Decoding the symphony : illuminating the mammalian mitochondrial RNA binding proteome with a novel interactome approach

Mitochondria, colloquially known as the "powerhouses of the cell" are crucial for generating cellular energy through a process known as oxidative phosphorylation (OXPHOS). These minute but mighty organelles are distinct and semi-independent entities, containing their own DNA, which is circular in nature. This DNA has the capacity for replication, transcription, and translation, processes collectively referred to as mitochondrial gene expression. Out of the OXPHOS system's peptides, mitochondria encode for 13, with the remainder being produced in the nucleus and then transported into the mitochondria. The complex system of gene expression within mitochondria relies on an intricate interaction among DNA, RNA, and proteins. However, disruptions in this system have been linked to a variety of diseases, such as diabetes, neurodegenerative disorders, and cancer. RNA-binding proteins (RBPs) are essential in regulating various aspects of RNA's lifecycle, including its synthesis, stability, processing, epitranscriptomic modification, translation, and degradation. Yet, the specifics of how these proteins operate remain partially understood.

My doctoral research has been devoted to uncovering new RBPs and elucidating their roles in mitochondrial gene expression. Specifically, in paper 1, I introduced a technique named ‘mt-TRIzol-RBP-Ex’ for the efficient and unbiased enrichment of mitochondrial RBPs from HEK293T cells, leading to the identification and partial characterization of a potential novel RBP, C8orf82, in mitochondrial translation. Subsequent papers focused on other RBPs such as C6orf203 (in paper 2) and GTPBP8 (in paper 3), as well as translation initiation factors (in paper 4), revealing their indispensable roles in mitochondrial post-transcriptional pathways. The depletion of these proteins resulted in significant impacts on translation.

This doctoral thesis provides a comprehensive review of current knowledge on mitochondrial RBPs and presents both preliminary and published findings on the roles of specific, previously uncharacterized, or novel RBPs in mitochondrial gene expression.

List of scientific papers

I. Shreekara Gopalakrishna, Ilian Atanassov, Aishe Angeletti Sarshad, Daniel Ben Halevy, Shadi Bavafa, Benedikt Beckmann, Joanna Rorbach†. (2024). Enrichment of mammalian mitochondrial RNA-binding proteins using TRIzolEx reveals novel uncharacterised mt-RNA interacting proteins. †Corresponding author. [Manuscript]

II. Shreekara Gopalakrishna*, Sarah F Pearce*, Adam M Dinan, Florian A Rosenberger, Miriam Cipullo, Henrik Spåhr, Anas Khawaja, Camilla Maffezzini, Christoph Freyer, Anna Wredenberg, Ilian Atanassov, Andrew E Firth, Joanna Rorbach†. (2019). C6orf203 is an RNA-binding protein involved in mitochondrial protein synthesis. Nucleic Acids Res. 2019 Sep 26;47(17):9386-9399. *These authors contributed equally. †Corresponding author.
https://doi.org/10.1093/nar/gkz684

III. Miriam Cipullo, Genís Valentín Gesé, Shreekara Gopalakrishna, Annika Krueger, Petra Palenkova, Dmitrii Shiriaev, Yong Liu, Jelena Misic, Yu Cai, Minh Nguyen, Abubakar Abdelbagi, Xinping Li, Michal Minczuk, Ilian Attanasov, Martin Hällberg, Joanna Rorbach†. (2024). GTPBP8 is essential for mitoribosome formation in human mitochondria. †Corresponding author. [Submitted]

IV. Cristina Remes, Anas Khawaja, Sarah F Pearce, Adam M Dinan, Shreekara Gopalakrishna, Miriam Cipullo, Vasileios Kyriakidis, Jingdian Zhang, Xaquin Castro Dopico, Olessya Yukhnovets, Ilian Atanassov, Andrew E Firth, Barry Cooperman, Joanna Rorbach†. (2023). Translation initiation of leaderless and polycistronic transcripts in mammalian mitochondria. Nucleic Acids Res. 2023 Jan 25;51(2):891-907. †Corresponding author.
https://doi.org/10.1093/nar/gkac1233