reviews (4, 5, 19, 20, 48, 51, 53, 54, 58, 66). RNA editing changes the coding sequence of mRNAs and can be so extensive that it produces most of the mRNA sequence. All RNA processing involves catalytic activity and recognition of the molecule and site that is processed. These activities include specific endonucleolytic cleavage as in rRNA maturation (27) or cleavage combined with RNA ligation to excise introns in RNA splicing (38). Other catalytic activities add nucleotides to RNA termini as in polyadenylation (37) or modify nucleotides as in the case of tRNA maturation (9). The recognition and catalytic activities may occur in a single molecule such as poly(A) polymerase and may even be inherent to the processed molecule as in the case of self-splicing RNA (14). Other RNA processing employs a macromolecular complex, such as the spliceosome, which is composed of several RNAs and proteins (36). The mechanism of RNA editing is not known but appears to employ small guide RNAs (gRNAs) to direct the editing, perhaps within a macromolecular complex.

kDNA The mitochondrial DNA of trypanosomatids, kinetoplast DNA (kDNA), is the hallmark of the order Kinetoplastida and has unusual characteristics that reflect the presence of RNA editing. The kDNA is composed of 20-50 maxicircles and 5000-10,000 minicircles. These numerous molecules are catenated together into a single DNA network that is located across from the base of the flagellum within the single large mitochondrion. Maxicircles are between 20 and 39 kb in size, depending on species, but are all identical within each network. The minicircles are between about 0.5 and 2.5 kb in size, depending on species, and are heterogeneous in sequence within each network; the total heterogeneity varies widely among species. The signifi­ cance of the organization of kDNA into a network is not known, but if related to editing, it may keep newly synthesized pre-mRNAs and gRNAs in physical proximity and enhance their opportunity for interactions that occur during RNA editing, or it may serve as a scaffold that binds other components of the editing machinery. The location of the kDNA network opposite the flagellar basal body may allow the basal body to function analogously to a centriole and segregate the duplicated kDNA at cell division. Several authors have reviewed the characteristics of kDNA (3, 42, 47, 52).

RNA EDITING

329

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Maxicircles Maxicircles encode mitochondrial rRNAs and components of the mitochon­ drial respiratory system, as do all other mitochondrial DNAs that have been examined (Table 1). However,some maxicircle genes are compact,overlap at their ends,contain frame-shifts,or have pronounced G vs C strand bias that reflects the presence of RNA editing. Several genes of the maxicircle encode proteins whose functions are not yet identified, in some cases because of extensive editing of the mRNA. These include the maxicircle unidentified reading frames (MURFs) 1 and 2 that are conserved among spccics, an open reading frame (ORF) adjacent to the ND7 gene,and six G vs C strand-biased sequences (CRI-6) whose positions but not sequences are conserved among species (31, 50). These genes may specify proteins encoded by the mitochondrial genomes of other organisms such as subunits of the ATPase (or ATP synthase) complex,the NADH dehydrogenase complex,or mitochon­ drial ribosomal proteins. More tantalizing, from the perspective of RNA editing, is the possibility that they may encode components of the RNA editing machinery. This would not be unprecedented since some mitochondria encode RNA processing activities such as maturase and self-splicing RNA. The maxicircle also contains a large variable region (VR) that differs in size and sequence among species and isolates (18, 41,57). The VR is primarily composed of repeated A + T-rich sequences,but the VR sequence adjacent to the rRNA genes is not repeated. The VR does not appear to encode proteins but encodes guide RNAs (gRNAs) as discussed below. Minicircles Minicircle function was a mystery until the discovery that these DNA mole." cules encode gRNAs (8,35,40, 60). Minicircles have approximately 120 bp of sequence that is conserved among minicircles within a single species and 12 bp that are conserved in minicircles of all kinetoplastid species. This conserved region is probably the origin of DNA replication because it is single stranded in replicating molecules and a potential RNA primer for replication has been identified (47). The size,organization,and total sequence diversity of minicircles vary among species. The significance of the variation in size and organization among species is not yet apparent. Among different species, however, minicircle diversity is correlated with the extent of RNA editing,as discussed below. Minicircle kinetic complexity is high ( 300 kb) in Trypano­ soma brucei in which extensive editing occurs and low ( 3 kb) in Leishmania tarentolae in which editing is less extensive (52). L. tarentolae contains six CR regions, which if extensively edited might exceed the gRNA coding capacity of the minicircles estimated from the kinetic complexity. These analyses do not provide a precise measure of the number of different minicir�



Table 1

Summary of RNA editing

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T. hllJCC'i U's editedd

sizeC genea st r andb

r!

