Kinetoplast DNA-encoded ribosomal protein S12: a possible functional link between mitochondrial RNA editing and translation in Trypanosoma brucei

Abstract

Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, which are encoded by the kinetoplast genome, and more than 150 proteins encoded in the nucleus and imported from the cytoplasm. However, a single ribosomal protein RPS12 is encoded by the kinetoplast DNA (kDNA) in all trypanosomatid species examined. As typical for these organisms, the gene itself is cryptic and its transcript undergoes an extensive U-insertion/deletion editing. An evolutionary trend to reduce or eliminate RNA editing could be traced with other cryptogenes, but the invariably pan-edited RPS12 cryptogene is apparently spared. Here we inquired whether editing of RPS12 mRNA is essential for mitochondrial translation. By RNAi-mediated knockdowns of RNA editing complexes and inducible knock-in of a key editing enzyme in procyclic parasites, we could reversibly downregulate production of edited RPS12 mRNA and, by inference, synthesis of this protein. While inhibition of editing decreased edited mRNA levels, the translation of edited (Cyb) and unedited (COI) mRNAs was blocked. Furthermore, the population of SSU-related 45S complexes declined upon inactivation of editing and so did the amount of mRNA-bound ribosomes. In bloodstream parasites, which lack active electron transport chain but still require translation of ATP synthase subunit 6 mRNA (A6), both edited RPS12 and A6 mRNAs were detected in translation complexes. Collectively, our results indicate that a single ribosomal protein gene retained by the kinetoplast mitochondrion serves as a possible functional link between editing and translation processes and provide the rationale for the evolutionary conservation of RPS12 pan-editing.

Keywords: RNA editing; TUTase; kDNA; mitochondria; polyadenylation; translation.

Figure 1. Inhibition of RNA editing by dual RNAi knockdowns of critical subunits in the RNA editing core (MP18 and MP24) and guide RNA binding (GRBC1 and GRBC2) complexes, and RNAi-based knock-in of catalytically inactive RET2 TUTase. Change in relative abundance for mitochondrial mRNAs and rRNAs was determined by real-time RT-PCR analysis; thick line at “1” reflects no change in relative abundance upon RNAi induction; bars above or below represent an increase or decrease, respectively. RNA levels were normalized to β-tubulin mRNA. Error bars represent the standard deviation from at least three replicates. P, pre-edited mRNA; E, edited mRNA; n/d, not determined.

Figure 2. MRNA editing and polyadenylation states in genetic backgrounds lacking RNA editing. (A) Expression of dual RNAi cassettes targeting subunits of the RNA editing core complex (MP18 and MP24, left panels) or guide RNA biding complex (GRBC1 and GRBC2, right panels) was induced by addition of tetracycline and cells were collected for RNA purification at 24 h intervals. Total RNA was separated on 1.7% agarose /formaldehyde gel and subjected to hybridization with DNA probe for unedited COI mRNA. [dT], RNA was treated with RNase H in the presence of 18-mer [dT] to remove poly(A) tails. ST and LT, mRNA forms terminating with short (A) or long (A/U) tails, respectively. (B) Pre-edited and edited forms of RPS12 mRNA were analyzed in RET2-CODA WT and RET2-CODA D97A genetic backgrounds. RNAi targeting the endogenous RET2 mRNA and expression of RNAi-resistant variants of the same were induced with tetracycline for indicated periods of time. Total RNA was separated on 5% polyacrylamide/ 8M urea gels and transferred onto membrane. Cytosolic 5.8S rRNAs served as loading control (C). Same RNA samples as in (B) were separated on 1.7% agarose/formaldehyde and probed for moderately edited Cyb, pan-edited A6 and unedited COI mRNAs. In addition, mitochondrial rRNAs (9S and 12S) were visualized on the same membrane.

Figure 3. Loss of the 80S mRNA-associated ribosomal population upon inactivation of RNA editing. (A) Native gel analysis of ribosomal complexes. Total cell lysates obtained from parental and RET2 knock-in cell lines were fractioned on glycerol gradients and each fraction was further separated on 3–12% Bis-Tris NuPAGE native gel and transferred onto nylon membrane. NativeMARK Unstained Protein Standards (Invitrogen) were separated alongside. Hybridizations were performed with the same probes as in Figure 2 to detect fully-edited RPS12 mRNA, 9S, and 12S rRNAs. (B) Sedimentation of rRNAs. RNA was extracted from indicated fractions, separated on 5% polyacrylamide/8M urea gel and simultaneously probed for 9S and 12S rRNAs.

Figure 4. Reduction of the 45S SSU-related complex (SSU*) and the 80S complex upon inactivation of RNA editing. (A) Ribosomal complexes in RET2 RNAi uninduced cells (tet[-]), as well as in cells upon induction of the RNAi at specified time points. Total cell lysates were fractionated in sucrose gradients; 9S and 12S rRNAs in gradient fractions were analyzed by slot-blot hybridization. (B) Ribosomal complexes in RET2-CODA WT and RET2-CODA D97A knock-in cells.

Figure 5. Mitochondrial translation in parasites depleted of guide RNA binding complex (GRBC1/2), RNA editing core complex (MP18/24), RNA editing TUTase 2 (RET2), and knock-in cell lines expressing catalytically active (RET2-CODA WT) or inactive (RET2-CODA D97A) TUTases. RNAi and protein expression were induced for indicated periods of time and translation products were metabolically labeled with EasyTag mix (PerkinElmer Life Sciences) in isotonic buffer supplemented with 0.1 mg/ml of cycloheximide. Cells were collected by centrifugation, dissolved in SDS gel loading buffer, and fractionated by two-dimensional electrophoresis. Gels were stained with Coomassie brilliant blue R250 (inset panels) and exposed to X-ray film (large panels). Based on previous protein identifications, major spots represent COI and Cyb proteins encoded by unedited and edited mRNAs, respectively.

Figure 6. Comparative sedimentation analysis of RPS12, A6 and COI mRNAs between procyclic and bloodstream developmental forms of T. brucei. Total cell lysates were obtained from parental cell lines routinely used for RNAi studies in procyclic (29–13) and bloodstream (Lister 427 “single marker”) parasites were fractioned on glycerol gradients. RNA was extracted from indicated fractions and separated on 5% polyacrylamide/8M urea gel (for RPS12 and rRNA detection) or 1.7% agarose/formaldehyde (for A6 and COI mRNA detection).