Pentatricopeptide repeat poly(A) binding protein KPAF4 stabilizes mitochondrial mRNAs in Trypanosoma brucei

Figures

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  Fig. 1. Repeat organization, subcellular localization, and complex association of KPAF4. a Schematic repeat organization of kinetoplast polyadenylation factor 4 from Trypanosoma brucei (Tb) and Leishmania infantum (Li). Repeat boundaries were determined using the TPRpred online tool (https://toolkit.tuebingen.mpg.de/#/tools/tprpred) and adjusted according to Cheng et al. 17. Amino acids in positions 5 and 35/last potentially involved in adenosine recognition are indicated in separate columns. b Mitochondrial targeting of KPAF4-TAP fusion protein. Crude mitochondrial fraction was isolated by hypotonic lysis and differential centrifugation (crude mito), and further purified by renografin density gradient (pure mito). The latter preparation was extracted under conditions that separate matrix from membrane-bound proteins45. Protein profiles were visualized by Sypro Ruby staining and KPAF4-TAP was detected with an antibody against the calmodulin-binding peptide. The mitochondrial enrichment was calculated by quantitative western blotting vs. total protein loading. Representative of two experiments is shown. c Tandem affinity purification of KPAF4. Final fraction was separated on 8–16% SDS gel…

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  Fig. 2. KPAF4 interactions and proximity networks. a Model of the interactions between KPAF4, KPAP1 poly(A) polymerase, KPAF1-2, and KPAF3 polyadenylation factors, RNA editing substrate-binding complex (RESC), and MRP1/2 RNA chaperones. KPAP1, KPAF1, KPAF2, KPAF3, KPAF4, MRP2, MERS1, and Tb927.3.2670 proteins (encircled in blue) were affinity purified from mitochondrial lysates. The network was generated in Cytoscape software from bait–prey pairs in which the prey protein was identified by five or more unique peptides. The edge thickness correlates with normalized spectral abundance factor (NSAF) values ranging from 2.9 × 10−3 to 4.4 × 10−5 (Supplementary Data 1). Edges between tightly bound RESC modules (GRBC, REMC, and PAMC) were omitted for clarity25. All purifications were performed in parallel under uniform conditions. b KPAF4 proximity network. Spectral counts derived from BioID experiments with KPAP1, KPAF4, GRBC2, and MERS1 fusions with BirA* biotin ligase (encircled in blue) were processed as in (a) to build a proximity network. The edge thickness correlates with normalized spectral abundance factor (NSAF) values ranging from 2.9 × 10−3 to 2.6 × 10−5 (Supplementary Data 2). Edge…

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  Fig. 3. KPAF4 repression effects on cell growth and polyadenylation complex. a Northern blotting analysis of KPAF4 mRNA downregulation by inducible RNAi. b Growth kinetics of procyclic parasite cultures after mock treatment and KPAF4 RNAi induction with tetracycline. Data representative of three independent experiments are shown as mean ± s.d. c Quantitative real-time RT-PCR analysis of RNAi-targeted KPAF4 mRNA, and mitochondrial rRNAs and mRNAs. The assay distinguishes edited and corresponding pre-edited transcripts, and unedited mRNAs. RNA levels were normalized to β-tubulin mRNA. RNAi was induced for 55 h. Error bars represent 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. d Cell lysates prepared at indicated time points of KPAF4 RNAi induction were sequentially probed and re-probed by quantitative immunoblotting on the same membrane in the following order: antigen-purified rabbit polyclonal antibodies against KPAP1, KPAF1, KPAF3, GRBC1/2, and mouse monoclonal antibodies against RET1 TUTase. Signals…

