Ribosome profiling reveals features of normal and disease-associated mitochondrial translation

Abstract

Mitochondria are essential cellular organelles for generation of energy and their dysfunction may cause diabetes, Parkinson's disease and multi-systemic failure marked by failure to thrive, gastrointestinal problems, lactic acidosis and early lethality. Disease-associated mitochondrial mutations often affect components of the mitochondrial translation machinery. Here we perform ribosome profiling to measure mitochondrial translation at nucleotide resolution. Using a protocol optimized for the retrieval of mitochondrial ribosome protected fragments (RPFs) we show that the size distribution of wild-type mitochondrial RPFs follows a bimodal distribution peaking at 27 and 33 nucleotides, which is distinct from the 30-nucleotide peak of nuclear RPFs. Their cross-correlation suggests generation of mitochondrial RPFs during ribosome progression. In contrast, RPFs from patient-derived mitochondria mutated in tRNA-Tryptophan are centered on tryptophan codons and reduced downstream, indicating ribosome stalling. Intriguingly, long RPFs are enriched in mutated mitochondria, suggesting they characterize stalled ribosomes. Our findings provide the first model for translation in wild-type and disease-triggering mitochondria.

Figure 1. A modified ribosomal profiling protocol improves detection of mitochondrial translation.

(a) A plot showing the TE as a function of mRNA abundance in a standard ribosomal profiling protocol. Mitochondrial and histone genes (in green and red, respectively) appear as outside groups. (b) Western blot showing the abundance of mitochondrial (mtRPL11) and cytosolic (RPL10a, RPL7 and RPS6) ribosomal proteins in different sucrose gradient fractions after RNAse I treatment. (c) Size distribution of RPFs in the standard ribosome profiling. The upper panel shows all reads, the lower panel shows nuclear-encoded genes (red) and mitochondrially encoded genes (green) separately. (d) Overview of our modified ribosome profiling protocol. (e) Size distribution of RPFs obtained by our modified ribosome profiling. (f) Same as in a for modified protocol. Marked improvement in the detection of TE in mitochondrial genes is observed.

Figure 2. Reproducibility and quality control of modified ribosome profiling protocol.

(a) Correlation between ribosome profiling data set replicates in the modified ribosome profiling protocol. (b) Correlation between RNAseq data sets in the modified ribosome profiling samples. (c) In-frame RPF abundance for nuclear-encoded genes in two modified ribosome profiling replicate samples. Error bars indicate standard deviations across the genes with at least 100 RPFs (n=5579 for sample #1 and n=7108 for sample #2). (d) Abundances of mRNA and contamination in the conventional and modified ribosome profiling protocol samples. (e) Coverage around START and STOP codons in modified ribosome profiling sample #1.

Figure 3. Size distribution of mitochondrial RPFs suggests mechanism of action.

(a) The normalized abundance of short and long RPFs along the 13 protein-coding mitochondrial genes. (b) Correlation between the 27 nt length (short) and 33 nt length (long) mitochondrial RPFs averaged over all mitochondrial protein-coding genes (n=13) in the control sample #1. Error bars indicate s.d.’s. (c) Normalized frame abundance of short and long RPFs on all mitochondrial protein coding genes. Error bars indicate s.d. across the mitochondrial protein-coding genes. (d) A schematic model showing how the long and short fragments might be produced during mitochondrial ribosome procession.

Figure 4. tRNA(Trp)^5556G>A causes mitochondria malfunctioning and an increase of RPFs at tryptophan codons.

(a) Screenshots from the UCSC genome browser showing RPFs of MT-CO2 gene in wt and tRNA(Trp)^5556G>A mutant cybrids. (b) Differential codon abundance in the tRNA(Trp)^5556G>A mutant cybrids compared with control wt. (c). The tRNA(Trp)^5556G>A mutant cybrid (red line) shows increased RPF density with respect to wt controls (gray line) centered at tryptophan codon but not at a phenylalanine codons, nor at nuclear encoded codons (right panels). (d) Quantitative reverse transcriptase PCR (qRT–PCR) of tRNA(TRP) and control tRNA(GLU) from total RNA extracts from cybrids with either (Trp)^5556G>A mutant or wt mitochondria (upper panel). Extracts from cybrids were immunoprecipitated by anti-mt-RPL11 or control IgG antibodies. Immunoprecipitated RNAs were detected by qRT–PCR using primers for mt-tRNA(TRP) and mt-tRNA(GLU) (lower panel). Mean values and s.d.’s were calculated from three independent experiments. (e) Log-transformed translational efficiencies in the (Trp)^5556G>A mutant versus the wt control. Mitochondrially encoded genes are highlighted in green.

Figure 5. tRNA(Trp)^5556G>A stalls ribosomes at tryptophan codons and causes a change in RPF size distribution.

(a) RPF density from the 5′ end of transcripts up to the first tryptophan codon occurrence averaged over the 13 protein-coding mitochondrial transcripts. (b) Cumulative RPF density normalized over the 13 protein-coding mitochondrial transcripts. (c) Example of the cumulative density of RPF density along the mitochondrial genes MT-ND4L (having no Trp codons), MT-ND3 and MT-ND5, in wt and tRNA(Trp)^5556G>A mutant cybrids. (d) The relative abundance of RPF length in control and tRNA(Trp)^5556G>A mutant cybrids.

Figure 6. Model for ribosome stalling.

(a) Schematic model for ribosome stalling at codons translated by affected tRNAs, explaining the unbalanced generation of long RPFs in mutant mitochondria.