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. 2008 Jun 4;27(11):1596-608.
doi: 10.1038/emboj.2008.87. Epub 2008 May 8.

3' adenylation determines mRNA abundance and monitors completion of RNA editing in T. brucei mitochondria

Ronald D Etheridge  1 , Inna Aphasizheva, Paul D Gershon, Ruslan Aphasizhev
Affiliations

3' adenylation determines mRNA abundance and monitors completion of RNA editing in T. brucei mitochondria

Ronald D Etheridge et al. EMBO J. .

Abstract

Expression of the mitochondrial genome in protozoan parasite Trypanosoma brucei is controlled post-transcriptionally and requires extensive U-insertion/deletion mRNA editing. In mitochondrial extracts, 3' adenylation reportedly influences degradation kinetics of synthetic edited and pre-edited mRNAs. We have identified and characterized a mitochondrial poly(A) polymerase, termed KPAP1, and determined major polypeptides in the polyadenylation complex. Inhibition of KPAP1 expression abrogates short and long A-tails typically found in mitochondrial mRNAs, and decreases the abundance of never-edited and edited transcripts. Pre-edited mRNAs are not destabilized by the lack of 3' adenylation, whereas short A-tails are required and sufficient to maintain the steady-state levels of partially edited, fully edited, and never-edited mRNAs. The editing directed by a single guide RNA is sufficient to impose a requirement for the short A-tail in edited molecules. Upon completion of the editing process, the short A-tails are extended as (A/U) heteropolymers into structures previously thought to be long poly(A) tails. These data provide the first direct evidence of functional interactions between 3' processing and editing of mitochondrial mRNAs in trypanosomes.

