A multiprotein, high molecular weight complex active in both U-insertion and U-deletion as judged by a pre-cleaved RNA editing assay was isolated from mitochondrial extracts of Leishmania tarentolae by the tandem af®nity puri®cation (TAP) procedure, using three different TAP-tagged proteins of the complex. This editing-or E-complex consists of at least three protein-containing components interacting via RNA: the RNA ligase-containing L-complex, a 3¢ TUTase (terminal uridylyltransferase) and two RNA-binding proteins, Ltp26 and Ltp28. Thirteen approximately stoichiometric components were identi®ed by mass spectrometric analysis of the core L-complex: two RNA ligases; homologs of the four Trypanosoma brucei editing proteins; and seven novel polypeptides, among which were two with RNase III, one with an AP endo/exonuclease and one with nucleotidyltransferase motifs. Three proteins have no similarities beyond kinetoplastids. Keywords: editosome/RNA editing/TAP/TUTase Introduction Uridine insertion/deletion RNA editing is a post-transcriptional RNA modi®cation phenomenon that occurs in the mitochondrion of kinetoplastid protists . The mechanism involves the initial hybridization to an mRNA of a complementary guide RNA (gRNA) which guides a speci®c endonuclease cleavage at the ®rst editing site . This is followed by either deletion of the unpaired uridines from the cleavage fragment or the 3¢ addition to the mRNA 5¢ cleavage fragment, hybridization of the added Us to the guiding nucleotides in the gRNA, and religation of the two mRNA cleavage fragments. Each gRNA speci®es the 3¢ to 5¢ editing of a small number of sites and, in the case of a multiple gRNA-mediated editing domain, creates the anchor sequence for hybridization of the adjacent upstream gRNA, thus producing an overall 3¢ to 5¢ progression of editing. A minimal non-progressive editing activity at one or two sites has been demonstrated in vitro using crude or partially puri®ed mitochondrial extract, and the reaction was shown to involve high molecular weight RNP complexes (Byrne et al., 1996;Cruz-Reyes and Sollner-Webb, 1996;Kable et al., 1996;Seiwert et al., 1996). The mechanism described above was proposed >12 years ago , and was veri®ed experimentally in 1996 for both Trypanosoma brucei and Leishmania tarentolae (Byrne et al., 1996;Cruz-Reyes and Sollner-Webb, 1996;Seiwert et al., 1996). However, progress in the identi®cation of speci®c proteins involved in editing has been hampered by their low abundance and by the low ef®ciency of the in vitro editing assays. A seven polypeptide complex from T.brucei mitochondria that supported in vitro insertion and deletion editing was isolated by two chromatographic steps and was proposed to represent a core editing complex (Rusche et al., 1997). An~20 polypeptide complex with similar activities was isolated in another laboratory by a similar fractionation (Panigrahi et al., 2001a,b).The genes for several of the major components of these complexes have been identi®ed, but only a few proteins so far have been ascribed ...

A number of mitochondrial proteins have been identified in Leishmania sp. and Trypanosoma brucei that may be involved in U-insertion/deletion RNA editing. Only a few of these have yet been characterized sufficiently to be able to assign functional names for the proteins in both species, and most have been denoted by a variety of species-specific and laboratory-specific operational names, leading to a terminology confusion both within and outside of this field. In this review, we summarize the present status of our knowledge of the orthologous and unique putative editing proteins in both species and the functional motifs identified by sequence analysis and by experimentation. An online Supplemental sequence database (http://164.67.60.200/ proteins/protsmini1.asp) is also provided as a research resource.

Uridine insertion͞deletion RNA editing in trypanosomatid mitochondria is a posttranscriptional RNA modification phenomenon required for translation of mitochondrial mRNAs. This process involves guide RNA-mediated cleavage at a specific site, insertion or deletion of Us from the 3 end of the 5 mRNA fragment, and ligation of the two mRNA fragments. The Leishmania major RNA ligase-containing complex protein 2 expressed in insect cells has a 3-5 exoribonuclease activity and was therefore renamed RNA editing exonuclease 1 (REX1). Recombinant REX1 specifically trims 3 overhanging Us and stops at a duplex region. Evidence is presented that REX1 is responsible for deletion of the 3 overhanging Us from the bridged mRNA 5 cleavage fragment and that RNA editing ligase 1 is responsible for the ligation of the two mRNA cleavage fragments in U-deletion editing. The evidence involves both in vivo down-regulation of REX1 expression in Trypanosoma brucei by RNA interference and the reconstitution of precleaved U-deletion in vitro editing with only two recombinant enzymes: recombinant REX1 and recombinant RNA editing ligase 1.ligase ͉ trypanosomes ͉ REX1 ͉ editing U ridine insertion͞deletion RNA editing is a posttranscriptional RNA modification phenomenon that occurs in the mitochondria of kinetoplastid protists (1). The insertion and deletion of Us into transcripts of 12 mitochondrial-encoded cryptogenes is mediated by guide RNAs (gRNAs) that hybridize downstream of the editing sites and recruit several protein complexes that interact via RNA. The RNA ligase-containing complex (L-complex) from both Leishmania sp. and Trypanosoma brucei contains Ϸ16 proteins, which have been labeled LC-X (for L-complex protein) or MP-X (for mitochondrial protein), respectively (2, 3), by relative gel mobility. These proteins include the LC-2 (MP100) and LC-3 (MP99) ''Exoendophos''-Pfam motif proteins (4), the RNA editing ligase 1 (REL1) (5-7) and RNA editing ligase 2 (REL2), and the RNA editing 3Ј terminal uridylyltransferase (TUTase) 2 (RET2) proteins, among others. Editing involves an initial cleavage of the mRNA transcript just upstream of the mRNA-gRNA anchor duplex, which is followed by either an addition of Us to the 3Ј end of the 5Ј fragment or a deletion of non-base-paired Us from the 3Ј end of the 5Ј fragment. The two fragments are then ligated, thereby extending the mRNA-gRNA duplex. The process repeats at the next upstream editing site, and, after completion of the editing mediated by a single gRNA, in some cases, additional gRNAs hybridize to the edited sequences and mediate overlapping blocks of editing, extending the edited region further upstream (8). The RET2 TUTase in the L-complex was shown to be responsible for the gRNA-mediated addition of Us to the editing sites, and the RET1 TUTase, which is not a component of the L-complex but interacts via RNA, is responsible for the addition of Us to the 3Ј end of the gRNAs (9, 10).Heterologous expression of properly folded active editing enzymes has proven difficult. The only recombinant...