RNA Proximity Labeling: A New Detection Tool for RNA–Protein Interactions

Cellular stress can induce DNA lesions that threaten the stability of genes. The DNA damage response (DDR) recognises and repairs broken DNA to maintain genome stability. Intriguingly, components of nuclear paraspeckles like the non-POU domain containing octamer-binding protein (NONO) participate in the repair of DNA double-strand breaks (DSBs). NONO is a multifunctional RNA-binding protein (RBP) that facilitates the retention and editing of messenger (m)RNA as well as pre-mRNA processing. However, the role of NONO in the DDR is poorly understood. Here, we establish a novel human U2OS cell line that expresses NONO fused to the engineered ascorbate peroxidase 2 (U2OS:NONO-APEX2-HA). We show that NONO-APEX2-HA accumulates in the nucleolus in response to DNA damage. Combining viability assays, subcellular localisation studies, coimmunoprecipitation experiments and in vivo proximity labeling, we demonstrate that NONO-APEX2-HA is a stably expressed fusion protein that mimics endogenous NONO in terms of expression, localisation and bona fide interactors. We propose that in vivo proximity labeling in U2OS:NONO-APEX2-HA cells is capable for the assessment of NONO interactomes by downstream assays. U2OS:NONO-APEX2-HA cells will likely be a valuable resource for the investigation of NONO interactome dynamics in response to DNA damage and other stimuli.

Section: Resultsmentioning

confidence: 99%

In vivo Proximity Labeling of Nuclear and Nucleolar Proteins by a Stably Expressed, DNA Damage-Responsive NONO-APEX2 Fusion Protein

Trifault

Mamontova

Burger

2022

“…Combining puromycylation ( Tom Dieck et al, 2015 ), to tag newly synthesised proteins, with specific antibodies (i.e. RPs) could help understand the spatial location of these newly translated RPs, to further understand RNA/protein interactions ( Weissinger et al, 2021 ) in different brain cells and neuronal compartments in vitro . Obtaining functional in vivo data remains the gold standard for understanding molecular mechanisms linked to behaviour, including sleep.…”

Section: Discussionmentioning

confidence: 99%

Are there roles for heterogeneous ribosomes during sleep in the rodent brain?

Buchanan

Smith²,

Gerber³

et al. 2022

The regulation of mRNA translation plays an essential role in neurons, contributing to important brain functions, such as brain plasticity and memory formation. Translation is conducted by ribosomes, which at their core consist of ribosomal proteins (RPs) and ribosomal RNAs. While translation can be regulated at diverse levels through global or mRNA-specific means, recent evidence suggests that ribosomes with distinct configurations are involved in the translation of different subsets of mRNAs. However, whether and how such proclaimed ribosome heterogeneity could be connected to neuronal functions remains largely unresolved. Here, we postulate that the existence of heterologous ribosomes within neurons, especially at discrete synapses, subserve brain plasticity. This hypothesis is supported by recent studies in rodents showing that heterogeneous RP expression occurs in dendrites, the compartment of neurons where synapses are made. We further propose that sleep, which is fundamental for brain plasticity and memory formation, has a particular role in the formation of heterologous ribosomes, specialised in the translation of mRNAs specific for synaptic plasticity. This aspect of our hypothesis is supported by recent studies showing increased translation and changes in RP expression during sleep after learning. Thus, certain RPs are regulated by sleep, and could support different sleep functions, in particular brain plasticity. Future experiments investigating cell-specific heterogeneity in RPs across the sleep-wake cycle and in response to different behaviour would help address this question.

“…Aptamer-based PDB exploits the tagging of the RNA of interest with the MS2 or BoxB aptamers that are specifically recognized and bound by the MS2 coat protein (MCP) and λN peptide fused in frame with the labeling enzyme, respectively ( Weissinger et al, 2021 ). An example is represented by the RNA-protein interaction detection—mass spectrometry (RaPID-MS) strategy ( Ramanathan et al, 2018 ), where the RNA of interest is expressed in living cells tagged with three BoxB aptamers located both at the 5′ and 3′ ends.…”

Section: Rna Centric Methodsmentioning

confidence: 99%

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

Giambruno

Nicassio

2022

Proximity ligation technologies are extremely powerful tools for unveiling RNA-protein interactions occurring at different stages in living cells. These approaches mainly rely on the inducible activity of enzymes (biotin ligases or peroxidases) that promiscuously biotinylate macromolecules within a 20 nm range. These enzymes can be either fused to an RNA binding protein or tethered to any RNA of interest and expressed in living cells to biotinylate the amino acids and nucleic acids of binding partners in proximity. The biotinylated molecules can then be easily affinity purified under denaturing conditions and analyzed by mass spectrometry or next generation sequencing. These approaches have been widely used in recent years, providing a potent instrument to map the molecular interactions of specific RNA-binding proteins as well as RNA transcripts occurring in mammalian cells. In addition, they permit the identification of transient interactions as well as interactions among low expressed molecules that are often missed by standard affinity purification strategies. This review will provide a brief overview of the currently available proximity ligation methods, highlighting both their strengths and shortcomings. Furthermore, it will bring further insights to the way these technologies could be further used to characterize post-transcriptional modifications that are known to regulate RNA-protein interactions.