Research
Ribonucleoprotein Particles in Polarized Cells
The molecular function and fate of mRNAs are controlled by RNA-binding proteins (RBPs) that assemble with the mRNAs to messenger ribonucleoprotein particles (mRNPs). However, the identification of all interacting proteins of a specific mRNA is still very challenging. Based on the widely-used RNA tagging with MS2 aptamers for RNA visualization, we developed a novel method called RNA proximity biotinylation (RNA-BioID). Here, a proximity labeling enzyme (a biotin ligase or an ascorbate peroxidase) is tethered to the 3’-UTR of endogenous MS2-tagged RNAs. RNA-associated proteins can then be biotinylated in vivo and subsequently isolated using the biotin label. We have demonstrated the feasibility of this approach by characterizing the dynamic interactome of the conserved β-actin mRNA in mouse embryonic fibroblasts. Currently, we apply this approach to various mRNAs and long non-coding RNAs in polarized cells, iPSCs, and neurons to compare and understand the composition of different RNP complexes.
Proximity Labelling in Yeast
Proximity labeling methods to identify interacting biomolecules are widely used in cell culture. However, its application in bacteria and yeast has been impaired by differences in the cellular metabolism and composition, in particular the cell wall. We have overcome these obstacles and established a protocol for APEX2-mediated biotinylation. It allows quantitative proteomic mapping of subcellular compartments and protein networks in living yeast cells using high resolution mass spectrometry. The method can also extract data about dynamic cellular processes, for example changes upon DNA-damage or within the cell cycle. Quantitative proteomic mapping by the histone APEX2-H2B led to the identification of novel chromatin-associated proteins. We are currently characterizing a DNA-binding protein of unknown function that is highly conserved in evolution.
RNA-binding proteins in translation and protein aggregation
The large RNA-binding protein Scp160p is the yeast homolog of the conserved vigilin protein family. These proteins influence a variety of cellular functions both nuclear and cytoplasmic. Vigilins contain 14 RNA-binding domains and can affect translation of their target mRNAs, including translation speed and consequently co-translational folding of nascent peptides. Using polyglutamine (polyQ) reporters mimicking disease variants of the aggregation-prone Huntingtin protein, we demonstrated that the absence of Scp160p diminishes aggregation of such polyQ reporters. Furthermore, the naturally occurring aggregation of many endogenous glutamine/asparigine (Q/N) -rich proteins was reduced. As aggregation mediated by these regions can be important for the functions of all these proteins, Scp160p may thus impact many processes via aggregation of Q/N-rich proteins. We are currently analyzing the impact of various mutations in Scp160p to reveal the role of the individual domains in these processes.
Mitochondrial targeting of nucleus-encoded mRNAs
Targeting of mitochondrial proteins is generally accepted to occur post-translationally. However, several studies indicate the existence of an import pathway involving the local translation of nuclear-encoded mitochondrial transcripts at the mitochondrial outer membrane (MOM). Several RNA-binding proteins (RBPs) recruiting nuclear-encoded mitochondrial transcripts have been suggested to be - at least temporarily - located at the outer mitochondrial membrane, including human PINK1 and CLUH, or yeast Puf3p. mRNAs including human ATP6 and SOD2 were reported to harbor specific signals that mediate their targeting or localization to the mitochondrial surface. Using proximity labeling approaches we want to identify mRNAs located and translated at the major mitochondrial import complex (TOM) as well as RBPs regulating these mRNAs.