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Figures

Figure 1
Figure 1
KPAP1 is a mitochondrial PAP. (A) Purification of the recombinant KPAP1 from E. coli. M: mol. mass marker; MAC: metal affinity chromatography; CEX: cation exchange column. Proteins were separated on 8–16% gradient SDS–PAGE. (B) NTP specificity of KPAP1. The 5′-labelled 24-mer 6[A] RNA was incubated with KPAP1 at increasing concentrations of NTPs (1, 10, and 100 μM). The mutant D97A protein was tested in the presence of ATP. C: control RNA. The products were resolved on a 15% polyacrylamide/8 M urea gel. (C) Primer challenge assay. The 5′-labelled 6[A] RNA and KPAP1 were pre-incubated at final concentrations of 25 and 50 nM, respectively, for 10 min at 27°C. Reactions were started by simultaneous addition of ATP to 100 μM and unlabelled 6[A] RNA to 0, 25, 50, 100, 200, 400, and 800 nM. Incubation was continued for 30 min. (D) Subcellular fractionation of procyclic T. brucei. Approximately 10 μg of protein from hypotonic cell extract, cytosolic fraction, and purified mitochondria was separated on SDS–PAGE. Western blotting was performed with antibodies against KPAP1 and mitochondrial (MP81), cytoskeletal (β-tubulin), and cytosolic (HSP70.4) proteins. (E) Intracellular distribution of KPAP1. The C-terminal KPAP1–eYFP fusion was expressed in pLew79-based vector (Wirtz et al, 1999). Fluorescent images were captured in the presence of MitoTracker Red CMX ROS and DAPI stains.
Figure 2
Figure 2
KPAP1 is an essential gene required for mitochondrial function. (A) Cumulative cell growth after KPAP1 RNAi induction in procyclic T. brucei. (B) Depletion of KPAP1 protein in PF T. brucei. Cell lysates from the parental cell line (29-13) and tet-induced cells were separated on SDS–PAGE and probed with anti-KPAP1 antibody. (C) Subcellular fractions obtained from KPAP1 RNAi cell line were probed with anti-KPAP1 antibody. The Coomassie blue staining was used as loading control.
Figure 3
Figure 3
Inhibition of KPAP1 expression does not affect RNA editing complexes. (A) Co-immunoprecipitation of KPAP1 with 20 S editosome and RET1. Mitochondrial extract (200 μl, ∼5 mg protein/ml) from T. brucei was incubated for 1 h with 10 μl of magnetic beads pre-coated with antigen-purified anti-KPAP1 antibodies. Additional 30 min washes with 1% Triton or RNase A (0.1 mg/ml) in PBS buffer, or with 0.5 M KCl, were performed to assess the stability of KPAP1 interactions. Immunoprecipitated material was adenylated on beads, separated on SDS–PAGE, and probed for RET1 and MP81 on immunoblotting. Co-IP with antibodies against glutamate dehydrogenase (GDH) served as a negative control. (B) Sedimentation of the 20 S editosome from KPAP1-depleted mitochondrial extracts. Mitochondrial extract was fractionated on a 10–30% glycerol gradient. Fractions were incubated with [α-32P]ATP to detect editing ligases REL1 and REL2 and separated on SDS–PAGE. Sedimentation standards (catalase (11 S), thyroglobulin (19 S), and E. coli 30 S ribosome subunit) migrated as indicated by arrows. (C) U-insertion editing activity in the peak gradient fraction. RNA substrates for the pre-cleaved editing assay were assembled as follows: (1) 5′ fragment, no proteins added; (2) 5′ fragment; (3) 5′ fragment+‘guide' RNA; (4) 5′ fragment+3′ fragment; (5) fully assembled substrate for +2 addition, 5′ fragment+3′ fragment+‘guide' RNA. Positions of +2 guided U-insertions and ligation of edited product are shown by arrows. (D) gRNA labelling. Total RNA was isolated from the parental cell line (29-13), KPAP1, and RET1 RNAi cells. Guide RNAs were 5′ labelled with [α-32P]GTP in the presence of vaccinia virus guanylyltransferase and separated on 10% polyacrylamide/urea gel. The unidentified cytosolic RNA labelled with [α-32P]GTP was used as a loading control.
Figure 4
Figure 4
Purification of the KPAP1 complex. (A) Glycerol gradient fractionation. Mitochondrial extract from T. brucei cells expressing C-terminally TAP-tagged KPAP1 was fractionated on 10–30% glycerol gradients. Fractions were analysed for the presence of RELs (adenylation) and tagged protein (TAP detection). (B) Tandem affinity purification of the KPAP1 complex. The final fraction from the calmodulin column was separated on 8–16% SDS–PAGE and stained with Sypro Ruby. Carryover of TEV protease is indicated by an asterisk. The same fraction was probed along with the mitochondrial extract with antibodies against KPAP1, RET1, and MRP1. Before gel separation, samples were incubated with [α-32P]ATP to detect editing ligases REL1 and REL2. MRP1 has been shown to dimerize even under denaturing conditions (Zikova et al, 2008). (C) PAP and TUTase activities of the purified KPAP1 complex. Left panel: the 89-mer fragment of pre-edited RPS12 mRNA (100 nM) was incubated with KPAP1 complex (∼10 nM KPAP1, as estimated for the KPAP1-CBP band in Sypro Ruby-stained SDS gel) for 5, 15, and 45 min in the presence of 100 μM ATP or UTP, or both. Radiolabelled [α-32P]ATP and [α-32P]UTP were added as indicated. C: RPS12 RNA labelled at the 5′ end. Right panel: recombinant KPAP1 (20 nM) and RET1 (20 nM) were incubated with RPS12 RNA under the same conditions. Reaction products were separated on 8% polyacrylamide/urea gel. (D) Synthesis of a short A-tail by the recombinant KPAP1. The 5′-labelled RPS12 mRNA fragment (20 nM) was incubated with 100 μM ATP and increasing concentrations of KPAP1 (5, 10, 20, 50, 100, and 200 nM) for 30 min.
Figure 5
Figure 5
Polyadenylation determines the abundance of never-edited and edited mRNAs. (A) ‘Poisoned' primer extension analysis. DNA oligonucleotides were hybridized with total RNA and reverse transcription was performed in the presence of ddGTP (Missel et al, 1997). Reaction products were separated on 10% polyacrylamide/urea gel. Single-extension products were observed with never-edited CO1 mRNA and β-tubulin. In the A6 panel, primers for β-tubulin, A6, and CO1 mRNA were used in a single reaction. Only extension products are shown, and 5′-labelled DNA primers are omitted. The relative decrease in abundance was calculated assuming the mitochondrial mRNA/β-tubulin mRNA ratio in mock-induced cells as 100%. (B) Quantitative RT–PCR analysis of edited mRNAs. The RNA levels averaged from three replicates were normalized to β-tubulin mRNA. The thick line at 1 indicates no change in relative abundance. (C) Relative abundance of never-edited mRNAs and 12 S ribosomal RNA. (D) Northern blotting of the never-edited (CO1), cis-edited (CO2), 5′-edited (Cyb), and pan-edited (A6) mRNAs. Gel bands corresponding to mRNAs with long-tail (LT) or short-tail (ST) adenylation patterns are shown by arrows. Total RNA (25 μg) isolated from uninduced (−) and induced (+) KPAP1 RNAi cells was separated on 1.4% agarose-formaldehyde gel. (E) RPS12 mRNA decay in the course of KPAP1 RNAi. Total RNA was separated on 6% polyacrylamide/urea gel and sequentially probed for edited and pre-edited RPS12 transcripts, and 9 S mitochondrial ribosomal RNA. Long-tail (LT), short-tail (ST), and non-adenylated (NA) forms are shown by arrows. C: untreated RNA; (−), RNase H digestion minus DNA oligo.
Figure 6
Figure 6
Short A-tails are synthesized by KPAP1. (A) cRT–PCR amplification of pre-edited RPS12 mRNA. PCR products were separated on 2% agarose gel. The pre-edited adenylated (ST) and non-adenylated (NA) forms are shown by arrows. (B) Distribution of A's per tail in pre-edited RPS12 mRNA. Box plot shows the distribution of A's per tail with possible outlying points in the 25–75 percentile range. The middle line identifies the median sample value. The upper and lower ‘whiskers' highlight the range of non-outlying data points. The diamond illustrates the sample mean and 95% confidence interval. The line across each diamond represents the group mean. The vertical span of each diamond represents the 95% confidence interval for each group. (C) cRT–PCR amplification of the CO1 mRNA. (D) Distribution of A's per short A-tail in CO1 mRNA after KPAP1 RNAi.
Figure 7
Figure 7
The A/U extension constitutes the ‘long A-tail' of fully edited RPS12 mRNA. (A) cRT–PCR amplification of the long A-tail (LT) and short A-tail (ST) forms of the fully edited RPS12 mRNA. (B) Distribution of A's per tail in edited RPS12 mRNA following RNAi knockout of KPAP1. (C) Schematic representation of the A/U extensions in fully edited RPS12 mRNA derived from LT PCR products. (D) Northern blotting of pre-edited and partially edited RPS12 mRNAs in RNA isolated from RET1 and KPAP1 RNAi cells. The schematic positioning of DNA probes (solid line) within mRNA (dotted line) is shown under each panel; the sequences are provided in Supplementary Figure S3. [dT]: RNase H treated; C: control RNA; 9 S rRNA: mitochondrial small subunit rRNA. (E) Hybridization of the same membrane as in (D) with a probe for the fully edited RPS12 mRNA (Supplementary Figure S3).
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