An in vitro system to silence mitochondrial gene expression
In brief
The mitochondrial genome encodes core subunits of the OXPHOS system. Imported precursor-morpholino chimera efficiently blocks the translation of mitochondrial mRNAs. Translation of bicistronic mRNAs was shown to depend on translation of the upstream open reading frame. IGF2BP1 represents a mitochondrial RNA- and ribosomeinteracting protein required for mitochondrial translation.
|核心内容：
人类线粒体基因组编码氧化磷酸化系统的13个核心亚基，线粒体基因表达缺陷导致严重的神经肌肉疾病。
然而，由于缺乏分析这些过程的实验方法，线粒体基因表达的机制尚不清楚。
在这里，我们提出了一个体外系统来沉默纯化线粒体的翻译。
在体外输入化学合成的前体-吗啉杂交体，使我们能够靶向单个线粒体mrna的翻译。通过应用这种方法，我们得出结论，双反子、重叠的ATP8/ATP6转录本通过单个核糖体/mRNA参与进行翻译。
我们发现，COX1组装因子的招募到翻译核糖体依赖于新生链的形成。
通过定义COX1和COX2的mrna特异性相互作用组，我们揭示了胞质癌胎IGF2BP1在线粒体翻译中的一个意想不到的功能。
我们的数据为线粒体翻译和研究线粒体基因表达的创新策略提供了见解。原文摘要：
The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders.
However, the mechanisms of mitochondrial gene expression remain poorly understood due to a lack of experimental approaches to analyze these processes.
Here, we present an in vitro system to silence translation in purified mitochondria.
In vitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs.
By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement.
We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation.
Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression.
INTRODUCTIONMitochondria shape their proteome through the import of nuclear-encoded proteins and expression of their own genome.
The mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation (OXPHOS) system in the inner mitochondrial membrane that converts energy from reduction equivalents into ATP.
Mitochondrial gene expression requires transcription of the mitochondrial genome into two polycistronic transcripts by the mitochondrial RNA (mRNA) polymerase POLRMT.
The primary transcripts are processed to form eleven mRNAs, two rRNAs, and 22 tRNAs. Two of the transcripts, ATP8/ ATP6 and ND4L/ND4, are bicistronic. mtPAP, the mitochondrial poly(A) polymerase, mediates polyadenylation of mRNAs (Ha¨ llberg and Larsson, 2014; Pearce et al., 2017).
These maturation steps are thought to occur at defined foci termed ''mitochondrial RNA granules,’’ which also harbor stages of ribosome maturation and RNA turnover (Antonicka and Shoubridge, 2015; Pearce et al., 2017).
After processing, mitochondrial mRNAs are translated by membrane-associated mitochondrial ribosomes that enable co-translational insertion of synthesized polypeptide chains into the lipid bilayer.
For this, ribosomes are bound to the conserved OXA1L insertase in the membrane (Itoh et al., 2021; Ott et al., 2016; Richter-Dennerlein et al., 2015).
The newly synthesized mitochondrial-encoded proteins have to associate with assembly factors in the inner membrane, which stabilize these polypeptides and maintain them competent to receive cofactors and imported, nuclear-encoded OXPHOS complex subunits.
To balance mitochondrial translation with the availability of nuclear-encoded subunits, translation responds to the influx of these proteins (Couvillion et al., 2016; Richter-Dennerlein et al., 2016).
Stalled ribosome nascent chain complexes seem to play a key role in this regulatory process, but the mechanisms underlying mitochondrial translation regulation remain open (Mai et al., 2017; Pearce et al., 2017; Richter-Dennerlein et al., 2016).
Considering the importance of OXPHOS for cellular function, it is not surprising that defects at every step of the mitochondrial gene expression process have been linked to human disorders (Brischigliaro and Zeviani, 2021; Fontanesi and Barrientos, 2013).
Most of the patients display neuromuscular pathologies, which have been attributed to the especially high energy demands of these tissues.
Nevertheless, basic principles of mitochondrial gene expression are still not understood.
While recent structural analyses of mitochondrial POLRMT (Hillen et al., 2017a, 2017b) and ribosomes (Amunts et al., 2015; Greber et al., 2015; Itoh et al., 2021) provide us with insight into the machineries of gene expression, we still lack understanding of the mechanisms of these processes and how gene expression is integrated into the cellular and mitochondrial contexts.
A lack of appropriate techniques to target the individual steps of gene expression represents a major obstacle to advancing our understanding.
A major problem in the development of new strategies is the transport of RNAs into mitochondria that would allow us to target mitochondrial RNAs and thereby interfere with gene expression processes.
Here, we present an in vitro approach to target translation of mitochondrial mRNAs.
Chemically synthesized protein-morpholino chimera, which are imported into purified mitochondria, allow us to specifically stall translation of selected mRNAs.
Utilizing this strategy, we show that ribosome association to proteinspecific assembly factors in the inner membrane occurs through nascent chain translation intermediates.
In addition, we investigated the mechanism of translation of the bicistronic, overlapping ATP8/ATP6 mRNA.
Our findings indicate that a single ribosome/RNA encounter mediates expression of ATP8 and ATP6.
Silencing translation of individual mRNAs allowed us to define early assembly intermediates of the OXPHOS system.
Lastly, purification of chimera-associated mRNAs enabled us to define mRNA-specific interactomes and identified the cytosolic RNAbinding IGF2BP1 as an unidentified mitochondrial protein that interacts with mitochondrial ribosomes and mRNAs to regulate mitochondrial translation.
参考文献：An in vitro system to silence mitochondrial gene expression Luis Daniel Cruz-Zaragoza,1 Sven Dennerlein,1 Andreas Linden,2,3 Roya Yousefi,1 Elena Lavdovskaia,1,5 Abhishek Aich,1,5 Rebecca R. Falk,4 Ridhima Gomkale,1 Thomas Scho¨ ndorf,1 Markus T. Bohnsack,4,5 Ricarda Richter-Dennerlein,1,5 Henning Urlaub,2,3 and Peter Rehling1,5,6,7,* 1Department of Cellular Biochemistry, University Medical Center Go¨ ttingen, 37073 Go¨ ttingen, Germany 2Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Go¨ ttingen, Germany 3Department of Clinical Chemistry, University Medical Center Go¨ ttingen, 37073 Go¨ ttingen, Germany 4Department of Molecular Biology, University Medical Center Go¨ ttingen, 37073 Go¨ ttingen, Germany 5Cluster of Excellence, Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells (MBExC), University of Go¨ ttingen, Go¨ ttingen, Germany 6Max Planck Institute for Biophysical Chemistry, 37077 Go¨ ttingen, Germany 7Lead contact *Correspondence: peter.rehling@medizin.uni-goettingen.de
https://doi.org/10.1016/j.cell.2021.09.033
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