LRPPRC-mediated folding of the mitochondrial transcriptome

Abstract

The expression of the compact mammalian mitochondrial genome requires transcription, RNA processing, translation and RNA decay, much like the more complex chromosomal systems, and here we use it as a model system to understand the fundamental aspects of gene expression. Here we combine RNase footprinting with PAR-CLIP at unprecedented depth to reveal the importance of RNA-protein interactions in dictating RNA folding within the mitochondrial transcriptome. We show that LRPPRC, in complex with its protein partner SLIRP, binds throughout the mitochondrial transcriptome, with a preference for mRNAs, and its loss affects the entire secondary structure and stability of the transcriptome. We demonstrate that the LRPPRC-SLIRP complex is a global RNA chaperone that stabilizes RNA structures to expose the required sites for translation, stabilization, and polyadenylation. Our findings reveal a general mechanism where extensive RNA-protein interactions ensure that RNA is accessible for its biological functions.

Fig. 1

High-throughput RNase footprinting. a Schematic showing the principle of high-throughput RNA footprinting. b A circular representation of the mitochondrial genome (centre track) displaying LRPPRC footprints (exterior tracks) colored by the log₂ fold change of F score (scale: 0.0–2.5). Control samples were scanned for footprints and compared to Lrpprc knockout samples to identify LRPPRC-binding sites. In the control samples we identify regions with F scores <2 that indicate lower RNase accessibility in the central footprint relative to the flanking regions. The control F scores are compared to the F scores identified for each footprint in the Lrpprc knockout samples and the significant log₂ fold changes between the control and Lrpprc knockout mice are shown

Fig. 2

PAR-CLIP analysis of LRPPRC-binding sites. a Circular representation of the mitochondrial genome (centre track) displaying the PAR-CLIP-binding site regions (interior tracks) and normalized coverage (log₁₀ (reads per million); scale: 1–100,000) across positions with at least a 95% posterior probability of being cross-linked (exterior tracks), as identified by BMix. b RNA EMSA of the LRPPRC–SLIRP complex, LRPPRC only or SLIRP only incubated with a RNA probe identified as a strong LRPPRC–SLIRP-binding motif in vivo. c Schematic of shared binding sites among footprinting and PAR-CLIP data sets; the significance (P) of the overlap is indicated. d A sequence logo shows a predicted consensus binding motif for LRPPRC based on the binding sites that overlap between footprinting and PAR-CLIP data sets

Fig. 3

LRPPRC footprints show an increase in secondary structure propensity with its loss. a Circular representation of the mitochondrial genome (centre track) displaying footprints that are present in the Lrpprc knockout mice but not the controls (exterior tracks) colored by the log₂ fold change of the F score (scale: 0.0–−2.5). The negative values indicate that we have compared the footprints present when LRPPRC is lost to control samples. b A footprint located in mt-Nd5, showing the increased C score (scale: −4.3–3.7) and R score (scale: −0.5–1.0) across the footprint region in the knockout. c Violin plot showing the distribution of the log₂ fold change in average R score across all LRPPRC footprints that overlap PAR-CLIP-binding sites across the mitochondrial transcriptome, compared to the left and right 10 nt flanking regions. Loss of LRPPRC causes an increase in secondary structure propensity of mitochondrial RNAs. d LRPPRC demonstrates RNA chaperone activity in RNA annealing assays. Complementary oligoribonucleotides were hybridized in the presence or absence of the mouse LRPPRC–SLIRP complex

Fig. 4

LRPPRC–SLIRP complex binding is conserved in humans and mice. a Transcriptome-wide distribution of binding sites in HeLa and MEFs expressing LRPPRC-FLAG determined by PAR-CLIP. b The locations of binding sites of the LRPPRC–SLIRP complex within mt-Cyb are well conserved in human and mouse. c RNA EMSA indicating the preference of the LRPPRC–SLIRP complex for its target within each species