612



3 64h

309h

247k

0

I-,ORF

1171

27

912

882

32

2

I,ORF

0 28

0

ORF

ND

NO

ND

22 5

39

0 0

0

I,ORF

NO

6431

1117

ND

NO

6391

1089

NO

251 h

ORF

NO

NO

191 h

NO

NO

1812

NO

acRl-6, G versus C strand biased gene sequences 1-6; ORF, open reading frame whose position but no t sequence is conserved among species; NDIA,5, and 7, NADH dehydrogenase subunits 1,4,5 and 7; COI,II and III, cytochrome

c

oxidase subunits I, II and III; CYb, apocytochrome b; A6, ATP synthase subunit 6; MURFI and 2, maxicircle unidentified reading frames I and 2 with

homol ogy conserved among species.

beading stra nd the same as ribosomal RNA (R) or opposite to ribosomal RNA

(0).

cSizes of edited (ed) and unedited (uned) mR NAs (not inc lu d ing the most Y A or U nucleotides). dNumber of Us added (add) and removed (rem) in fully edited mRNA compared to DNA. eDevelopmental regulation of editing where editing occurs in animal host (A), insect vector (V), or both animal fResult of editing creating

(1)

or

deleting (1-)

and insect (8).

initiation and tennination (T) codons and creating or extending an open reading frame (ORF).

gNE, no editing detected: +, editing detected but not fully characterized; P, editing is probable from G versus C strand bi as but not yet demonstrated; NO, gene sequence not yet completely determined, unable to assess extent of editing. hEstimated from G versus C strand bias and location of surrounding genes.

is' end from RNA sequ enci ng and 3' end from ORF analys i s (stop codon). JS' end from ORF a naly sis (start codon) and 3' end from eDNA. kBoth S' and 3 I end s from ORF analysis.

(/l



1198

NE 28

w w o

flisciculaLa

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RNA EDITING

331

cles and probably underestimate minicircle diversity; the 1 4. 5-kb VR of L. tarentolae may also encode numerous gRNAs. Minicircle sequence organization varies substantially among species. The minicircles of T. brucei and L. tarentolae are organized quite differently. T. brucei minicircles contain three gRNA coding cassettes between 18-bp in­ verted repeats in addition to the conserved region (33, 34). The 0.9-kb minicircles of L. tarentolae do not have this repeat organization and may only encode one gRNA per molecule (60). The l . 4-kb minicircles of Trypanosoma cruzi are organized into four-fold repeats (47), but RNA editing and gRNAs have not yet been reported in this spccics. The significancc of the variation in minicircle organization among species is not known, but the differences may reflect the substantial differences in the developmental regulation of the respiratory system. For example, cytochromes and Krebs cycle enzymes are present in L. tarentolae in both the animal and insect phases of the life cycle but are only present in the insect stages in T. brucei (65). kDNA Mutations Mutations that delete or modify kDNA occur spontaneously in kinetoplastids and can be induced with intercalating agents such as ethidium bromide and acridines (30, 52). Mutants that are devoid of kDNA or have substantial kDNA alterations are conditionally lethal in African trypanosomes; they survive in the animal host where mitochondrial respiration is not essential but not in the insect host where this respiration is essential. Trypanosoma evansi, Trypanosoma equiperdum, and Trypanosoma equinum are classified as sepa­ rate species but may be considered variants or mutants of T. brucei. These three species are morphologically indistinguishable from T. brucei but lack developmental stages in an insect host, probably because of partial or total kDNA deletions, and are transmitted venereally or mechanically. T. evansi lacks maxicircles but retains a single class of microheterogeneous minicircles, and mutants of T. evansi are devoid of kDNA ( 12). Similarly, T. equiperdum retains a single class of microheterogeneous minicircles but also retains maxicircles (26). Some sequences are deleted in these maxicircles. These mutants of African trypanosomes may be useful tools for the study of RNA editing. Intercalators can also induce kDNA loss in Leishmania spp. and other kinetoplastids, but these mutants are not viable for extended periods of time ( 61), presumably because of the requirement for mitochondrial respiration. Editing has been most extensively studied in T. brucei, L. tarentolae, and Crithidiafasciculata, but it also occurs in other Leishmania species, including those causing human disease (55). It may occur in all species that contain kDNA minicircles. However, although T. equiperdum contains minicircles that encode gRNAs, we find maxicircle transcripts but not edited RNA in this species (K. Stuart, unpublished data). This lack of editing may result from

332

STUART

loss of specific maxicircle and/or minicircle sequences. It is uncertain if RNA editing occurs in Bodo caudatus, which has atypical kDNA that contains 1 0-1 2 kb circles that may be the equivalent of minicircles (1 0). The absence of minicirdes is not an inherent constraint since some gRNAs are encoded in maxicircles (1 0). No evidence yet indicates K-RNA-type editing outside the mitochondrion of kinetoplastids.

