A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression.
Bottom Line: 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.
Affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute for Ageing and Health.Show MeSH
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Mentions: Depletion of mtPAP, mediated by siRNAs targeting the MTPAP transcript, has been shown by several groups to result in transcript-specific effects on the steady-state levels of mt-mRNAs in cultured human cell lines (14–16). In the homozygote patient lines (Fig. 2A lanes 2 and 3), we also saw transcript-specific effects, where steady-state levels of RNA14 and all MTCO were decreased, MTND1 increased and MTND3 unaffected. A similar trend had been noted when mt-transcripts lacked polyadenylated termini as a consequence of the poly(A) tails being removed by a cytosolic poly(A)-specific ribonuclease that had been targeted to mitochondria (17). To determine what effect these altered steady-state levels, caused by the mutant mtPAP, had on the translation of these transcripts, de novo metabolic labelling was performed. Autoradiographic data analysis of the homozygote compared with the control indicated that there were varied but reproducible differences in the amount of translation products from different transcripts (Fig. 2B). There was an evident decrease in de novo synthesis of the components of the COX2/3 ATP6 triplet, which was in contrast to the increase in ND5 and ND2. Those transcripts that showed decreased stability, such as MTCO3, showed a concomitant decrease in translation product. However, there was no consistent correlation between transcript level and translation as the dramatic increase in stability of MTND1 (Fig. 2A) did not result in a concomitant increase in ND1 (Fig. 2B).Figure 2.
Affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute for Ageing and Health.