Functional proximity mapping of RNA binding proteins uncovers a mitochondrial mRNA anchor that promotes stress recovery

Abstract

Proximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, it should be possible to map specific protein subclasses within distinct compartments of living cells. Here we demonstrate this capability for RNA binding proteins (RBPs), by combining peroxidase-based PL with organic-aqueous phase separation of crosslinked protein-RNA complexes (“APEX-PS”). We validated APEX-PS by mapping nuclear RBPs, then applied it to uncover the RBPomes of two unpurifiable subcompartments - the nucleolus and the outer mitochondrial membrane (OMM). At the OMM, we discovered the RBP SYNJ2BP, which retains specific nuclear-encoded mitochondrial mRNAs during translation stress, to promote their local translation and import of protein products into the mitochondrion during stress recovery. APEX-PS is a versatile tool for compartment-specific RBP discovery and expands the scope of PL to functional protein mapping.

Neuronal mitochondria transport Pink1 mRNA via synaptojanin 2 to support local mitophagy

“…Thus the relocalization of the Pink1 transcript was likely to be a direct consequence of its inability to associate with mitochondria. SYNJ2BP itself has been suggested to have RNA-binding capabilities (Qin et al, 2020), but our observation that the mitochondrial localization of the Pink1 transcript was neuron-specific led us to explore the expression patterns of its interactors. Unlike the SYNJ2BP-interacting ER protein RRBP1 (Hung et al, 2017), which was much more abundant in fibroblasts (Fig.…”

“…Overexpression of the mitochondrial outer membrane protein SYNJ2 Binding Protein (SYNJ2BP, also known as Outer Membrane Protein 25, OMP25) leads to an increase in mito-ER contact sites that are enriched for ribosomes (Hung et al, 2017). A recent preprint now also connects SYNJ2BP with the localization of mitochondrial RNAs to the outer mitochondrial membrane (Qin et al, 2020).…”

Proximity-dependent biotinylation technologies for mapping RNA-protein interactions in live cells

“…Protein centric PDB methods allow the characterization of the RNA transcripts that are bound to or in proximity of a protein of interest through next generation sequencing (NGS) approaches. Differently from standard protein centric methods, this strategy does not require the use of an antibody for the protein target and, hence, can be applied to any protein or protein isoform (Qin et al, 2021b). The protein bait is expressed in living cells as a fusion protein having either at its N-or C-terminus a spacer containing a tag used for detection (i.e.…”

“…Hence, the labeling intensity is not directly correlated with the strength of the interaction (Mair and Bergmann, 2022). A detailed summary of the various PDB enzymes currently used in molecular biology and their mechanism of actions can be found elsewhere (Samavarchi-Tehrani et al, 2020;Qin et al, 2021b).…”

A proximity-dependent biotinylation map of cytosol-facing organelle outer membrane proteome in living Arabidopsis cells

“…The proximity labeling (PL) approach has demonstrated great potential for probing spatiotemporal interactions between macromolecules and subcellular compartments (Antonicka et al, 2020;Gingras et al, 2019;Hung et al, 2017;Ke et al, 2021;Liu et al, 2018;Mair and Bergmann, 2022;Qin et al, 2021;Yang et al, 2021). In the PL approach, a biotin ligase is fused in-frame with a bait protein, which can be any protein of interest: a cytosolic or nuclear protein or a protein anchored to the membrane of a subcellular compartment.…”