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RNA EDITING IS TRANSCRIPT AND TRANSCRIPT-REGION SPECIFIC Editing of Restricted Regions Only certain mRNAs are edited, and the editing is restricted to specific regions of edited transcripts. The COIl genes of three kinetoplastid species contain homology to the COIl genes of other organisms in two different reading frames (1 7,2 9. 39). TheJinding of this frameshift set the stage for the discovery of RNA editing. Benne and colleagues (7) and others (2 4, 44) showed that COlI mRNA contains four Us that are not encoded in the COlI gene. These additional Us eliminate the frameshift in mRNA,implying that it is the edited mRNA that is translated and a candidate protein has been detected (46). Other mRNAs are edited near, but not at, their 5' terminus by the addition and in some cases deletion of Us. In most cases, the 5' editing creates an AUG in frame with an ORF that is homologous to those of other organisms. No AUG is encoded in the 5' region of the gene in most cases,and thus editing may function as a translational control mechanism because unedited transcripts would not be translated. Nevertheless, no editing has been detected in the 5' end of ND1 transcripts of T. brucei and C. Jasciculata that lack encoded AUGs; editing of ND7 transcripts in C.fasciculata shifts an AUG out of frame; and ND7 is edited in L. tarentolae, but no in-frame AUG is produced (45, 63, 64). This finding suggests that noncanonical initiation codons may be used as previously proposed (6) or that a small or undetected fraction of transcripts may contain the in-frame AUG. Many maxicirc1 e transcripts have been only partially sequenced but some have been entirely sequenced (summarized in Table 1 ). Editing has not been detected in the 5' region of the COl and II, ND l , 4, and 5,and MURFI transcripts; in rRNA of T. brucei and L. tarentolae; nor in rRNA of C. Jasciculata ( 54). Editing of NO 1 and N0 7 transcripts of C. fasciculata appears restricted to the regions indicated in Table 1 (62, 63), and we only detected RNA editing in the 5' regions of T. brucei CYb and L. tarentolae A6 transcripts (K. Stuart. un­ published results). Extensive Editing Perhaps most startling are the cases of extensive editing that occur in T. brucei se-

in which these transcripts are completely remodeled such that mRNA

'

---

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RNA EDITING

333

quence is determined as much by editing as it is by transcription. RNA editing changes all but the farthest 5' and 3' sequences of the COllI (21; K. Stuart, unpublished results) and A6 (8) transcripts of T. brucei. The added Us comprise 56 and 55% of the fully edited mRNA sequence, respectively, and editing also removes tens of encoded Us from these transcripts. The CRl , 2, 4, and 6 transcripts of T. brucei are edited to a similar extent (L. Simpson, unpublished data; K. Stuart, unpublished data). The 5' region of the A6 transcript of L. tarentolae is also extensively edited but to a lesser extent than in T. brucei (8). The most complex case to date is the ND7 transcript of T. brucei, which is completely edited except at the 5' and 3' terminal regions and in a 59-nucleotide region that separates two extensively and in­ dependently edited domains (35). All cases of editing examined create con­ tinuous ORFs, most create initiation codons, and many create termination codons. Interestingly, the 5' terminus of the two ND7 editing domains in T. brucei correspond in position to the regions of editing in L. tarentolae (45) and C. Jasciculata (64). Indeed, the same transcripts are edited in the same regions in all three species but the editing is more extensive in T. brucei. All of the extensively edited regions exhibit prominent G vs C strand bias in the DNA sequence such that the Gs are present in the transcripts (22, 31, 32, 50). Based on this observation, we predicted that transcripts from the six CR regions are extensively edited (58). This hypothesis was confirmed for four CR transcripts of T. brucei (K. Stuart, unpublished data). Edited CRI mRNA appears to encode a protein with two iron sulfur binding domains and thus may be a component of the respiratory chain, perhaps complex I (A. Sousa, unpublished data). Preliminary studies indicate the CR2, 4, and 6 transcripts of T. brucei (K. Stuart, unpublished data) and the CR5 transcript of L. tarentolae (L. Simpson, personal communication) are edited. Several cDNAs corresponding to a G vs C strand-biased region of T. brucei COl that were cloned using polymerase chain reaction are not edited, implying that this region of the RNA is not edited (K. Stuart, unpublished data). Thus, the strand bias is a useful but not infallible indicator of RNA editing. In all cases, at least 16 encoded 3' and at least 32 encoded 5' nuc1eotides are not edited in the final RNA, probably reflecting the requirements of gRNA utilization as discussed below. The definition of the fully edited mRNA sequence is somewhat operational. The fully edited mRNA sequence is the consensus of several eDNA sequences that are in agreement with sequence obtained through direct RNA sequencing. All these fully edited transcripts appear to be the functional mRNAs. They contain continuous ORFs that predict proteins that are homologous to proteins encoded by mRNAs that are edited less or not edited in other species. In addition, a protein of the predicted size is detected with antibodies prepared with a synthetic peptide predicted from the edited COIl mRNA of L. tar­ entolae (44). Small sequence variations occur in discrete regions of the fully

334

STUART

edited RNA sequence of some mRNAs as detected by cD NAs and/or RNA sequencing (8, 35). These variations may not affect protein function because they predict conservative replacements of a few amino acids or occur up­ stream of the initiation codon sequences (8, 35). These variations might reflect the use of multiple gRNAs (59) or some flexibility in the editing process.

