Trypanosoma brucei RNA editing protein TbMP42 (band VI) is crucial for the endonucleolytic cleavages but not the subsequent steps of U-deletion and U-insertion

Abstract

Trypanosome mitochondrial mRNAs achieve their coding sequences through RNA editing. This process, catalyzed by approximately 20S protein complexes, involves large numbers of uridylate (U) insertions and deletions within mRNA precursors. Here we analyze the role of the essential TbMP42 protein (band VI/KREPA2) by individually examining each step of the U-deletional and U-insertional editing cycles, using reactions in the approximately linear range. We examined control extracts and RNA interference (RNAi) extracts prepared soon after TbMP42 was depleted (when primary effects should be most evident) and three days later (when precedent shows secondary effects can become prominent). This analysis shows TbMP42 is critical for cleavage of editing substrates by both the U-deletional and U-insertional endonucleases. However, on simple substrates that assess cleavage independent of editing features, TbMP42 is similarly required only for the U-deletional endonuclease, indicating TbMP42 affects the two editing endonucleases differently. Supplementing RNAi extract with recombinant TbMP42 partly restores these cleavage activities. Notably, we find that all the other editing steps (the 3'-U-exonuclease [3'-U-exo] and ligation steps of U-deletion and the terminal-U-transferase [TUTase] and ligation steps of U-insertion) remain at control levels upon RNAi induction, and hence are not dependent on TbMP42. This contrasts with an earlier report that TbMP42 is a 3'-U-exo that may act in U-deletion and additionally is critical for the TUTase and/or ligation steps of U-insertion, observations our data suggest reflect indirect effects of TbMP42 depletion. Thus, trypanosomes require TbMP42 for both endonucleolytic cleavage steps of RNA editing, but not for any of the subsequent steps of the editing cycles.

FIGURE 1.

Mechanism of RNA editing and the editing proteins. (A) Editing cycles, as described in the text, with the responsible enzymes indicated. The G and A represent either purine and the C a pyrimidine. G-U pairing is allowed between the gRNA and the pre-mRNA. (B) Various nomenclatures for editing proteins, including the original “band” designations of T. brucei proteins from Rusche et al. (1997) (see Law et al. 2007 for additional verification of the reproducibility of this purification), the “TbMP” designation (representing T. brucei mitochondrial protein followed by the molecular weight of its cytoplasmic precursor) from Panigrahi et al. (2001a), and a more recent kinetoplastid RNA editing protein nomenclature (“KREPX#,” representing kinetoplastid RNA editing protein followed by a letter for the class of protein and a number indicating approximate ascending size within the group), that is being replaced with a KREØ# functional nomenclature (KRE as above, followed by a letter for the biological role of protein and a number indicating approximate ascending size within the group) from the Stuart laboratory (see references in the text; for reviews, see Worthey et al. 2003; Simpson et al. 2004; Stuart et al. 2005; Kang et al. 2006; Panigrahi et al. 2006). The proteins shown in brackets appear quite unabundant and/or are only obtained from some purification procedures (e.g., Panigrahi et al. 2006). In some references, the initial “K” is omitted, and, additionally, TbMP52 has also been referred to as DREL (Cruz-Reyes et al. 2002) and TbMP48 as IREL (Cruz-Reyes et al. 2002). Purification of editing complexes from L. tarentolae yields many homologous proteins, initially named LmLC# (Simpson et al. 2004) then shortened to LC# (Kang et al. 2006). For uniformity with the bulk of the editing literature, after their initial mention, we use the TbMP# designation. The final column summarizes the demonstrated (and proposed) roles in editing, as cited in the Introduction and the above references. mHel61p helicase of the ∼20S complex may aid removal of gRNAs after editing (Missel et al. 1997); also proteins separate from the ∼20S complex can affect editing, including TbMP108/KRET1 (that adds U-tails onto gRNAs) (Aphasizhev et al. 2002, 2003c), TbgBP21 and TbgBP25 (that stimulate RNA annealing) (Müller et al. 2001; Müller and Göringer 2002; Aphasizhev et al. 2003b; Schumacher et al. 2006), REAP1 (a putative mRNA-binding protein) (Madison-Antenucci et al. 1998), and RBP16 (a CYb RNA factor) (Pelletier and Read 2003).

