Mitochondrial Polyadenylation Is a One-Step Process Required for mRNA Integrity and tRNA Maturation

Abstract

Polyadenylation has well characterised roles in RNA turnover and translation in a variety of biological systems. While polyadenylation on mitochondrial transcripts has been suggested to be a two-step process required to complete translational stop codons, its involvement in mitochondrial RNA turnover is less well understood. We studied knockdown and knockout models of the mitochondrial poly(A) polymerase (MTPAP) in Drosophila melanogaster and demonstrate that polyadenylation of mitochondrial mRNAs is exclusively performed by MTPAP. Further, our results show that mitochondrial polyadenylation does not regulate mRNA stability but protects the 3' terminal integrity, and that despite a lack of functioning 3' ends, these trimmed transcripts are translated, suggesting that polyadenylation is not required for mitochondrial translation. Additionally, loss of MTPAP leads to reduced steady-state levels and disturbed maturation of tRNACys, indicating that polyadenylation in mitochondria might be important for the stability and maturation of specific tRNAs.

Fig 1. CG11418 is an essential mitochondrial poly(A) polymerase.

(A) Immunocytochemistry of HeLa cells (top panel) and Schneider 2R+ (S2R+, bottom panel) expressing a GFP-tagged CG11418 fusion protein (DmMTPAP-GFP). The mitochondrial network was visualised with TOM20 antibodies in HeLa cells or Mitotracker Red in S2R+ cells, respectively. Scale bars represent 20 μm (top panel) and 5 μm (bottom panel). (B) Ubiquitous silencing of CG11418 resulted in significant reduction of CG11418 transcript levels in both DmMTPAP KD lines (black and checked bars) in contrast to the control lines (white, grey and striped bars). Transcript levels were normalised to cytosolic ribosomal protein 49 (RP49) mRNA levels in 5 days after egg laying (ael) larvae (n = 5). Data are represented as mean ± SD (***P < 0.001, *P < 0.05). (C) Body sizes of DmMTPAP KD (black and checked bars) were significantly larger than control 6-day-ael larvae (white, grey and striped bars) (n = 20). Data are represented as mean ± SD (***P < 0.001). (D) The relative eclosure rates of DmMTPAP KD flies (black and checked bars) were significantly lower, when compared to control flies (white, grey and striped bars) (n = 5). Data are represented as mean ± SD (***P < 0.001). Individual mitochondrial (E) mRNAs or (F) rRNAs from 5 days ael larvae were cloned and sequenced to determine poly(A) tail length of various transcripts in DmMTPAP KD (red, DmMTPAP RNAi #1) and control (grey; daGAL4 control). Mean poly(A) tail length in control samples varied between 35 and 49 adenines (grey; n = 4–14), while DmMTPAP KD samples had mainly reduced poly(A) tail lengths. The annotated 3' termini of the indicated transcripts was set to zero to determine poly(A) tail length. Data are represented as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0,001), using a Mann-Whitney test.

Fig 2. MTPAP is the only mitochondrial adenylase in flies and is required to protect the 3' termini of mRNAs.

(A) Body size comparison in control (wt and FM7,Tb) and DmMTPAP KO larvae (DmMTPAP^KO) at 4 days ael. (B) qRT-PCR analysis of DmMTPAP transcript levels in 1 day heterozygous DmMTPAP^KO flies (DmMTPAP^KO/FM7,Tb) and 4-day-old hemyzygous DmMTPAP^KO larvae (DmMTPAP^KO) and their corresponding age-matched controls (wt, FM7,Tb). Histone 2B transcript was used as endogenous control. Data is represented as mean ± SEM (*P < 0.05, ***P < 0.001, n = 5). (C) mRNA and poly(A) tail length in individually sequenced clones after transcript circularisation (MTATP6/8, MTND4/4L, MTND1 and MTND5) or 3' RACE (MTCOX1 and MTCYTB) in DmMTPAP^KO (red, n = 14–26) and control larvae (grey, n = 11–25) at 4 days ael. The annotated 3' termini of the indicated transcripts was set to zero to determine poly(A) tail length. (D) rRNA and poly(A) tail length in individually sequenced clones after transcript circularisation in DmMTPAP^KO (red, n = 17–25) and control larvae (grey, n = 24–29) at 4 days ael. Data are represented as mean ± SEM. (***P < 0,001), using a Mann-Whitney test.

Fig 3. Impaired 3' termini do not affect the stability of most mtDNA-encoded transcripts.

