Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins

Abstract

Uridine (U)-insertion/deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model U-deletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNA-editing nuclease 1.

Fig. 1.

Conditional RNAi down-regulation of TbMP90 expression in T. brucei procyclic cells. (A Upper) Growth curve of noninduced and tet-induced cells. The cultures were diluted daily to maintain log-phase growth. Tetracycline (1 μg/ml) was added to initiate RNAi. (A Lower) RT-PCR of TbMP90 mRNA from cells at days 3, 6, and 9. D, day. (B) Full-round editing and cleavage activities of the 20S gradient fraction from cells down-regulated for TbMP90 expression for 6 days and 9 days by using 5′-end-labeled U-deletion substrate. Numbers below the lanes show percent of editing. (C) Glycerol gradients of mitochondrial lysates from the RNAi cells at days 0, 6, and 9. (Upper) Fractions were autoadenylated to show the location of the REL1 and REL2 ligases. The L-complex-containing fractions are circled. (Lower) Western blot analysis of the same fractions using antibodies against MP81, MP63, and MP42. (D) Diagrams of the U-deletion substrates (20). The nt 22 and nt 25 cleavage sites are indicated in S1 and the nt 11 cleavage site in S2. The ³²P-label is indicated with an asterisk.

Fig. 2.

Requirement for gRNA and stimulatory effect of AMP-CP on cleavage at the U-deletion site during full-round editing in vitro. (A) The 20S gradient fraction from noninduced cells was assayed for full-round U-deletion editing and cleavage activity by using 5′-end-labeled U-deletion substrate. The arrows indicate the −3U edited product and the cleavage fragment. The size marker lane contains partially hydrolyzed 5′-labeled RNA. Addition of PPi to the reaction to inhibit ligation (right lane) prevented formation of the edited product and slightly increased the yield of cleavage fragments. (B) The 20S gradient fraction from noninduced cells was assayed for cleavage activity in the presence of PPi to inhibit ligase activity. (Upper) AMP-CP stimulates cleavage activity. The percent cleavage is shown below each lane. (Lower) Cleavage requires the presence of annealed cognate gRNA.

Fig. 3.

Expression of TAP-tagged LmMP90 in L. tarentolae. (A) Western blot analysis using PAP reagent of expressed LmMP90-TAP in cytosol (Cyto) and mitochondrial (Mito) fractions. (B Left) Sedimentation of mitochondrial lysates from transfected L. tarentolae. Fractions were assayed for the L-complex by autoadenylation of REL1 and REL2. The ≈20S L-complex fractions are circled. (B Center) Western blot analysis of fractions 11 and 9 from both gradients, using antibodies against LC1, LC4, and LC7B. (B Right) Western blot analysis of the same fractions using the PAP reagent. Arrows indicate intact LmMP90 and LmMP90-D bands. (C Left) Comparison of cleavage activities of peak fractions from gradients in B. Control lanes have input RNA. PPi and AMP-CP were present. The same amount of protein was used for each reaction. (C Center) Stimulation of cleavage activity of purified LmMP90-TAP pull-down by AMP-CP. (C Right) Comparison of cleavage activities of pull-downs using LmMP90-TAP and LmMP90-D-TAP cells. The numbers below the lanes show relative ratios of cleavage.

Fig. 4.

U-deletion cleavage activity of rTbMP90-CBP. (A) Stained gels and Western blots of the purified rTbMP90-CBP (wt) and rTbMP90-D-CBP (D) proteins. (B) Cleavage of −3U-deletion (Fig. 1D, S2) and +3U-insertion (Fig. 4E) substrates using rTbMP90-CBP. Marker is 3′-end-labeled partially hydrolyzed RNA. The slight lack of correspondence between marker bands and fragment bands is a known artifact (39). rTb20S, peak L-complex gradient fraction. U-deletion cleavage reactions were performed in the presence of PPi and AMP-CP, and U-insertion cleavage reactions were performed in the presence of PPi and UTP. (C) Comparison of cleavage activities of rTbMP90-CBP and rTbMP90-D-CBP. Percent cleavage is indicated below each lane. Effects of AMP-CP and proteinase K treatment are also shown. (D) Titration of Mg²⁺ requirement for cleavage of U-deletion substrate. (E) Sequence of 3′-end-labeled +3U-insertion substrate used in B Right.

Fig. 5.

U-deletion cleavage activity of rLmMP90-CBP and mutant LmMP90 (E/K)-CBP obtained from expression in L. tarentolae. (A) Isolation of rLmMP90-CBP. (Left) Sypro-stained gels of the final preparations of LmMP90-CBP and LmMP90-CBP (E/K). (Right) Western blot analysis with anti-CBP antibody. The location of the rLmMP90-CBP band is indicated. (B) U-deletion cleavage activity of rLmMP90-CBP and rLmMP90-CBP (E/K). Equal amounts of protein (30 nM) were used. The percent cleavage is shown below each lane. (C) Stimulation of cleavage activity by 3 mM AMP-CP. The relative ratio of cleavage is shown below each lane. (D) Alignment of RNase III motifs from E. coli, S. cerevisiae, LmMP90, and TbMP90. Absolutely conserved residues are in dark gray. E378 in LmMP90 is indicated with an arrow.

Fig. 6.

Reconstitution of full-round editing with three recombinant proteins. The 3′-end-labeled U-deletion substrate was incubated with a mixture of recombinant LtREL1, LmREX1, and LmMP90 proteins. (A) Diagram of proposed reaction steps involving cleavage by LmMP90, deletion of 3′ Us by REX1, and ligation by REL1. (B) Full-round −3U deletion editing. The positions of the edited products containing no Us (−3), one U (−2), and two Us (−1) are indicated. The percent edited product is indicated below each lane.

Fig. 7.

Effect of adjacent upstream nucleotide on full-round editing and cleavage at U-deletion site. (A) Diagrams of RNA substrates used. The standard substrate has a U upstream of the cleavage site, and the other has a C. (B Upper) full-round U-deletion editing. The control lanes have the input RNA. F10 indicates the peak L-complex fraction from a gradient of mitochondrial lysate from WT cells. U indicates the upstream U RNA and C the upstream C RNA. The reactions in the final two lanes contained a mixture of rLtREL1, rLmREX1, and rLmMP90. The edited product is indicated. (B Lower) Cleavage activity. The reactions were performed in the presence of AMP-CP and the absence of ATP to inhibit ligase activity. The cleavage product is indicated.