FIGURE 2.

TbMP42 RNAi cell lines. (A) Northern blot from noninduced (−) or 24 h induced (+) RNAi cells showing the ∼1.5-kb TbMP42 mRNA and the (denatured) ∼0.7-kb RNA from the RNAi construct. (B) Growth curves of independent, clonal RNAi cell lines (RNAi) and control 29.13 cells (Co), following Tet addition. (C,D) Western blots using antibodies against TbMP42 and an hsp70 load control protein (see Materials and Methods; Law et al. 2005, 2007) to analyze (C) rapid cell extracts prepared from control cells or RNAi cells at day 3 (d3) and day 6 (d6) of induction and (D) traditional extracts prepared from control cells or RNAi cells at day 4 (d4) of induction. The protein amount analyzed is indicated, with 1× = 3 μg; the numbers below the lanes indicate the relative protein abundances, quantified as by Law et al. (2007), with TbMP42 values reported relative to lane loading (determined by the mitochondrial control protein hsp70) (see Materials and Methods). Horizontal dash marks on these and subsequent gels indicate the position of the examined protein.

FIGURE 3.

Retention of other editing proteins upon depletion of TbMP42. Western blot analyses, as in Figure 2C,D, using antibodies against the indicated proteins (left), including load control proteins (hsp and lip) used for the indicated gels, to probe extracts of control 29.13 cells and RNAi cells, prepared by the rapid extract protocol at (A) day 3 and (B) day 6 of tet administration and (C) by the traditional extract protocol at day 4. For each antibody and type of extract, the RNAi and control samples were run in the same gel and blotted together. The protein amount analyzed is indicated, with 1× = 3 μg. The approximate abundance of each editing protein relative to the control level, after correction for the signal of a load control antibody, is presented in the approximately equals (≅) column to the right of each panel.

FIGURE 4.

TbMP42 is essential for both U-deletional and U-insertional cleavages. Assessment of (A,B) U-deletional cleavage activity and (C,D) U-insertional cleavage activity using the editing substrates diagrammed below. (B,D) Verification of adenosine nucleotide specificity (see Cruz-Reyes et al. 1998b), using the nonhydrolyzable ADP analog, AMP-CP. These assays, as those of Figures 5 and 6, use the rapid extracts prepared from control or RNAi cells induced for 3 or 6 d. The weaker upstream cleavages are routinely observed (see Huang et al. 2002; Law et al. 2005, 2007). The analyzed protein amount is indicated, with 1× = 2.4 μg. G and nt indicate ladders showing guanosine residues and all nucleotides, from treatment of this mRNA with RNase TI and NaOH; − indicates no extract; arrowheads indicate the expected product (size shown below). In substrate diagrams of this and subsequent figures, * represents the ³²P label of the pre-mRNA strand.

FIGURE 5.

TbMP42 is required for retention of the basic U-deletional-like cleavage but not similarly for the basic U-insertional-like cleavage. (A) The basic U-deletion-like cleavage and (B) the basic U-insertion-like cleavage activities of the respective editing endonucleases, with products marked by arrowheads in the figures and assessed using the indicated substrates. In A, a nonediting single-strand nuclease also cleaves 1- and 2-nt upstream from the indicated site, especially in the control extract, and other nuclease activities generate two additional upper bands that are enhanced upon TbMP42 depletion; this could arise because the editing complex is less stable (see Fig. 8) and protects that upstream region less well from attack by single-strand-specific nucleases present in these crude cell extracts. In B, there are two main products due to heterogeneity in the gRNA length (Law et al. 2005). Control experiments confirm that the cleavages shown by the arrowheads retain their normal AMP-CP sensitivities (data not shown).

