Poly(A) binding KPAF4/5 complex stabilizes kinetoplast mRNAs in Trypanosoma brucei

Figures

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  Figure 1.. Kinetoplast polyadenylation factors 4 and 5 form a stable heterodimer involved in higher-order interactions. (A) Tandem affinity purification of KPAF4 and KPAF5. Final fractions were separated on 8–16% SDS gel and stained with Sypro Ruby. Bait proteins retaining calmodulin binding peptide and 6-His tags after release by TEV protease (21) are indicated by arrows. (B) Mitochondrial extract was separated for 5 h at 178 000 g in a 10–30% glycerol gradient. Each fraction was resolved on 3–12% Bis–Tris native gel (upper panels) and 8–16% SDS gel (lower panels). Positions of native protein standards are indicated. KPAF5 and RESC1/2 were visualized by immunoblotting with polyclonal antibodies. Thyroglobulin (19S) and bacterial ribosomal subunits were used as apparent S-value standards. (C) Affinity co-purification (left) and in vivo proximity biotinylation (BioID (22), right) interaction networks involving KPAF4/5. Rapid pulldowns were performed with TAP-tagged KPAF1, KPAF2, KPAF3, KPAF4, KPAF5, KPAP1 and MERS2 proteins. BioID experiments were carried out with BirA*-tagged MERS1, MERS2, KPAP1, KPAF3, KPAF4, KPAF5, RESC2, RESC5, RESC7, RESC3, RESC18 and RESC19 proteins (41). KPAP1 poly(A)…

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  Figure 2.. KPAF5 is essential for viability of procyclic and bloodstream parasite forms. (A) Growth kinetics of procyclic (PF) and bloodstream (BF) parasite cultures after mock treatment and RNAi induction with tetracycline. Data from three independent experiments are shown as mean ± s.d. (B) Northern blotting analysis of KPAF4 mRNA downregulation by inducible RNAi in BF. (C) Real-time RT-PCR analysis of RNAi-targeted KPAF5 mRNA, mitochondrial rRNAs and mRNAs. RNA levels were normalized to β-tubulin mRNA. RNAi was induced for 72 h. Error bars show the standard deviation from at least three biological replicates. The thick line at ‘1’ reflects no change in relative abundance; bars above or below represent an increase or decrease, respectively. P, pre-edited mRNA; E, edited mRNA.

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  Figure 3.. Stage-specific effects of KPAF4 and KPAF5 knockdowns on pan-edited and moderately-edited mRNAs. (A) Northern blotting of pre-edited (Pre-E) and fully-edited (E) RPS12 and A6 mRNA variants. Total RNA was separated on a 5% polyacrylamide/8M urea gel and sequentially hybridized with radiolabeled DNA probes. Parent: Lister 427 29-13 strain; (dT), total RNA from parental cell line was hybridized with 20-mer oligo(dT) and treated with RNase H to locate non-adenylated molecules. Zero time point: mock-induced RNAi cell line. Cytosolic 5.8S rRNA was used as loading control. (B) Northern blotting of moderately-edited Cyb mRNA. Total RNA was separated on a 1.7% agarose/formaldehyde gel and hybridized with oligonucleotide probes for pre-edited and fully-edited sequences. Loading control: cytosolic 18S rRNA.

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  Figure 4.. Stage-specific effects of KPAF4 and KPAF5 knockdowns on never-edited mRNAs, ribosomal and guide RNAs. (A) Northern blotting of never-edited CO1 mRNA. Total RNA was separated on a 1.7% agarose/formaldehyde gel and sequentially hybridized with oligonucleotide probes. Loading control: cytosolic 18S rRNA. (B) Northern blotting of never-edited Murf5 (uS3m) mRNA. Total RNA was separated on 5% polyacrylamide/8M urea gel and hybridized with radiolabeled DNA probe. Loading control: cytosolic 5.8S rRNA. (C) Northern blotting of mitochondrial rRNAs. Total RNA was separated on a 1.7% agarose/formaldehyde gel and hybridized with oligonucleotide probes for 9S and 12S rRNAs. Loading control: cytosolic 18S rRNA. (D) Guide RNA northern blotting. Total RNA was separated on a 10% polyacrylamide/8M urea gel and sequentially hybridized with oligonucleotide probes specific for minicircle-encoded gA6(14) and gCyb(560) gRNAs that participate in editing of pan-edited and moderately-edited mRNAs, respectively. Maxicircle-encoded gMurf2(II) gRNA was detected on the same membrane. Samples from RNAi cell line targeting the gRNA binding and stabilizing factor RESC1 (GRBC1, GAP2) typify gRNA loss, which causes…

