A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression

Abstract

The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.

TEFM regulates both transcription elongation and RNA processing in mitochondria

“…As expected, our results confirmed that TEFM interacts with POLRMT (Fig ), as previously reported . In addition, we identified a number of RNA processing‐related proteins that interacted with TEFM, including the RNase P complex (MRPP1‐3), GRSF1, FASTKD5, ELAC2, mtPAP, and the components of mitochondrial RNA degradosome (SUPV3L1‐PNPase) complex (Fig ), which are all reported to be localized in or adjacent to RNA granules . Western blot analyses of the eluates confirmed the association between RNA processing factors (ELAC2, GRSF1, and MRPP1) and TEFM (Fig EV5B).…”

Mitochondrial RNA Granules Are Centers for Posttranscriptional RNA Processing and Ribosome Biogenesis

“…The proteins we identified in this analysis corroborate recent studies showing that the subunits of RNase P Jourdain et al, 2013), RNase Z and some structural subunits of the mitochondrial ribosome , an rRNA methyltransferase (RNMTL1) (Lee et al, 2013), mtPAP, the mitochondrial poly(A) polymerase (Wilson et al, 2014), and components of the mRNA degradation complex SUPV3L1 and PNPase also localize to GRSF1/BrU-positive foci (or foci adjacent to nucleoids). Immunoprecipitation analysis of GRSF1, DHX30, DDX28, FASTKD2, and FASTKD5 from UVcrosslinked cells (Table S3) further reinforces the notion that these proteins form a core interactome of the RNA granules.…”

The Pseudouridine Synthase RPUSD4 Is an Essential Component of Mitochondrial RNA Granules

“…The newly identified proteins are implicated in mtRNA processing (FASTKD1, PTCD1, and PTCD2), mtRNA modification (TFB1M, TRUB2, RPUSD3, and RPUSD4), mitoribosome assembly (ERAL1 and NOA1/C4orf14), and mitoribosome structure (MRPL47, MRPS7, and MRPS9). This expands the previously established list of MRG-associated proteins that includes GRSF1 (23, 24), FASTK (20), FASTKD2 (19, 20), FASTKD5 (19), MRPP1 (23, 25), DDX28 (26), MRM1–3 (27, 28), and MTPAP (29). Thus, we provide further evidence that MRGs are intimately involved in all stages of mitochondrial gene expression from the accumulation of newly synthesized RNA (14, 23) to the assembly of the mitoribosomes (19, 26, 30).…”