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CONSEQUENCES OF EDITING Edited genes can be compact, especially in T. brucei in which editing is extensive. These genes are so encrypted that homology to related genes cannot be detected without knowing the edited sequence. The CR l-6 genes that are G vs C strand biased and conserved in position but not sequence among species are compact and may encode proteins that are highly homologous among kinetoplastids (but this possibility cannot be discerned from the D NA sequence) (50). The G vs C and A vs T strand bias and the general lack of Ts in the coding sequences also reflect the U addition and deletion and use of wobble base-pairing (see below) aspects of RNA editing. The predicted consequences of editing at the protein level are intriguing, although few studies have directly examined the protein products of edited mRNA. In most cases, unless noncanonical initiation codons are used, un­ edited versions of edited RNAs would produce no product because they lack an initiation codon. However, initiation codons occur in unedited transcripts of COIl in T. brucei, C.Jasciculata, andL. tarentolae; CYb in T. brucei; and ND7 in C. Jasciculata, and thus these transcripts could conceivably be translated, although their association with mitochondrial ribosomes has not been tested. Particularly intriguing is that editing of these three transcripts is developmentally regulated in T. brucei (23,24,35) raising the possibility that unedited transcripts may be translated to produce truncated proteins perhaps with modified functions. RNA editing is posttranscriptional and proceeds in the 3' to 5' direction. A variety of approaches have shown that unedited RNA is invariably present along with fully edited RNA, and partially edited RNA is abundant. Numer­ ous cD NAs, probably derived from RNA that was in the process of editing when isolated, have edited 3' and unedited 5' sequences (1, 16,21,35,58, 59). The presence of both edited and unedited sequences on the same mole­ cules is strong evidence that editing is posttranscriptional and indicates that editing proceeds in the 3' to 5' direction. The detection with probes for 5' edited sequences of transcripts that are larger and hence more edited than those with 3' probes reinforces this conclusion (35). In addition, PCR analy­ sis of total cellular RNA detects only partially edited RNA that is edited on the 3' and not the 5' side (1). The partially edited RNAs from exten-

RNA EDITING

335

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sively edited mRNAs are more abundant than the fully edited mRNA accord­ ing to Northern analysis results (1,8,21,35). This finding is not surprising given the numerous events that must occur during the editing of these molecules. Us are present in mRNA poly(A) tails (21, 25, 64). Both the positions and numbers of these U additions vary even in transcripts from the same gene, suggesting that their addition is not directed by gRNA. These Us may be added by the enzyme that adds Us during editing and/or by poly(A) polymerase. GUIDE RNA gRNA Characteristics The discovery of gRNA (10) revealed the likely repository of the edited sequence and provided substantial insight into the RNA editing process. The gRNAs (8, 10, 11, 35, 40) are small (�60 nucleotides) and are com­ plementary to the edited RNA sequence. The predicted complementarity at the 5' end of the gRNA with mRNA is primarily, if not entirely, Watson­ Crick base-pairing, while the remaining complementarity entails some wob­ ble (G-U) base-pairing. It is important to recognize that the wobble base­ pairing in gRNA-mRNA interactions indicates that gRNA is not functioning as a conventional template because gRNA Gs would specify Cs and gRNA Us would specify As; however, Us are specified during editing by Gs and As. The 5' Watson-Crick complementarity suggests that a duplex initially forms between mRNA and the 5' region of the gRNA. Approximately 15 noncoded Us are added to the 3' end of the gRNAs. These Us may stabilize the gRNA-mRNA association during editing (11). An alternate possibility is that the 3' Us create a secondary structure that leaves the 5' end of the gRNA single stranded and hence available to duplex with mRNA (U. Goeringer, personal communication). Of the numerous gRNAs identified directly or as coding sequences, only those for the farthest 3' and hence initial region of an editing domain can form a duplex between the gRNA 5' end and unedited RNA (8, 10, 35; K. Stuart, unpublished data). The other gRNAs must be edited to provide the sequence with which the 5' region of the gRNA can duplex, which suggests that the order in which gRNAs are utilized is de­ termined by the ability of their 5' ends to form a duplex with the mRNA. This suggestion explains the overall 3' to 5' direction of editing and provides a mechanism for the order of gRNA utilization. gRNA Coding Sequences The gRNAs are encoded in both maxicircles and minicircles, but the number of gRNA coding sequences and their distribution among the maxicircles and minicircles differs between T. brucei and L. tarentolae. Most gRNAs identi-