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  Fig. 4. Divergent effects of KPAF4 knockdown on mitochondrial RNAs. a Northern blotting of pre-edited (Pre-E), partially edited (Part-E), and fully edited RPS12 mRNA variants. Total RNA was separated on a 5% polyacrylamide/8 M urea gel and sequentially hybridized with radiolabeled single-stranded DNA probes. Zero-time point: mock-induced RNAi cell line. Cytosolic 5.8S rRNA was used as loading control. Parent, RNA from parental 29-13 cell line; (dT), RNA was hybridized with 20-mer oligo(dT) and treated with RNase H to show positions of non-adenylated molecules in parental cell line. Pre-edited RNA length increase in KPAF4 RNAi is shown by brackets. Representative of four experiments for edited and three experiments for pre-edited RPS12 mRNA forms are shown. b Alignment of representative RPS12 mRNA 3′ ends in KPAF4 RNAi cells. RNA termini were amplified by cRT-PCR, cloned and sequenced8. A fragment of 3′ untranslated region, short A-tail, and U-extensions are indicated. c Northern blotting of pan-edited A6 mRNA. Total RNA was separated on a 1.7% agarose/formaldehyde gel and sequentially hybridized with oligonucleotide probes for pre-edited and fully edited sequences. Loading control:…

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  Fig. 5. Sequencing of mRNA and rRNA 3′ extensions in KPAF4 RNAi background. a Length distribution of short mRNA and 12S rRNA tails. Non-encoded 3′ end extensions (MiSeq instrument, Illumina, single biological replicate) were individually binned into 10-nt length groups. Mock-induced and RNAi datasets, indicated by blue and red bars, respectively, represent percentage of the total number of reads. b Length distribution of long mRNA tails. Non-encoded 3′ end extensions (PacBio RS II instrument, two biological replicates) were individually binned into 10-nt length groups before 100 nt, and in 50-nt groups thereafter. Mock-induced and RNAi datasets are indicated by blue and red bars, respectively, that represent percentage of the total number of reads. c Positional nucleotide frequencies in short mRNAs and 12S rRNA tails. A nucleotide percentage was calculated for each position that contained at least 5% of the total extracted sequences. The nucleotide bases are color-coded as indicated. Arrows show positions of equal adenosine and uridine frequencies. d Positional nucleotide frequencies in long mRNA tails. A nucleotide percentage was calculated for each position that contained at least 5%…

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  Fig. 6. Distribution of KPAF4 in vivo-binding sites between pre-edited and edited mRNAs. a Isolation of in vivo KPAF4-RNA crosslinks. Modified TAP-tagged fusion protein was purified by tandem affinity pulldown from UV-irradiated (+) or mock-treated (−) parasites. The second purification step was performed under fully denaturing conditions and resultant fractions were subjected to partial on-beads RNase I digestion and radiolabeling. Upon separation on SDS–PAGE, RNA–protein crosslinks were transferred onto nitrocellulose membrane. Protein patterns were visualized by Sypro Ruby staining (left panel), and RNA–protein crosslinks were detected by exposure to phosphor storage screen (right panel). RNA from areas indicated by brackets was sequenced. Representative of six biological replicates is shown. b KPAF4 in vivo-binding sites. Crosslinked fragments were mapped to the maxicircle’s gene-containing region. Annotated mitochondrial transcripts encoded on major and minor strands are indicated by blue and red arrows, respectively. c Position-specific nucleotide frequency in partially mapped KPAF4 CLAP-Seq reads. In reads selected by partial mapping to maxicircle and edited mRNAs, the unmapped 3′…

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  Fig. 7. KPAF4-bound adenylated RNA is partially resistant to uridylation and degradation in vitro. a Western blotting of affinity purified KPAF4-WT and KPAF4-Mut samples. Protein samples were purified from mitochondrial fraction by rapid affinity pulldown with IgG-coated magnetic beads. KPAF4 polypeptides were detected with an antibody against the calmodulin-binding peptide. Single experiment performed. b Electrophoretic mobility shift assay with KPAF4-WT. Increasing amounts of affinity-purified KPAF4 were incubated with 5′ radiolabeled RNAs and separated on 3–12% native PAGE. Representative of six experiments is shown. c Electrophoretic mobility shift assay with KPAF4-Mut was performed as in (b). Representative of two experiments is shown. d RNA adenylation and uridylation. KPAP1, RET1, or in combination, were incubated with 5′ radiolabeled RNA and ATP, UTP, or ATP/UTP mix, respectively, in the absence or presence of KPAF4. Recombinant enzymes were purified from bacteria as described8,48. Reactions were terminated at indicated time intervals and products were resolved on 10% polyacrylamide/ 8 M urea gel. Representative of seven experiments are shown. e RNA degradation. The same RNA…