RNA editing Barbara The Johns Hopkins

Sollner-Webb

University,

Baltimore,

Maryland,

USA

Since its discovery, RNA editing in kinetoplastid mitochondria has proven a fascinating topic of study, and the last one and a half years have witnessed enormous advances in our understanding of this unprecedented form of RNA processing. The information flow in this RNA editing, once considered a candidate for defying the central dogma, is now known to conform to the DNA-to-RNA-to-protein paradigm, with the novel feature that the sequence of an edited region is not actually present in any DNA segment, but instead derives by a novel micro-interdigitating of information encoded in multiple DNA regions. Current Opinion in Cell Biology 1991, 3:105&1061 Introduction The term ‘RNA editing’ is frequently used to refer to the RNA processing that involves post-transcriptional insertion (and occasional deletion) of U residues in mitochondrial transcripts of kinetoplastid organisms, as well as the less understood insertion of C residues (and occasionally A and U residues) into mitochondrial transcripts of Pbparum. These processing events are the subject of this review. In its broader definition, the term ‘RNA editing’ encompasses all processing events in which the primary sequence of an RNA is altered in ways other than the generally recognized 5’ capping, 3’ polyadenylation, intron removal, 3’ CCA addition, or tRNA- and rRNA-like nucleotide modifications. Such events include two C-to-U base changes in the mammalian nuclearencoded apolipoprotein B mRNA ([1**,2*] and references therein), multiple C-to-U and occasional U-to-C changes in the mitochondrial (and less frequently chloroplast) RNAs of plants [3,4,5*,6*], and A-to-l base changes in nuclear-encoded vertebrate RNAs that can result from an RNA unwinding activity [7]; the definition can even be extended to encompass specific nucleotide insertion in homo-oligomer regions that can occur during paramyxoviral RNA production ( reviewed in [8.,9*] 1. These latter forms of editing will not be covered in this review.

Identification

of kinetoplastid

RNA editing

Kinetoplastids are parasitic protozoa classified by the presence of a kinetoplast [lo] - a concatenated network of DNA molecules, contained within their single large mitochondrion, consisting of 50 ‘maxicircles’ (most closely corresponding to the mitochondrial genome of other organisms) and up to tens of thousands of ‘minicircles’ (numerous classes of w I-kb DNA molecules which will

be discussed further below). The most popularly studied kinetoplastids are T?ypanosoma brucei, Leishamu tarc~toluc~and Crithidia fasciculuta. These primitive eukary otes are well known for exhibiting numerous molecular and biochemical peculiarities, including several mitochondrial genes that lack their AUG initiation codons or contain internal frameshifts, and an apparent absence of other normal mitochondrial genes (e.g., [ I1 I and references therein ). The process by which functional mRNA is derived from such abnormal mitochondrial genes constitutes what is generally regarded as the most exciting aspect of these interesting organisms discovered to date: RNA editing. Kinetoplastid RNA editing was lirst reported in 1986 b) Benne and colleagues [ 121 who showed that the sequence of cytochrome oxidase subunit 11 (COII) mRNA in 7: hrucei and C Jirsciculuta differs from its corresponding gene by having a total of four Ll residues inserted at three adjacent intemucleotide sites, thereby correcting a genomically encoded frameshift within the CO11 coding region. Subsequently, other kinetoplastid mitochondrial transcripts were shown to similarly correct frameshifts as well as to alter sequence and gain AUG initiation codons by such U insertion and by less frequent U deletion ( [ 13,141 and reviewed in [ 15,161). A number of transcripts have also been shown to edit to different extents in the bloodstream and the procyclic lifeforms of 7: hrrrcei, indicating a developmental regulation [ 17,181. An amazing example of RNA editing that riveted the attention of biologists worldwide was reported by Stuart’s group in 1988 [ 191. This is the CO111RNA of 7: brucei, where over 50% of the + 103 nucleotides of the mature mRNA are U residues that were added by editing at more than 150 separate sites, while at least 19 II residues were deleted from at least nine separate sites. This massive editing explains why a CO111gene had not been detected previously in T. brtrcei. Furthermore, rather than CO111

Abbreviations COII-cytochrome

1056

@ Current

oxidase

Biology

subunit

II; gRNA-guide

Ltd ISSN 0955-0674

RNA.

RNA editing

being unique in its extent of editing, current work indicates that 7: brucei has about eight other such massively edited transcripts, and that C: fmciculutu and L furento lae each have around six (e.g. [ 20=,21=] >.