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336

STUART

fied to date in L. tarentolae are encoded in the maxicircle (10),but some are also encoded in minicircles (60; K. Stuart, unpublished data). All of the known gRNA coding sequences in T. brucei are present in minicircles (8,3 5, 40). Potential gRNA coding sequences have been identified in the maxicircle ofT. brucei, but the gRNAs have not been demonstrated. Some gRNA coding sequences identified in the maxicirc1e of L. tarentolae do not appear to be encoded in the maxicircle of T. brucei. For example, the gRNA genes for CYb are not in T. bruce; maxicircles, at least not at the same position as in L. tarentolae (K. Stuart,unpublished data). This absence may reflect either the developmental regulation of editing that occurs in T. brucei and not L. tarentolae or the more extensive editing in T. brucei. T. brucei kDNA has adequate coding capacity for all gRNAs needed for all editing detected and predicted in this species. The total number of Us known to be added/removed by editing in T. brucei and L. tarentolae is 1870/208 and 256/24,respectively (Table 1). These changes approximately double the sizes of the edited regions. Net increases of about 750 and 1000 Us in T. brucei and L. tarentolae, respectively, are required to account for the remaining editing predicted if the CR transcripts are extensively edited in both species. The large minicircle diversity of T. brucei can easily encode sufficient gRNAs to direct this editing. Approximately 175 gRNAs, each adding 15 Us, could account for all the observed and predicted editing in T. bruce;, allowing about 5 copies of each gRNA gene, since at least 300 different minicircles each encode 3 gRNAs. The repetition frequency could be higher because this minicircle complexity estimate is based on minimal minicirc1e sequence diversity. The lower minicircle diversity of L. tarentolae at first glance cannot account for the approximately 80 gRNAs estimated for the anticipated editing in this species. Only single gRNA coding sequences have been found on each L. tarentolae minicircle to date. However, editing of L. tarentolae CR RNAs may be less extensive than anticipated, thus requiring fewer gRNAs. Minicir­ c1e diversity may also be greater than estimated by renaturation kinetic and other analyses, and the -14-kb VR of L. tarentolae may encode several gRNAs. Thus,L. tarentolae can encode all its gRNAs in kDNA. Multiple gRNAs with different sequences that can specify the same editing, because of wobble base-pairing,have been detected (K. Stuart,unpublished data). These occur in minicirc1es from two different stocks of T. bruce;, which indicates minicirc1e divergence and/or redundancy of gRNA coding sequences. Thus,minicircle abundance and diversity,especially in T. brucei reflect gRNA gene copy number, different gRNAs that specify the same edited sequence,and different gRNAs that specify editing to similar but not identical sequences, i.e. conservative differences. Little is known about the transcription of maxicirc1es or minicirc1es. No promoters have been identified in either molecule, but both strands of both ,

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RNA EDITING

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molecules are transcribed. Detection of transcripts that span more than one maxicircle gene in steady-state RNA indicates that polycistronic maxicircle transcripts occur with some abundance (56),but no polycistronic minicircle transcripts have been reported. All T. brucei minicircle gRNAs are encoded in the same minicircle DNA strand and between inverted 18-bp repeats, includ­ ing cases in which mUltiple gRNAs are encoded in the same minicircle (8, 35, 40; K. Stuart, unpublished data). This strand is opposite to that which encodes transcripts from near the conserved sequence and opposite to the strand encoding gRNAs in L. tarentolae (K. Stuart, unpublished data). Several transcripts from maxicircles and minicircles can be radiolabeled using guanyl­ transferase,indicating that the most-5' nucleotide has a 5' di- or triphosphate and suggesting that these are primary transcripts (11,40,49). Thus,maxicir­ cles and minicircles may have multiple promoters,but the compactness of the genes and the presence of apparent polycistronic transcripts suggest that each maxicirc1e gene does not have its own promoter. Polycistronic precursors may be processed in the mitochondrion of kinetoplastids as in other organisms. The inverted 18-bp repeats that flank gRNA coding sequences in T. brucei may have a role in transcript processing. Further study is needed to identify promoters and primary transcripts and to determine if kDNA transcripts undergo processing in addition to RNA editing and polyadenylation. PARTIALLY EDITED mRNAs Junctions Several studies have examined partially edited mRNAs in detail (1, 16,21, 35,58,59; D. J. Koslowsky,unpublished data) because they are likely to be molecules undergoing the process of editing and thus may provide insight into this process. All partially edited RNAs,or their editing domains,are edited on their 3' but not their 5' side. Their unedited sequence is identical to thc gene sequence,and the fully edited sequence is identical among edited cDNAs and to the RNA sequence determined directly from RNA sequencing. In some molecules, the RNA sequence switches directly from edited to unedited sequence. Importantly,the great majority of molecules contains a short region of edited sequence that does not match the fully edited RNA sequence at the junction of the edited and unedited sequence (Figure 1). The simplest in­ terpretation is that the junction is the region of active editing. Most partially edited RNAs are probably end products of an editing cycle at a site rather than of editing of reaction intermediates. Such intermediates with 5' Us have been observed as cDNAs (1,23) and may be products of the cleavage associated with editing, or some may reflect cDNA cloning artifacts (64). The junctions (sequences that are edited but do not match fully edited RNA) vary in size,and their positions often overlap. Junction size varies from

