Kinetoplastid RNA editing involves a 3' nucleotidyl phosphatase activity

Abstract

Mitochondrial pre-messenger RNAs (pre-mRNAs) in African trypanosomes require RNA editing in order to mature into functional transcripts. The process involves the addition and/or removal of U nucleotides and is mediated by a high-molecular-mass complex, the editosome. Editosomes catalyze the reaction through an enzyme-driven pathway that includes endo/exoribonuclease, terminal uridylate transferase and RNA ligase activities. Here we show that editing involves an additional reaction step, a 3' nucleotidyl phosphatase activity. The activity is associated with the editing complex and we demonstrate that the editosomal proteins TbMP99 and TbMP100 contribute to the activity. Both polypeptides contain endo-exonuclease-phosphatase domains and we show that gene ablation of either one of the two polypeptides is compensated by the other protein. However, simultaneous knockdown of both genes results in trypanosome cells with reduced 3' nucleotidyl phosphatase and reduced editing activity. The data provide a rationale for the exoUase activity of the editosomal protein TbMP42, which generates nonligatable 3' phosphate termini. Opposing phosphates at the two pre-mRNA cleavage fragments likely function as a roadblock to prevent premature ligation.

Figure 1.

Nucleotide analysis of the r42-mediated trimming reaction. (A) Schematic representation of the input RNA (RNA55). RNA55 was co-transciptionally labeled using α-[³²P]-CTP (radioactive phosphates are depicted in gray) and arrows indicate potential cleavage sites of pN- and Np-specific ribonucleases. (B) 2D thin-layer chromatography of the hydrolysis products of RNA55 generated by digestion with r42 or with pN- and Np-specific ribonucleases. (C) Gelelectrophoretic analysis of r42-derived cleavage fragments in comparison to RNA fragments derived from alkaline hydrolysis (OH⁻). Panel to the left: The input RNA is 5′ [³²P]-labeled (*) and was incubated with four different r42 preparations (left to right) at limiting protein concentrations. The generated fragments co-migrate with their corresponding OH ladder fragments and thus must carry 3′ phosphates. Panel to the right: Complementary analysis of the corresponding 3′ cleavage fragments (radiolabeled at their 3′ ends through the ligation of 5′-[³²P] pCp). These fragments do not co-migrate with their OH-treated counterparts, thereby indicating the presence of 5′ OH groups. Schematic representations of the different RNAs are given on the left and right. Mock represents an RNA sample incubated in the absence of r42.

Figure 2.

Comparison of exoUase reaction products derived from incubations with r42 and 20S editosomes. (A) Sketch of the input precleaved deletion mRNA/gRNA hybrid molecule (5′ cleavage fragment (CF), black, 3′ CF, grey). An asterisk designates the radioactive phosphate group. OH and NH₂ indicate 3′ terminal hydroxyl or amino groups. (B) Electrophoretic analysis of the reaction products of the pre-mRNA/gRNA hybrid shown in (A) after incubation with r42 or 20S editosomes. A schematic representation of the processed RNAs is given on the right. Mock represents an RNA sample incubated in the absence of 20S editosomes or r42.

Figure 3.

Characterization of the 3′ nucleotidyl phosphatase activity of 20S editosomes. (A) A 31 nt ssRNA molecule (RNA31) was radioactively labeled (*) at its 3′ end using 5′-[³²P]-pCp and incubated for up to 3 h with 20S editosomes or alkaline phosphatase (AP). Reaction products were resolved in urea containing polyacrylamide gels and are depicted in the margin to the right. (B) Plot of the dephosphorylation (dfos) activity of 20S editosomes at different pH values. (C) Dephosphorylation of RNA-editing substrate molecules as part of an RNA-editing reaction cycle. Left panel: Precleaved insertion editing of 20S editosomes using a 3′ phosphorylated 5′ pre-mRNA cleavage fragment (5′ CF). Mock, mock control; T4 RNA ligase, control ligation with T4 RNA ligase. Right panel: Precleaved insertion editing of 20S editosomes using a 3′ dephosphorylated 5′ CF. Reaction products and intermediates are sketched in between the two gels. The mock sample contains a single band and degradation was measured <1%.

Figure 4.

RNAi-mediated downregulation of TbMP99 and/or TbMP100. (A) RT-PCR analysis of TbMP99 and TbMP100 single knockdown (99 skd and 100 skd) and TbMP99/TbMP100 double knockdown cells (99/100 dkd). Upon induction (6 days) with tetracycline (+tet) the mRNA level of either TbMP99, TbMP100 or of both transcripts was ≤5%. tub, α-tubulin. (B) Growth behavior of 99 skd, 100 skd and 99/100 dkd trypanosomes. (C) Autoadenylation of the editing ligases TbMP52 and TbMP48 in gradient fractionated mitochondrial extracts from wild-type (WT), 99 skd, 100 skd and 99/100 dkd trypanosomes prepared 6 days postinduction. Sub-20S and 20S fractions are indicated.

Figure 5.

RNA-editing activity of TbMP99/100-minus trypanosomes. (A) Precleaved insertion editing assay of mitochondrial 20S and sub-20S fractions of wild-type (WT) and TbMP99/100 double knockdown (99/100 dkd) cells. The assay monitors the gRNA-dependent insertion of 3U nucleotides. A representation of the pre-mRNA/gRNA hybrid is depicted on the right. 5′ pre-mRNA cleavage fragment (CF). black; 3′ CF, grey. The chemical identities of the 5′ and 3′ ends are as in Figure 2A. An asterisk indicates the radiolabel. Reaction products were separated in denaturing polyacrylamide gels and visualized by phosphorimaging. A schematic representation of the reaction products is given on the right. The graph shows a quantitative analysis of the editing activity. (B) Precleaved deletion editing assay of mitochondrial sub-20S and 20S fractions of WT and 99/100 dkd cells. The assay monitors the gRNA-dependent deletion of 4Us. All annotations are as in (A).

Figure 6.

3′ Nucleotidyl phosphatase activity of wild-type (WT) and TbMP99/100-depleted (99/100 dkd) sub-20S and 20S editosomes. (A) Autoradiograph of a phosphatase assay using an 8-nt single-stranded RNA substrate molecule (RNA8). An asterisk represents the position of the radioactive label. Reaction products are given in the margin to the right. Mock: RNA8 incubated in the absence of 20S editosomes. AP, alkaline phosphatase (B) Quantitative analysis of the assay shown in (A).

Figure 7.

Reaction cycle of an RNA-editing deletion reaction including a nucleotidyl phosphatase activity (altered from ref. 64). Base pairing between the pre-edited mRNA and the gRNA are indicated by vertical lines. Colons represent G:U bp.