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, 109 (13), 4840-5

Correcting Human Mitochondrial Mutations With Targeted RNA Import

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Correcting Human Mitochondrial Mutations With Targeted RNA Import

Geng Wang et al. Proc Natl Acad Sci U S A.

Abstract

Mutations in the human mitochondrial genome are implicated in neuromuscular diseases, metabolic defects, and aging. An efficient and simple mechanism for neutralizing deleterious mitochondrial DNA (mtDNA) alterations has unfortunately remained elusive. Here, we report that a 20-ribonucleotide stem-loop sequence from the H1 RNA, the RNA component of the human RNase P enzyme, appended to a nonimported RNA directs the import of the resultant RNA fusion transcript into human mitochondria. The methodology is effective for both noncoding RNAs, such as tRNAs, and mRNAs. The RNA import component, polynucleotide phosphorylase (PNPASE), facilitates transfer of this hybrid RNA into the mitochondrial matrix. In addition, nucleus-encoded mRNAs for mitochondrial proteins, such as the mRNA of human mitochondrial ribosomal protein S12 (MRPS12), contain regulatory sequences in their 3'-untranslated region (UTR) that confers localization to the mitochondrial outer membrane, which is postulated to aid in protein translocation after translation. We show that for some mitochondrial-encoded transcripts, such as COX2, a 3'-UTR localization sequence is not required for mRNA import, whereas for corrective mitochondrial-encoded tRNAs, appending the 3'-UTR localization sequence was essential for efficient fusion-transcript translocation into mitochondria. In vivo, functional defects in mitochondrial RNA (mtRNA) translation and cell respiration were reversed in two human disease lines. Thus, this study indicates that a wide range of RNAs can be targeted to mitochondria by appending a targeting sequence that interacts with PNPASE, with or without a mitochondrial localization sequence, providing an exciting, general approach for overcoming mitochondrial genetic disorders.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
H1 RNA import sequence regulates mitochondrial import of mt-tRNA precursors. (A) Hepatocyte-specific Pnpt1 knockout (HepKO) in 6-wk-old mice (6). Immunoblot from 6-wk-old WT and HepKO mouse livers shows ∼50% reduction in PNPASE expression. (B) Radiolabeled mt-tRNA precursors with (Upper) a 5′ H1 20-ribonucleotide predicted stem-loop sequence (designated RP, marked with loop in schematic) were in vitro transcribed and incubated with WT or HepKO liver mitochondria. Nonimported RNA was digested with added nuclease, followed by RNA isolation, separation on a urea acrylamide gel, and autoradiography. Import reactions were repeated with 1× and 2× amounts of mt-tRNA. (Lower) Loading control showing equivalent amounts of mitochondria used in the imports, as revealed by total mitochondrial nucleic acids separated on an agarose gel. (C) As in B, but the mt-tRNA precursor lacks the 5′ H1 20-ribonucleotide predicted stem-loop sequence.
Fig. 2.
Fig. 2.
Import mt-tRNA precursors with the RP sequence partially rescue the translation defect of isolated MERRF and MELAS mutant mitochondria. (A) mt-tRNA precursors with or without RP were imported into isolated WT or MERRF mitochondria from cybrid lines for 2 min at RT, followed by an additional 5 min with rNTP supplementation. Following RNase A digestion of the nonimported mt-tRNA, mitochondria were pelleted and resuspended in an in organello translation buffer with radiolabeled methionine and cysteine for 30 min at 37 °C. The autoradiograms are shown in Fig. S1. Individual lanes were quantified, and total radioactivity was calculated and normalized to total protein amounts. The WT control in the presence of assay buffer was set at 100%. n = 3 independent experiments. *P < 0.01 with Student's t test. (B) As in A with WT and MELAS cybrid cell lines. n = 3 independent experiments. *P < 0.01 with Student's t test.
Fig. 3.
Fig. 3.
In vivo import of mitochondrial-coded COX2 into mitochondria, using the RP sequence. (A) Diagrams of mCOX2 expression vectors. (B) Mitochondria were isolated from HeLa cells expressing mCOX2 or RP-mCOX2. Mitoplasts were made with digitonin, followed by treatment with nuclease. RNA was then isolated from total cell lysates (Input) or from nuclease-treated mitoplasts (Mito) and analyzed by primer-specific RT-PCR. hCOX1 is a control for total and mitochondria-isolated RNAs. (C) Mitochondria were isolated from mouse embryonic fibroblasts stably expressing hCOX2 or RP-hCOX2. hCOX2-specific expression was analyzed by Western blot from isolated mitochondria.
Fig. 4.
Fig. 4.
Three elements are required for mt-tRNA precursors encoded in the nucleus to rescue mt-tRNA respiratory defects in vivo. (A) Schematic of the mt-tRNA precursors generated for the in vivo rescue assay. The single step loop at the 5′ of the second row structures is the H1 RNA import sequence, RP. The shaded box indicates ribonucleotides that were changed to make tRNA precursors less susceptible to processing in the nucleus. The solid box is the 3′-UTR of MRPS12 that localizes RNA to the vicinity of mitochondria (32). (B) tRNALys precursors lacking one or two of the three elements do not rescue the MERRF respiratory defect. (C) tRNALys or tRNALeu precursors with all three elements rescue respiration in MERRF and MELAS cells. n = 3 independent experiments. *P < 0.01 with Student's t test.
Fig. 5.
Fig. 5.
Rescue of respiration is due to restoration of mitochondrial translation. (A) Analysis of mitochondrial translation in vivo with stable rescue cell lines. Mitochondrial translated proteins were separated by SDS/PAGE and visualized by autoradiography. (B) Quantification of specific bands on gels from A. Autoradiogram counts were normalized to protein amounts and expressed relative to WT control samples. (C) Steady-state levels of nucleus-encoded and mitochondrial-encoded proteins in WT, MERRF, and MELAS cells. TOMM40 and PNPASE also serve as loading controls.

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