338

STUART

A DNA RNA

TGTCC A GUCC A

CA GC A CCC GTTTC A CA GC AuCCC G C A

G G

C C

A A

C C

A G A uG

TTG G

G A uG uuuuA

G G uG uuG

5M10 5M21

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L303

B DNA RNA

AA GG G G G A G GCA A GCG A AA G ATTTTGAA ACTTTCCG AG GuGCAuuuAuGCGuuuAuuAAuuGuA****GAAuuuAC***CCGuAGuuuuAAuGGuuuGuuGuGuA

gND7-398 Figure 1 edited

1111: : III: 11111111 : : I

AAAUGUGCAGAUAAUUAAUGU

1111: III

II: 1111111111 :

CUUAGAU�UAUCAAAAUUAU

(A) Junctions of partially edited RNA (boxed) compared to gene (DNA) and fully

(RNA) sequences. Shaded regions in the junctions match the fully edited sequence while

unshaded regions do not.

(8) A gRNA sequence compared to fully edited (RNA) and unedited

(DNA) sequences. The underlined site contains, 3' to a C, Ts that are removed by editing.

3 to 109 nuc1eotides. The sizes of some junctions exceed the size of any gRNA detected to date, implying that a junction may represent the utilization of more than one gRNA or that some gRNAs may be large. Numerous junctions have been found for limited regions of editing such as the 5' regions of CYb and COllI (16, 58, 59; K. Stuart, unpublished data). Numerous junctions have also been observed for extensively edited RNAs. Some of these junctions may represent molecules that are not destined to become fully edited, but some, perhaps many and possibly all, must be edited to the final mRNA sequence. This observation indicates an extensive diversity of partial­ ly edited molecules, suggesting that many events must transpire in the editing of mRNAs, espccially in thc extensively edited RNAs. The relationship between junctions and gRNAs, where both have been characterized, has not been determined. The 3' boundary of most junctions is usually identical to that of one or more other junctions, but the 3' boundaries can occur at adjacent editing sites (see Figure 1). The junctions contain numerous sites with the same number of Us (16,35, 58, 59; K. Stuart, unpublished data) as the final RNA sequence interspersed with sites that have either more or fewer Us than present in the final RNA and thus require further editing (Figure 1 ). Commonly, many sites contain Us where none are present in unedited or fully edited mRNA. This finding suggests that most, if not all, sites in an editing domain undergo editing to achieve the final edited sequence. It also suggests that Us are added to many

RNA EDITING

339

sites and subsequently removed, implying that sites are reedited. The distribu­ tion of the sites requiring further editing among those that match the final edited sequence does not have a regular pattern progressing precisely 3' to 5' . Thus, the characteristics of junctions may reflect gRNA diversity, overlap­ ping gRNAs, and/or an editing process that does not proceed precisely 3' to 5' .

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Models of gRNA Usage No studies have yet been published that demonstrate that gRNAs determine the edited sequence. However, the existence of this class of small RNAs that matches the edited sequence strongly indicates this role. Analyses of partially edited RNAs led to two different hypotheses to explain how gRNAs are used to produce the edited sequence. One hypothesis (0), which I term mismatch recognition, proposes rounds of (a) cleavage of the mRNA at the mismatch immediately 5' (mRNA perspective) of the gRNA-mRNA duplex, (b) U addition or deletion to eliminate the mismatch, and (c) religation; this process continues until the mRNA region matches the gRNA. The other hypothesis (16), which I term match p rotection, proposes random U addition and dele­ tion within a restricted region of the mRNA until the gRNA and mRNA match, thus protecting the mRNA from further editing. Probably neither model is correct exactly as proposed. For example, neither model can ade­ quately explain how to accommodate sequences of the form 5' CU 3' in mRNA where Us are either deleted or added 3' to the C. For example, the gRNA in Figure I contains a G at a position where three encoded Us are deleted, which would prevent recognition of the mismatch according to the first model and could produce inappropriate protection of a sequence accord­ ing to the second model. The mismatch recognition model does not fully explain the distribution of sites that require further editing in junctions of numerous partially edited mRNAs, although utilization of gRNAs that do not produce the final RNA sequence may explain some of these molecules. The match protection model does not provide insight into the enzymology of the process. Thus, substantial modification of these models or an alternate model is required. Editing may proceed precisely 3' to 5' but use a variety of gRNAs covering the same region, thus explaining the characteristics of the junction sequences. Another possibility, which does not preclude multiple gRNAs, is that editing does not proceed precisely 3' to 5'. The secondary structures assumed by gRNA and/or mRNA could explain the order of editing site selection because sites could be presented to the catalytic machinery in a non-3' -to-5' order. This mechanism would allow for reediting of sites using the same gRNA. The progressive change of the mRNA sequence during editing might result in a succession of secondary structures that could be very specific. Studies of the means of gRNA utilization would be aided by an in vitro editing system.

