Structural studies of the human mitochondrial RNA life cycle

Project Information

MitoRNA

Grant agreement ID: 101116869

DOI

10.3030/101116869

EC signature date 12 September 2023

Start date 1 January 2024

End date 31 December 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 499 754,00

EU contribution

€ 1 499 754,00

Coordinated by

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

Periodic Reporting for period 1 - MitoRNA (Structural studies of the human mitochondrial RNA life cycle)

Reporting period: 2024-01-01 to 2026-06-30

Summary of the context and overall objectives of the project

Mitochondria contain their own genome, whose expression is essential for cellular energy metabolism and human health. Although mitochondrial gene expression relies on specialized molecular machineries, the underlying regulatory principles remain incompletely understood. In particular, the processes governing mitochondrial RNA metabolism and the coordination of gene expression steps within the mitochondrial matrix are poorly characterized. The ERC Starting Grant project MitoRNA aims to advance a mechanistic understanding of mitochondrial gene expression by focusing on mitochondrial RNA biology as a central and previously underexplored regulatory layer. Using an integrated structural biology framework, the project seeks to elucidate key mechanisms of mitochondrial RNA metabolism, how it is organised and how it contributes to the functional coordination of gene expression in human mitochondria. By addressing these fundamental questions, MitoRNA aims to establish a molecular framework for mitochondrial RNA regulation and to open new directions in the study of mitochondrial biology.

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

A central goal of MitoRNA is to elucidate the molecular mechanisms underlying different steps of RNA metabolism. To this end, we have made substantial progress during the reporting period.
In particular, we have uncovered the mechanism of canonical RNA processing in human mitochondria. Mitochondrial transcription produces polycistronic transcripts that contain ribosomal RNA (rRNA) sequences and messenger RNA (mRNA) sequences interspersed by transfer RNA (tRNA) sequences. The primary transcripts are then processed by the endonucleases mitochondrial RNase P and Z, which excise the tRNA segments to liberate all RNAs. During the reporting period, we have determined the structural and molecular basis of 3’ processing of both mitochondrial and nuclear tRNAs by RNase Z (Bhatta et al., Nature Structure and Molecular Biology 2025). This work reveals why the responsible endonuclease ELAC2 requires the auxiliary factors TRMT10C and SDR5C1 for processing of structurally degenerate mitochondrial tRNAs but not for the processing of canonical nuclear-encoded tRNAs. These results not only provide the mechanism of tRNA 3’ processing in human cells but also clarify that RNase Z is a multi-subunit complex in mitochondria. They further reinforce the notion that TRMT10C and SDR5C1 act as a tRNA maturation platform in human mitochondria. Together with our previous work on mitochondrial RNase P (Bhatta et al., Nature Structural and Molecular Biology 2021), we have now defined the complete mechanism of canonical RNA processing in human mitochondria. Finally, we have also determined the mechanism of the next step of tRNA maturation, 3’ CCA-addition by the enzyme TRNT1. Using single-particle cryo-EM, we have determined high-resolution snapshots of TRNT1 in complex with substrate tRNAs at different steps of the reaction cycle. Together with biochemical data, this provides detailed mechanistic insights into tRNA maturation in both mitochondria and the nucleus and the molecular basis of TRNT1-associated diseases.
In addition, we have also provided insights into non-canonical RNA processing in human mitochondria during this reporting period. While most mRNAs and rRNAs are flanked by tRNAs in the primary transcript, some are not and it remains unclear how these non-canonical RNA junctions are processed. In collaboration with Eric Shoubridge (McGill University, Canada), we have shown that FASTKD5 is directly involved in processing of these junctions. This provides a framework for mechanistic studies of non-canonical RNA processing in human mitochondria.
Finally, we have also provided insights into the coupling of RNA biogenesis and translation in human mitochondria (Heinrichs et al., Nature Communications 2025). In particular, we could show that MTG3, an assembly factor for the small subunit (SSU) of the human mitochondrial ribosome, can remain bound to the SSU even during initiation complex formation. This suggests that MTG3 may act as a quality control factor and that ribosome biogenesis and translation initiation may be coupled in human mitochondria.

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 results obtained during this reporting period represent major advances beyond the state of the art for the field of mitochondrial gene expression as well as gene expression in general. In particular, the mechanistic elucidation of the complete pathway of canonical RNA processing in human mitochondria represents a major milestone, which provides unprecedented molecular and evolutionary insights into mitochondrial gene expression. Moreover, the identification of a direct player in non-canonical RNA processing is a major stepstone towards uncovering the mechanisms underlying this process. Finally, our results on the coupling of ribosome biogenesis and translation initiation represent an important step forward towards our goal of understanding the interplay between different gene expression steps in mitochondria.