A Novel Approach to Establish a Comprehensive Atlas of Human Mitochondrial mRNA Binding Proteins
Abstract
Mitochondria function requires the expression of structural subunits of the oxidative phosphorylation system encoded by the mitochondrial genome. Thus, mtDNA expression is essential for cellular energy production and its alteration leads to major energetic deficits in cells and tissues, which result in severe human diseases, namely mitochondrial disorders, such as encephalomyopathies and cardiomyopathies. Mitochondrial gene expression is predominantly regulated at the post-transcriptional level by the activity of nuclear-encoded mitochondrial RNA-binding proteins (mt-RBPs). A systematic study of mammalian mt-RBPs is missing and key questions regarding the mitochondrial gene expression process and the factors involved remain unanswered. The goal of our project is the development of a novel methodology to identify mt-mRNA-specific RBPs through the combination of single mt-mRNA immunoprecipitation and mass-spectrometry-based protein identification approaches. For this purpose, we aim to engineer mitochondrion-targeted RNA-binding domain (PUM1-HD) acting as selective mt-mRNA handles that can be used to isolate and characterize ribonucleoprotein complexes in human mitochondria. With the support of this DoD discovery award, we have generated a collection of 18 PUM1-HDs designed to target specific mt-mRNAs, purify there combinant proteins and demonstrated the specific RNA-binding capacity in vitro of six of them. Moreover, we have subcloned these domains in a mammalian inducible expression system and obtained one HeLa cell lines expressing in an inducible manner the collection ofPUM1-HDs targeted to mitochondria.
Document Details
- Document Type
- Technical Report
- Publication Date
- Mar 01, 2022
- Accession Number
- AD1173404
Entities
People
- Flavia Fontanesi
Organizations
- University of Miami