In Trypanosoma brucei RNA Editing, TbMP18 (Band VII) Is Critical for Editosome Integrity and for both Insertional and Deletional Cleavages

Trypanosomes are early diverging parasitic protozoa that cause debilitating diseases, such as African sleeping sickness, and exhibit numerous interesting biological properties. Notably, many of their mitochondrial transcripts undergo a unique form of processing, termed RNA editing, in which uridylate residues (U's) are inserted and deleted at specific locations within pre-mRNAs (reviewed in references 46, 48, and 49). This editing is essential for parasite viability (44) and is very extensive in certain transcripts, creating up to three-quarters of the codons and frequently the start and stop signals. Editing is directed by numerous short guide RNA molecules (gRNAs), which are complementary to regions of the edited transcript (5) and thus mismatch the unedited mRNA at each editing site. The first gRNA is also complementary to the segment just 3′ of the editing domain, so it can base pair to this region in the pre-mRNA, forming an “anchor duplex” that extends up to the first editing site. In each editing cycle (see Fig. ​Fig.1A),1A), the pre-mRNA is cleaved just upstream of the anchor duplex (5; see also reference 8), then U's are either added to or removed from the 3′ end of the upstream cleavage fragment by a terminal U transferase (TUTase) (4) or a 3′-U-exonuclease (3′-U-exo) (5; see also reference 38), and finally, the transcript is resealed by an RNA ligase (28, 38, 39, 44). Upon completion of an editing cycle, the anchor duplex extends up to the next mismatch to direct another cycle, and editing thus progresses 3′ to 5′ along the pre-mRNA. FIG. 1. Mechanism of RNA editing and the purified editing complex. (A) Editing cycles, as described in the text, with the responsible enzymes indicated. U-deletional endonuclease (del. endo; cleavage shown with a hollow arrowhead) or U-insertional endonuclease ... The editing cycles are catalyzed by a protein complex, or editosome (22, 37, 38, 45), that has been purified by several different procedures. The first reported purification (38) yielded complex that catalyzes full editing cycles in vitro (9) and reproducibly contains seven major staining proteins (38), temporarily designated bands I through VII (see Fig. ​Fig.1B).1B). However, subsequently reported purification procedures yield complexes that catalyze precleaved editing especially actively and contain ∼20 noted proteins, designated TbMP# (see the legend to Fig. ​Fig.1B)1B) (31, 33, 35) and subsequently KREX# (see the legend to Fig. ​Fig.1B)1B) (49). These proteins, whose relative abundance appears to vary in different preparations, include bands I through VII and several critical enzymes that are minor staining components of the seven major protein complex (e.g., TbMP57 TUTase) (see Fig. ​Fig.1B1B legend) (57). Curiously, all these preparations of the editing complex, with quite disparate numbers of major components, are reported to sediment at ∼20S in glycerol gradients. The basis for the differences in the major protein profiles between editing complex purifications remains unclear but does not appear to be due to a loss of important proteins during the former purification, as that purified complex catalyzes full U-deletional and U-insertional editing cycles the most actively of any preparation yet reported (11, 12) and at least TbMP57 is not selectively lost (57). Possibly the differences reflect the different cell lines used for the purifications, 667 in reference 38 and IsTar 1.7a in reference 31, 33, and 35, as 29.13 cells subjected to a latter kind of purification yielded complex with a pattern of major staining proteins (43) that appeared strikingly similar to the former preparations. Figure ​Figure1C1C summarizes various nomenclatures for these key proteins. For uniformity with the bulk of the editing literature, after their initial mention, we will use the TbMP# nomenclature. Most of the proteins of the editing complex have been examined using biochemical and genetic approaches, almost all utilizing RNA interference (RNAi). Some of these proteins are the requisite enzymes. TbMP90 and TbMP61 are the endonucleases for U deletion and U insertion, respectively (7, 23, 32, 52), TbMP57 is the TUTase that adds the U's in U insertion (2, 15), and TbMP52 (band IV) and TbMP48 (band V) (38, 39, 44; see also references 28 and 40) are the RNA ligases that preferentially seal in U deletion and U insertion, respectively (12; see also references 19 and 43). Additionally, several proteins have been implicated as the 3′-U-exo: TbMP99 (band I) (43; see also reference 56), the related protein TbMP100 (24, 56), and TbMP42 (band VI) (6). Finally, mHel61p is a helicase that can augment editing (29). Several of the other editing proteins have critical structural and auxiliary roles. TbMP81 (band II) is required for protein and/or substrate recognition at each step of U insertion (26), including TbMP48 ligase retention (14, 30), but not for U deletion or for an ∼15S association of the other editing proteins (26, 30). TbMP63 (band III) is required for editing complex integrity, TbMP52 ligase retention, and U-deletional and U-insertional cleavages (20). Also, TbMP24 (42) and TbMP44 (53) appear essential for retention of all other examined editing proteins of the ∼20S complex. Indeed, all these noted structural proteins, as well as TbMP18 (band VII), have potential interaction domains, including homologous oligonucleotide/oligosaccharide binding (OB fold) domains and zinc fingers (reviewed in reference 56). Furthermore, other proteins that are not components of the ∼20S complex can affect editing, including TbMP108 (which adds U tails onto gRNAs) (2, 3), TbgBP21 and TbgBP25 (annealing stimulators) (1), RBP16 (a cytochrome b mRNA factor) (36) and REAP1 (a possible pre-mRNA binding component) (27). The editing activities within the ∼20S editing complex appear functionally and spatially partitioned. The three parallel steps of U deletion and U insertion utilize distinct enzymes (7, 8, 10, 12, 19, 38, 52) and TbMP63 holds the U-deletional ligase (20, 34, 43) while TbMP81 holds the U-insertional ligase (14, 30, 43) and is also required for the other steps of U insertion (26). Notably, overexpressing affinity-tagged ligases enriches ∼5S to 10S subcomplexes that contain mainly TbMP81, TbMP57, and TbMP48 and catalyze precleaved U insertion or subcomplexes that contain mainly TbMP99, TbMP63, and TbMP52 and catalyze precleaved U deletion (43) (both types of subcomplexes also contain some TbMP18). Yeast two-hybrid analyses (43) also support these associations. Nonetheless, the U-deletional and U-insertional portions of the editing complex appear functionally interconnected, since RNAi of the U insertion-specific TbMP48 ligase allows the U-deletional TbMP52 ligase to also seal U insertion (14, 17, 30), and the U-insertional TUTase can act within the U-deletional cycle (57). The current study assesses the role of TbMP18 and shows it is essential for viability, integrity of the ∼20S editing complex, and both U-deletional and U-insertional cleavage activities. The other editing activities and major editing proteins are not similarly dependent on TbMP18, and without it, the complex disassociates into subcomplexes reminiscent of those described by Schnaufer et al. (43).