(A) Relative steady-state level of mitochondrial transcripts were determined by Northern Blot in 5 day ael control (white and grey bars) and DmMTPAP KD larvae (black bar) larvae (n = 5). Expression levels were quantified using a Typhoon phosphorimager and normalised to histone 2B mRNA. All data are represented as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0,001). (B) Northern blot analysis and (C) quantification of steady-state levels of mitochondrial transcripts in control (wt and FM7,Tb) and DmMTPAP KO larvae (DmMTPAP^KO) at 4 days ael. Histone 2B transcript was used as loading control. (D) De novo mitochondrial transcription in isolated mitochondria of control and DmMTPAP KO larvae at 4 days ael in the presence of radioactively labelled [³²P]-UTP. Loading of the gels and absence of RNA degradation was confirmed by Northern blotting against COX1 and 16S RNAs. Western blotting of VDAC in the input samples was used as a loading control. (E) qPCR of mtDNA steady-state levels DmMTPAP KO and control larvae at 4 days ael. Primers against the cytosolic ribosomal protein 49 (RP49) were used to normalise to nuclear DNA content of the samples.

Fig 4. tRNA^Cys is not maturing correctly in the absence of MTPAP.

(A) Neutral PAGE, Northern Blot analysis and (B) quantification of mitochondrial tRNA steady-state levels of DmMTPAP KO larvae (DmMTPAP^KO) and control (wt and FM7,Tb) larvae at 4 days ael. rRNA 5.8S was used as a loading control. Data is represented as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0,001, n = 5). (C) 3' end sequencing of individual clones of tRNA^Val and tRNA^Cys in 3' RACE experiments of control (wt) and DmMTPAP^KO RNA samples at 4 days ael. tRNAs were grouped according to the presence of the complete CCA tail (CCA, black), incomplete CCA (CC- or C-, pale grey) or incorrect tail (detailed in S3 Table, dark grey). (D) Aminoacylation status of mitochondrial tRNAs was analysed in acid-urea PAGE followed by Northern blotting in 4-day-old control (wt) and DmMTPAP^KO larvae. Basic conditions (OH-) deaminoacylate all of the analysed tRNAs. Cytosolic tRNA^Tyr (cit-tRNA^Tyr) was used as a control of tRNA aminoacylation in each sample.

Fig 5. Polyadenylation is not required for mitochondrial translation.

(A) In organello labelling of mitochondrial translation products on isolated mitochondria from DmMTPAP KD (DmMTPAP RNAi #1) and control (w;;daGAL4/+ and w;UAS-mtPAPRNAi#1/+) 5 days ael larvae. Labelling was performed for 60min (pulse), followed by a 15 or 45 min chase with cold methionine. Loading was normalised to VDAC levels. (B) In organello labelling of mitochondrial translation products on isolated mitochondria from DmMTPAP KO (DmMTPAP^KO) and control (wt and FM7,Tb) 4-day-old larvae. Coomassie staining of the gels and VDAC Western blotting of the input samples were performed to ensure equal loading of the samples. Western blot analysis (C) and quantification (D) of nuclear-encoded subunit of Complex I (NDUFS3) in isolated mitochondria from control (daGAL4 control, RNAi #1 control and RNAi#2 control) and DmMTPAP KD (DmMTPAP RNAi #1 and DmMTPAP RNAi #2) 5-day-old larvae. VDAC was used as a loading control. Western blot analysis (E) and quantification (F) of the steady-state levels of a nuclear-encoded subunit of Complex I (NDUFS3) and an mtDNA-encoded subunit of complex IV (COX3) in mitochondria of control (wt and FM7,Tb) and DmMTPAP^KO 4-day-old larvae. VDAC was used as a loading control. Data are represented as mean ± SD.

Fig 6. Mitochondrial respiration is affected due to incomplete OXPHOS assembly.

(A) BN-PAGE and in gel staining of Complex I and Complex IV activities in mitochondrial protein extracts from control (wt and FM7,Tb) and DmMTPAP KO (DmMTPAP^KO) 4-day-old larvae. Coomassie staining of the gel and VDAC western blot of the input samples was performed to ensure equal loading of the gel. (B) Complex V assembly was assessed in DmMTPAP KD (DmMTPAP RNAi #1) 5-day-old larvae by BN-PAGE, followed by Western blot analysis against the F1 subunit of Complex V. Coomassie staining was used to ensure equal loading. (C) Oxygen consumption rates in permeabilised 4-day-old control (wt) and DmMTPAP^KO larvae, using glutamate, malate and pyruvate (GMP + ADP), succinate (GMP + ADP + succ) or TMPD and ascorbate (TMP + asc) as electron donors. Data are normalized to the protein content in each sample and are represented as mean ± SEM (***P < 0,001, n = 8). (D) Relative enzyme activities of respiratory chain complexes in 4-day-old control (wt) and DmMTPAP^KO larvae. Data are represented as mean ± SD (*P<0.05, **P < 0.01, ***P < 0,001, n = 3). (E) Relative enzyme activities of respiratory chain complexes (Complex I-IV) from control (white, grey and striped bars) and DmMTPAP KD (checked and black bars) 5-day-old larvae. Data is represented as mean ± SEM (**P < 0.01, ***P < 0,001, n = 5).