The mechanism

of kinetoplastid

RNA editing

What directs these editing modifications? RNA analysis suggested that editing proceeds in a 3’-5’ direction along the transcript [ 221, and incorporation studies in isolated mitochondria confirmed that editing is a post-transcriptional process [23*]. No consensus sequence or consensus structure was detected for the editing sites, however, and no contiguous DNA or RNA template for edited sequence could be found (reviewed in [8*,15,16], but see also [24*] >. Last year Simpson and colleagues [ 25**] made a major advance by identifying members of a family of -4O-80.nucleotide-long RNA molecules that they termed ‘guide RNAS’(gRNAs). These gRNAs are encoded in bcth maxicircle [25**] and minicircle [26**,27*-31.1 DNA and they contain a segment complementary to -15-55 residues of the edited version of a transcript. Thus, they presumably act to guide the insertion and deletion of U residues in the mRNA [25*-l, with multiple gRNAs required for larger editing regions. The gRNAs are unlikely to function as templates for synthesis of an edited version of the mRNA region, because their complementarity involves G-U and occasional C-A, as well as A-U and G-C, base-pairing [ 25**,30*]. A 5-15. nucleotide sequence at the 5’ end of the gRNA’s complementary region has the sequence to allow its annealing with the editing substrate mRNA adjacent to the region to be edited; this ‘anchor segment’ could facilitate selective association of the not-yet-edited mRNA with the correct gRNA. At their 3’ ends, gRNA molecules have a tract of -5-15 post-transcriptionally added U residues that could serve as an additional anchor sequence [ 26.01 and/or as the U donor for editing (see below). Using gRNAs to guide editing modifications, one might envision editing to be an orderly process, proceeding 3’-5’, one nucleotide at a time, along the respective gRNA [ 25**,32**]. When cDNAs for partially edited L fure?ztolue mRNAs were cloned and analyzed, many were indeed of this type [ 32**]. However, other trypanosomatid cDNAs were not [ 15,32*0,33,34**,35*]. Sollner-Webb’s laboratory [34**] reported that almost aff pattktffy edited cDNAs from T. brucei were surprisingly complex, containing a (often large) region of aberrant sequence between the 3’.edited and the 5’.unedited portions. These aberrant sequences contain wrong numbers of U residues at numerous internucleotide positions, equally frequently at sites that ultimately will and will not show editing in the mature mRNA. This aberrant editing may reflect an inherent variability in the U insertion mechanism. One possibiliry is that gRNAs function not to direct the U additions precisely, but rather to base-pair with and thereby preserve sites that became correctly edited [34*-l. There is also strong evidence that some, and POSsibly all, unexpected editings result from using a gRNA

Sollner-Webb

‘incorrectly’ [ 32**], possibly in an iterative and not a precisely 3’-5’ fashion [36**]. It is striking that aberrant editing occurs only within the domain of the mRNA that requires editing and that it starts precisely at the 3’ end of the editing domain [ 34**]. This implies accurate recognition of the location in the pre-edited mRNA where editing modifications are to begin, regardless of the nature of the subsequent editing modifications. Recent studies (M Harris, C Decker, S Hajduk and B Sollner-Webb, unpublished data) suggest that regions where editing will initiate can be specifically recognized and cleaved by an endonuclease present in mitochondrial extracts of 7: brztcei Furthermore, additional studies by this group reveal that a heterologous single-strandspecific nuclease, mung bean nuclease, shows the same cleavage pattern. This suggests that regions where editing initiates can be recognized by the secondary structure of the substrate RNA and, surprisingly, that this recognition can occur independently of gRNAs. The identification of kinetoplast mitochondrial RNA ligase and terminal U transferase activities ( [37] and references therein), together with results described above, have led to models for editing in which the substrate RNA is enzymatically cleaved, U residues are then added and, finally, the RNA is religated (Fig. la; e.g., see [ 15,16,25**,34**,38*] ). Although it has generally been envisioned that the U residues would be added to the mRNA directly from UTP by terminal U transferase, alternative sources seem at least as likely (see below). A different mechanism has been suggested recently (Fig. 1b) [ 39**,40°*], based on transesterification reactions involved in intron removal [41]. This model envisions that the 3' OH of the LJ tail of the gRNA attacks within the mRNA, forming a chimeric molecule between that gRNA and the downstream portion of the mRNA, while liberating the upstream portion of the mRNA. In a second transesterilication reaction, the 3’OH of the upstream portion of the mRNA attacks the chimeric molecule within the U tail of the gRNk This would then rejoin the mRNA, bearing an added U residue(s), and it would liberate the gRNA which could participate in further rounds of editing. Removal of U residues could occur similarly by concerted transesterification reactions, using the mRNA sites in the reverse order [39-,40-1. This model is extremely pleasing because of its simplicity and its use of transesterification-based intron removal as a prototype, with protein factors presumably involved [ 39**]. Indeed, gRNA-mRNA chimenc molecules, precisely as hypothesized above, have been detected [36-•,40**] (R Benne, personal communication). Because such chimeric molecules are obligatory intermediates in the transesterification model for editing, their identification is strongly supportive of such a mechanism. The existence of gRNA-mRNA chimeric molecules, however, is not a smoking gun that proves the transesterification model, as may be thought (e.g. [42-l). In fact, an enzymatic mechanism of editing could well involve such chimeric molecules as intermediates in the transfer of U residues to the mRNA from the U tract at the 3’ end of the cognate gRNA. In this scheme, each round of editing

105

1058

Post-transcriptional

processes Region to beldited 5’ . ..AGAAACGCUCUA...

I I*II

3’ mRNA

II I

3’ Un...UCAUUCAUAUGUCACAU...

c\-.

V

(a)

(c) Alternate enzymatic model (Two cleavage and ligation cycles)

(b) Transesterification model (Two transesterification reactions)

Traditional enzymatic model (Cleavage, U addition, religaton) 5’ ...GAAACG... I I* 3’ . ..UUCAUAUGU...

5’ gRNA

n

n L;