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STUART

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The Editing Machinery The study of the mechanism of RNA editing is at such an early stage that no molecules that participate in the process have been identified, other than gRNA and mRNA. Other molecules probably catalyze this intricate process, and such molecules have been suggested to occur as a macromolecular complex, provisionally termed the editosome, although its existence has not been demonstrated. Nevertheless, by analogy to RNA splicing,RNA editing may be catalyzed by a macromolecular complex perhaps composed of RNA andlor protein. The precise reactions and the sequence in which they occur during editing have not been identified. Endoribonuclease, uridine addition and deletion, and RNA ligase activities have been predicted to account for addition and deletion of Us within mRNAs (4, 21,23, 51,53). Candidate 3' terminal uridylyltransferase (TUTase) and RNA ligase activities have been identified in lysates of whole cells (67) and isolated mitochondria (2, 28), but these activities have not been shown to be associated with editing. Chimeric molecules of gRNAs,joined by their 3' ends to internal regions of edited ND7 and COIl mRNAs of L. tarentolae (68) and ND7 and CR6 mRNAs of T. brucei (K. Stuart, unpublished data), document the association between gRNA and mRNA and suggest that uridines may be added and removed by transesterification as also suggested by Cech ( 69). The gRNAs may have a role in the recognition of the mRNA and region to be edited. The complex may contain the catalytic molecules and other molecules that function in stabilization and RNA translocation. The number of components of such a complex could be as numerous as those of the spliceosome, and their genes may reside in nuclear and/or mitochondrial DNA. A nuclear location of some editosome genes seems possible since most of the kDNA genes are identified. This possibility is intriguing because the products of such genes might have a role in processing nuclear transcripts. DEVELOPMENTAL REGULATION RNA editing is developmentally regulated in T. brucei, indicating the exis­ tence of a regulatory system that controls the editing activity. Interestingly, the editing is transcript specific: some transcripts are constitutively edited; others are only edited in the animal stage of the life cycle; while others are only edited in the insect stage of the life cycle. In the animal host, slender bloodstream form (Sl-BF) T. brucei have no cytochromes nor a Krebs cycle; they produce ATP by glycolysis that occurs at a prodigious rate in the glycosome, an organelle that contains glycolytic enzymes. The nondividing stumpy bloodstream forms (St-BF), a less discrete stage than the Sl-BF, have a somewhat enlarged mitochondrion and an additional mitochondrial oxido­ reductive enzyme activity that has been detected only through cytochemical staining with tetrazolium dyes. The insect procyclic forms (PF) have a full

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RNA EDITING

341

complement of cytochromes,a Krebs cycle,and produce ATP primarily by mitochondrial oxidative phosphorylation (65). L. tarentolae and C.fascicula­ ta have less complex life cycles and do not regulate their mitochondrial composition in such a dramatic fashion. The COllI, A6, and MURF2 mRNAs are edited in both Sl-BF and PF, indicating that editing activity is present in both life-cycle stages (8,21, 24). The CYb and COIl mRNAs are edited in PF and St-BF but not in Sl-BF (23, 24). The abundance of edited transcripts is less in St-BF than in PF. The actual amount appears to differ among stocks, presumably reflecting differ­ ences among stocks and among cell populations of the same stock in the production of these life-cycle stages. Editing of these transcripts in PF is consistent with the greater mitochondrial activity in PF and St-BF than in Sl-BF (K. Stuart, unpublished data). The CRI transcripts are edited in Sl-BF but not PF. Although the function of the CRI protein is unknown, its iron-sulfur center motif suggests that it may be part of the respiratory chain, perhaps complex I. The significance of its editing in Sl-BF and not PF is unclear. However,ND5 transcripts are more abundant in Sl-BF than in PF (32), indicating that components of respiratory complex I may be more abundant in BF than PF. The 5' domain of ND7 is edited in both BF and PF but the larger 3' domain is preferentially edited in BF (35). Thus,editing is developmentally regulated in a transcript-specific fashion. This regulation is not controlled at the gRNA-abundance level because gRNAs that have been identified for developmentally regulated regions are present in similar abund­ ance in life-cycle stages whether or not they are edited (K. Stuart, un­ published data). Hence, some system must control editing during the life cycle, but how it functions is unknown. PERSPECTIVE The raison d'etre and the selective value of such a baroque process as RNA editing are not intuitively obvious. However, editing does provide an addi­ tional genetic regulatory capability. It could be an evolutionary residuum of processes that originated in the era of the RNA genome but no longer exists or is not extensively utilized in many organisms. While it is distinct from other types of RNA processing,some activities appear superficially similar and thus may have an evolutionary origin common to these other types. RNA editing appears superficially similar to RNA splicing because both entail recognition of specific regions of transcripts, cleavage, and religation. RNA editing may be catalyzed by a complex analogous to the spliceosome. The RNA editing reported in Physarum polycephalum resembles RNA editing in kinetoplastids since it occurs in the mitochondrion and primarily involves addition of single Cs (D. Miller,personal communication). Elucidation of gRNA interactions with mRNA may extend our knowledge of the importance of RNA in-