FIGURE 6.

TbMP42 is not required for the 3′-U-exo or TUTase activities. (A) Specific 3′-U-exo activity and (B) specific TUTase activity assessed using the indicated precleaved editing substrates. The precleaved substrate in A (Law et al. 2005) guides removal of 3 U's; this assay is U specific, as the next residue in this substrate pre-mRNA, a single-stranded A, is not significantly removed. The precleaved substrate in B (Igo et al. 2000) guides insertion of 2 U's. The band below the input RNA was seen previously (Law et al. 2007) and may results from an unrelated single-strand nuclease acting on some small, unannealed fraction of the labeled upstream oligo. In this and subsequent experiments using precleaved substrates, the labeled input oligo (in) and U removal products (−1U, −2U, −3U) or U-addition product (+2U) are indicated.

FIGURE 7.

TbMP42 is not required for U-deletional or U-insertional ligation. (A) Precleaved U-deletion (3′-U-exo plus ligation) and (B) precleaved U-insertion (TUTase plus ligation) assessed using the substrates of Figure 6 and the traditional extracts from control and RNAi cells induced for 4 d. 1× = 1.2 and 0.8 μg protein in A and B, respectively. The fully edited (−3U lig and +2U lig) products are indicated. Three-day rapid extracts yield comparable results (data not shown).

FIGURE 8.

TbMP18 is critical for stability of the editing complex. Glycerol gradient sedimentation analysis using the traditional extracts shown in Figures 2D, 3C, and 7. The indicated fractions (Fr 16 = top) were assayed for (A) the ligase proteins, by adenylylation, (B) precleaved U-deletion, as in Figure 7A, and (C) precleaved U-insertion, as in Figure 7B. In B and C, blank middle regions of these gels are not shown. Assessment of the individual 3′-U-exo and TUTase activities in these gradient fractions (reactions with PPi; not shown) gave results similar to those shown in the lower regions of B and C.

FIGURE 9.

Supplementation of TbMP42 RNAi cleavage reactions with rTbMP42. (A) U-deletional cleavage, (B) U-insertional cleavage, and (C) basic U-insertional cleavage were assessed as in Figures 4 and 5, with the reactions catalyzed by the indicated amounts of control extract or RNAi extract (1× = 2.4 μg of extract protein) alone (lanes 1–5,10), or supplemented with rTbMP42 (“rMP42”; lanes 6,11,12), or a with control protein (lanes 8; a recombinant 50-kDa segment of TbMP99 in A and B, “rMP99”; BSA in C), or by rTbMP42 alone (lane 7). Lane 9 is a nucleotide ladder. Lanes 10–12 are from a different experiment than lanes 1–9; the mRNA used for the experiment of lanes 1–9 in panels A and C had a 1-nt 3′ length heterogeneity and thus generated two product bands from the 3′ end-labeled substrate. The recombinant TbMP42 protein preparations not only stimulate editing cleavages by the RNAi extract (lanes 6), but alone they also direct some cleavage in the single-stranded regions upstream of the editing sites (lanes 7; see also lanes 8 for such cleavage by nonspecific recombinant protein preparations; substrates diagrammed in Figs. 4 and 5). However, those upstream cleavages are unlikely to be editing related since they generate 5′OH termini (the fragments co-migrate with an alkali-generated ladder of the same sequence [lane 9] and are out of phase with fragments from the editing cleavage; see, especially, Fig. 9B, lane 7 vs. lanes 2–6); in contrast, editing intermediates have 5′-P termini (see Cruz-Reyes et al. 1998a, and references therein).

FIGURE 10.

Summary of the data from Figures 2–9. Retention of indicated protein or activity in RNAi extract at day 3 of induction (when cells are still growing) and at day 6 of induction (after growth ceased). “+” Indicates at least one-half as much as the control extract; “−” indicates less than one-fifth as much as the control extract. “*” represents the U-insertional ligase efficiency assessed in combination with the remaining TUTase product (as in Fig. 7).