Abstract
Mitochondrial gene expression is a tightly regulated process due to its importance for ATP production and cell homeostasis. In mammalian cells, mitochondrial transcription generates polycistronic RNAs that are later processed into individual mature RNA molecules. However, individual mRNAs accumulate at different steady state levels, suggesting that gene expression is mainly regulated posttranscriptionally. Moreover, mammalian mitochondrial mRNA molecules have no 5’-methylated CAP as their cytosolic counterparts, or 5’-untranslated regions (5’-UTRs) to which translation activators can bind to promote translation, and the process of translation initiation remains ill-characterized. Our research aligns with the hypothesis that post-transcriptional regulation of mitochondrial gene expression is mediated by RNA binding proteins (RBPs). Our objective is to develop a new approach to identify mitochondrial RBPs (mtRBPs), specific to each mRNA. For this purpose, we use the PUMILIO1 homology domain (PUM-HD), which binds RNA in a sequence-specific and modular manner, as a scaffold to design protein domains targeting RNA sequences of our choice. Our long-term goal is to create a collection of HeLa cell lines expressing in an inducible manner PUM-HDs binding individual mitochondrial mRNAs. Upon PUM-HD induction, mtRBPs can be isolated through a combination of crosslinking and immunoprecipitation approaches and identified by mass spectrometry. Here, we focus on the construction of PUM-HDs that selective bind to specific mt-mRNAs. We have successfully generated engineered PUM-HDs targeting the mt-mRNAs encoding for Complex I subunits and characterized their RNA-binding properties in vitro. With the identification of the mitochondrial mRNA binding proteins interactome the field of mitochondrial biogenesis will be able to expand as a new layer of regulation is elucidated.