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  Figure 5.. Impact of KPAF4/5 repression on polyadenylation, PPsome, and RESC complex components. Cells collected at indicated KPAF4 and KPAF5 RNAi time points in procyclic (A) and bloodstream (B) forms were lysed, separated on 8–16% SDS gel, and sequentially probed by quantitative immunoblotting in a top-to-bottom order. Antigen-purified rabbit polyclonal antibodies against KPAF4 and KPAF5 (this study), KPAF1 (12), KPAF3 (6), KPAP1 (10), MERS1 (5) and RESC1/2 (39) were generated in-house against respective recombinant proteins. Signals were normalized to β-tubulin.

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  Figure 6.. Messenger RNA 3′ modifications in KPAF5 RNAi knockdown. (A) Positional nucleotide frequencies in mRNA 3′ extensions. A nucleotide percentage was calculated for each position downstream from the encoded 3′ end set as zero. KPAF5 RNAi was induced for indicated time periods (mean of three biological replicates). The nucleotide bases are color-coded, and the areas of differential A-tail extensions are indicated for pre-edited and never-edited mRNAs. (B) Length distribution of mRNA 3′ tails. Non-encoded 3′ end extensions were binned into 10-nt length groups. Relative ratios at 0, 24 and 48 h of RNAi induction are shown as percentage of the total number of reads.

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  Figure 7.. In vivo positioning of individual polyadenylation factors in 3′ mRNA regions. The normalized aggregated eCLAP read coverage for each KPAF is represented by the grey area, and the non-encoded nucleotides in 3′ extensions are color-coded. The nucleotide frequency was calculated for each position 200 nt upstream and 100 nt downstream from the mature 3′ end, which was set as zero. The never-edited mRNAs include MURF5, ND1 and CO1; pan-edited profiles include RPS12, A6, ND3, ND7, ND9, CO3, CR3 and CR4; and moderately-edited combine Cyb and Murf2.

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  Figure 8.. Polyadenylation complex binding to 5′ end regions. The normalized aggregated eCLAP read coverage of the 100 nt downstream from the mature 5′ end is delineated by the grey area. Transcript categories defined as in Figure 7. In moderately-edited mRNAs, the edited domains are shown by red bars.

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  Figure 9.. RNA editing substrate binding complex (RESC) stabilizes never-edited mRNAs. (A) In vivo proximity network of KPAP1 poly(A) polymerase, KPAF3, 4 and 5, and RESC5, 13 (RGG2) and 18, and PPsome's pyrophosphohydrolase MERS1. The BioID experiments were performed in parallel under uniform induction, biotinylation, and purification conditions. The network was generated in Cytoscape software. The edge thickness correlates with normalized spectral abundance factor (NSAF) ranging from 1.13 × 10−4 (MERS1–KPAP1) to 1.5 × 10−3 (KPAF4–KPAF5) and reflects the predicted interaction strength (Supplementary Table S1). (B) Northern blotting of pre-edited (Pre-E) and fully-edited (E) RPS12 mRNA variants in RESC10, RESC13 (RGG2) and RESC1 RNAi knockdowns. Total RNA was separated on a 5% polyacrylamide/8M urea gel and sequentially hybridized with radiolabeled DNA probes. Parent: Lister 427 29–13 strain; (dT), RNA was hybridized with 20-mer oligo(dT) and treated with RNase H to remove A-tails and locate migration positions of a non-adenylated molecules. Short A-tailed and A/U-tailed mRNAs are indicated by arrows. Zero-time point: mock-induced RNAi cell line. Cytosolic 5.8S rRNA or tRNACys were used as…

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  Figure 10.. A circularization model of mitochondrial mRNA stabilization pathway in trypanosomes. We propose that mRNA stability is determined by PPsome and polyadenylation complexes that sequester 5′ and 3′ termini, respectively. It appears that distinct modalities within RNA editing substrate binding complex (RESC) interact with these end-bound particles to facilitate mRNA circularization and resistance to 3′-5′ degradation by the MPsome.