Mitochondrial gene eXpression

Project Information

MiXpress

Grant agreement ID: 101054637

DOI

10.3030/101054637

EC signature date 4 October 2022

Start date 1 May 2023

End date 30 April 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 913 968,00

EU contribution

€ 1 913 968,00

Coordinated by

UNIVERSITAETSMEDIZIN GOETTINGEN - GEORG-AUGUST-UNIVERSITAET GOETTINGEN - STIFTUNG OEFFENTLICHEN RECHTS
Germany

Periodic Reporting for period 1 - MiXpress (Mitochondrial gene eXpression)

Reporting period: 2023-05-01 to 2025-10-31

Summary of the context and overall objectives of the project

Mitochondrial gene expression is essential for cellular metabolism and energy supply since 13 core subunits of the OXPHOS system are encoded on the mitochondrial genome. Despite its importance for cellular function, mitochondrial gene expression (mitoGE) and its regulation are not understood at a mechanistic level. To this end, we demonstrated that mitochondrial translation is prone to regulation, responding to influx of nuclear-encoded proteins (Mick et al., Cell 2012; Richter-Dennerlein,et al. Cell 2016). However, the mechanisms that regulate gene expression in mitochondria remain unknown. A lack of suitable experimental approaches to modulate mitoGE hampers progress in our understanding. Our recent work on an in organello system to target mitoGE in a transcript-specific manner provides the bases for this project. We aim to solve long-standing questions on mitochondrial gene expression eventually providing insights into components and mechanism of mitoGE and reveal how two genetically independent systems cooperate to build a functional metabolic pathway able to respond to energetic requirements and challenges. Considering that dysfunction of mitoGE are linked to severe human disorders, our findings will also provide a better understanding of the underlying molecular pathologies.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The MiXpress project investigates mitochondrial gene expression by using a technology that silences specific mitochondrial mRNAs in purified mitochondria, blocking their translation within minutes to an hour.
This method, originally limited to in vitro assays, has now been adapted for living cells by creating new chimeras and developing broadly applicable transfection techniques. The approach allows multiplexed targeting of multiple mRNAs and enabled the study of mitochondrial proteome changes and the identification of new OXPHOS biogenesis factors. Silencing individual mitochondrial mRNAs in cells revealed that nuclear gene expression responses are highly specific to the targeted transcript.
We found that the construction of new and more efficient morpholino chimera enables us to multiplex our approach and to target selected mRNAs in combination. The time resolved block in translation of individual mRNAs has enabled us to monitor proteomic changes in mitochondria between 8 and 72 hours and thereby to follow the decline of individual OXPHOS complex functions and how they are linked to other mitochondrial functions. We are able to monitor metabolic adaptations in response to complex IV deficiency - the cells switch to glycolysis. (Cruz-Zaragoza et al, 2025). Together with the group of Stefan Jakobs we have been able to establish a method that allows us to detect individual mitochondrial mRNA molecules in cells (Stoldt et al., 2025).
We previously established a method that allows to identify mitochondrial translation products by microscopy in cells (mitoFUNCAT). We now used this approach to screen siRNA libraries and to identify kinases that are linked to mitochondrial translation (Yousefi et al., 2025).
We established ribosome profiling for mitochondrial transcripts. This approach enabled us to monitor and proof that the chimera inhibit translation initiation. Moreover, we found that in mitochondria the speed of decoding is linked to the membrane insertion process of the synthesized polypeptide chain. This manuscript is currently in revision.
Along the lines of mRNA specific protein interactors, we have now been able to establish a robust protocol for Chimera import, mRNA purification, and mass spectrometric identification. These analyses are ongoing and are very promising. In addition to the identified proteins, we also recognize mRNA interaction patterns that point towards different mRNA environments and potentially phase separating patterns.This aspect of the work is ongoing.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The development of peptide-morpholino chimera and the technology to silence mitochondrial gene expression in living cells is a breakthrough for the field of mitochondrial research. The goal of the project had been to establish this approach based on our previously published in vitro system. Our new technology represents a game changing advance for the research field. The establishment of an imaging approach to monitor mRNA molecules in cells is a central new approach for the research field.