An essential RNase III insertion editing endonuclease in Trypanosoma brucei

Abstract

RNA editing adds and deletes uridine nucleotides in many preedited mRNAs to create translatable mRNAs in the mitochondria of the parasite Trypanosoma brucei. Kinetoplastid RNA editing protein B3 (KREPB3, formerly TbMP61) is part of the multiprotein complex that catalyzes editing in T. brucei and contains an RNase III motif that suggests nuclease function. Repression of KREPB3 expression, either by RNA interference in procyclic forms (PFs) or by conditional inactivation of an ectopic KREPB3 allele in bloodstream forms (BFs) that lack both endogenous alleles, strongly inhibited growth and in vivo editing in PFs and completely blocked them in BFs. KREPB3 repression inhibited cleavage of insertion editing substrates but not deletion editing substrates in vitro, whereas the terminal uridylyl transferase, U-specific exoribonuclease, and ligase activities of editing were unaffected, and approximately 20S editosomes were retained. Expression of KREPB3 alleles with single amino acid mutations in the RNase III motif had similar consequences. These data indicate that KREPB3 is an RNA editing endonuclease that is specific for insertion sites and is accordingly renamed KREN2 (kinetoplastid RNA editing endonuclease 2).

Fig. 1.

Repression of KREN2 inhibits growth in PFs and BFs. (A) Growth of the PF ZJM-KREN2 cell line in the presence of KREN2 expression (without tet, circles) and after repression of KREN2 expression by RNAi (with tet, squares). (B) Growth of the BF RKO-KREN2 cell line in the presence of KREN2 expression (with tet, circles) and after repression of KREN2 expression (without tet, squares). Recovery of KREN2 expression (triangles) by the addition of tet to repressed cells at day 4 (arrow) is also shown.

Fig. 2.

Editosome sedimentation is unaltered by repression of KREN2 in PFs and BFs. (A) PF ZJM-KREN2 cells grown in the absence (KREN2 expressed) or presence (KREN2 repressed) of tet for 4 days were lysed, fractionated on glycerol gradients, and analyzed by Western blot. Preload lane is cell lysate before gradient fractionation. Western blots were simultaneously probed with monoclonal antibodies against editosome proteins KREPA1, KREPA2, KREL1, and KREPA3. (B) Similar analysis of BF RKO-KREN2 cells grown in the presence (KREN2 expressed) or absence (KREN2 repressed) of tet for 3 days.

Fig. 3.

Repression of KREN2 reduces editing in vivo. Real-time PCR analysis of total RNA isolated from PF ZJM-KREN2 (day 4) and BF RKO-KREN2 (day 3) cell lines. Analysis was performed in triplicate. For each target amplicon, the relative change in RNA abundance was determined by using either β-tubulin (left bar in each pair) or 18S rRNA (right bar in each pair) as an internal control. Open bars denote pre-mRNA, dark gray bars denote edited mRNA, black bars denote RNase III motif-containing mRNA, and light gray bars denote never-edited mRNA ND4. (A) Editing is reduced after repression of KREN2 by RNAi in PF cells. (B) Editing is reduced in BF RKO-KREN2 cells after repression of KREN2. Gray line marked by arrow represents no change in target RNA level; bars above or below this line represent an increase or decrease in RNA, respectively.

Fig. 4.

Repression of KREN2 in PF and BF decreases endonuclease activity on insertion substrates but does not alter other editing activities. All assays used peak ≈20S fractions from glycerol gradient sedimentation of lysates from PF ZJM-KREN2 RNAi cells grown for 4 days in the presence (repressed) or absence (expressed) of tet (A–D) or from BF RKO-KREN2 cells grown for 3 days in the presence (expressed) or absence (repressed) of tet (E–H). The amount (in μl) of peak fraction used in each reaction is shown above each lane. RNA substrates and products are shown in schematics; asterisk denotes radiolabel, and wedge denotes cleavage site. A6 insertion site cleavage product (black arrows) was significantly diminished by KREN2 repression (gray arrows) in PFs (A) and BFs (E). Cleavage is due to RNA editing endonuclease activity, as indicated by the requirement for gRNA (-guide). T1-digested substrate RNA was used as a marker to determine where substrate RNA was cleaved. A6 deletion site cleavage was unaltered by repression of KREN2 in PF (B) and BF (F). Cleavage product (arrows) is absent in reactions without gRNA (-guide). Precleaved insertion editing is unaltered by KREN2 repression in PFs (C) and BFs (G). The input lane has only radiolabeled substrate. Precleaved deletion editing is unaltered by KREN2 repression in PFs (D) and BFs (H).

Fig. 5.

Point mutations in the RNase III signature motif of KREN2 inhibit growth and reduce insertion endonuclease activity. (A) Growth of RKO-WT-β, RKO-E227V-β, and RKO-D234A-β cell lines with the Reg-KREN2 allele expressed (E) or repressed (R), as indicated in Inset.(B) A6 insertion site cleavage in RKO-WT-β, RKO-E227V-β, RKO-D234A-β, and parental RKO-KREN2 (RKO) cell lines. Arrow indicates cleavage product. Assays were performed by using peak ≈20S fractions from each cell line in which WT Reg-KREN2 allele was expressed (E) or repressed (R). (C) The same fractions assayed for insertion cleavage were also assayed for A6 deletion site cleavage. Arrow indicates cleavage product. For both endonuclease assays, cleavage was guide-dependent (-guide) and absent when water was substituted for gradient fraction (water). T1-digested substrate RNA acted as a marker to identify the cleavage product.