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.