342

STUART

teractions beyond their role in splicing ( 38). These interactions may include the postulated role of small RNAs in poly(A) site selection (15) and the significance of the recently discovered enzymes that specifically modify double-stranded RNA (13). RNA editing may be the harbinger of other as yet undiscovered RNA processing phenomena. ACKNOWLEDGMENTS

thank Drs. P. Myler, M. Parsons, and J. Feagin for helpful discussions and critical reading of the manuscript. This work received support from NIH AI14102 and GM42188. The author is a Burroughs-Wellcome Scholar in Molecular Parasitology.

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I

Literature Cited I. Abraham, J. M., Feagin, J. E., Stuart,

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K. 1988. Characterization of cyto­ chrome c oxidase III transcripts that are edited only in the 3' region. Cell 55:267-72 Bakalara, N., Simpson, A. M., Simp­ son, L. 1989. The Leishmania kineto­ plast-mitochondrion contains terminal uridylyltransferase and RNA ligase ac­ tivities. J. BioI. Chem. 264:18679-86 Benne, R. 1985. Mitochondrial genes in trypanosomes. Trends Genet. 1:11 7-21 Benne, R. 1989. RNA editing in tryp­ anosome mitochondria. Biochim. Bio­ phys. Acta 1007:131-39 Benne, R. 1990. RNA editing in try­ panosomes: Is there a message? Trends Genet. 6:177-81 Benne, R., van den Burg, J., Brakenhoff, 1., deVries, B., Neder.lof, P., et al. 1985. Mitochondrial genes in trypanosomes: abnormal initiator trip­ lets, a conserved frameshift in the gene for cytochrome oxidase subunit II and evidence for a novel mechanism of gene expression. In Achievements and Per­ spectives of Mitochondrial Research, ed. E. Quagliariello, E. C. Slater, F. Paimierei, C. Saccone, A. M. Kroon, pp. 325-36. Amsterdam: Elsevier Benne, R., van den Burg, J., Brakenhoff, 1. P., Sioof, P., Van Boom, 1. H., et al. 1986. Major transcript of the frameshifted coxIl gene from trypano­ some mitochondria contains four nucleo­ tides that are not encoded in the DNA. Cel l 46:819-26 Bhat, G. J., Koslowsky, D. J., Feagin, J. E., Smiley, B. L., Stuart, K. 1990. An extensively edited mitochondrial transcript in kinetop1astids encodes a protein homologous to ATPase subunit 6. Cell 61:885-94 Bjork, G. R., Erickson, J. U., Gustafs-

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son, C. E. D., Hagervall, T. G., Jons­ son, Y. H., et al. 1989. Transfer RNA modification. Annu. Rev. Biochem. 58: 263-87 Blum, B., Bakalara, N., Simpson, L. 1990. A model for RNA editing in ki­ netoplastid mitochondria: "guide" RNA molecules transcribed from maxicircle DNA provide the edited information. Cell 60:189-98 Blum, B., Simpson, L. 1990. Guide RNAs in kinetoplastid mitochondria have a nonencoded 3' oligo(U) tail in­ volved in recognition of the preedited region. Cell 62:391-97 Borst, P., Fase-Fowler, F., Gibson, W. C. 1987. Kinetoplast DNA of Trypano­ soma evansi. Mol. Biochem. Parasitol. 23:31-38 Cattaneo, R. 1990. Messenger RNA editing and the genetic code. Experientia 46:1142-48 Cech, T. R. 1990. Self-splicing of group I introns. Annu. Rev. Biochem. 59:54368 Cutten, M., Gick, 0., Vasserut, A., Schaffner, G., Birnstic1, M. L. 1988. Specific contacts between mammalian U7 SnRNA and histone precursor RNA are indispensible for the in vitro 3' RNA processing reaction. EMBO. J. 7:8018 Decker, C. J., Sollner-Webb, B. 1990. RNA editing involves indiscriminate U changes throughout precisely defined editing domains. Cell 61:1001-11 de la Cruz, V. F., Neckelman, N., Simpson, L. 1984. Sequences of six genes and several open reading frames in the kinetoplast maxicircle DNA of Leishmania tarentolae. J. Bioi. Chem. 259:15136-47 de Vries, B. F., Mulder, E., Braken­ hoff, J. P., Sloof, P., Benne, R. 1988.

RNA EDITING

19. 20.

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21.

22.

The variable region of the Trypano soma brucei kinetoplast maxicircle: sequenc

RNA editing in trypanosomatid mitochondria.

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