Using mitoribosomal profiling to investigate human mitochondrial translation

• Published in: Wellcome open research 2017, Vol.2, p.116
• Main Authors: Gao, Fei, Wesolowska, Maria, Agami, Reuven, Rooijers, Koos, Loayza-Puch, Fabricio, Lawless, Conor, Lightowlers, Robert N, Chrzanowska-Lightowlers, Zofia M A
• Format: Article
• Language: English
• Subjects: Experimental Biophysical Methods; Genomics
• ISSN: 2398-502X; 2398-502X
• DOI: 10.12688/wellcomeopenres.13119.1

: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNA whilst in humans it is mt-tRNA . We have previously shown that when a mutation in mt-tRNA causes very low steady state levels, there is preferential recruitment of mt-tRNA . We have investigated whether this altered mitoribosome affects intra-organellar protein synthesis. : By using mitoribosomal profiling we have revealed aspects of mitoribosome behaviour with its template mt-mRNA under both normal conditions as well as those where the mitoribosome has incorporated mt-tRNA . : Analysis of the mitoribosome residency on transcripts under control conditions reveals that although mitochondria employ only 22 mt-tRNAs for protein synthesis, the use of non-canonical wobble base pairs at codon position 3 does not cause any measurable difference in mitoribosome occupancy irrespective of the codon. Comparison of the profile of aberrant mt-tRNA containing mitoribosomes with those of controls that integrate mt-tRNA revealed that the impaired translation seen in the latter was not due to stalling on triplets encoding either of these amino acids. The alterations in mitoribosome interactions with start codons was not directly attributable to the either the use of non-cognate initiation codons or the presence or absence of 5' leader sequences, except in the two bicistronic RNA units, and where the initiation sites are internal. : These data report the power of mitoribosomal profiling in helping to understand the subtleties of mammalian mitochondrial protein synthesis. Analysis of profiles from the mutant mt-tRNA cell line suggest that despite mt-tRNA being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs.