In vivo discovery of RNA proximal proteins via proximity-dependent biotinylation

Abstract

RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely mediated by RNA binding proteins (RBPs). Here we present RNA proximity labelling (RPL), an RNA-centric method comprising the endonuclease-deficient Type VI CRISPR-Cas protein dCas13b fused to engineered ascorbate peroxidase APEX2. RPL discovers target RNA proximal proteins in vivo via proximity-based biotinylation. RPL applied to U1 identified proteins involved in both U1 canonical and noncanonical functions. Profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs and provided additional evidence for 5ʹ-3ʹ proximity and unexplored subcellular localizations of poly(A) + RNA. Our results suggest that RPL allows rapid identification of target RNA binding proteins in native cellular contexts, and is expected to pave the way for discovery of novel RNA-protein interactions important for health and disease.

Hybridization-proximity labeling reveals spatially ordered interactions of nuclear RNA compartments

“…These tasks can be accomplished by proximity biotinylation of proteins and/or RNAs colocalizing with a “bait” molecule in the cell ( Li et al., 2018 ; Mukherjee et al., 2019 ; Qin et al., 2021 ; Ramanathan et al., 2018 ). For instance, ascorbate peroxidase APEX2 genetically fused with compartment-specific localization signals or RNA interaction domains has been recently used to examine several RNA-containing complexes ( Benhalevy et al., 2018 ; Fazal et al., 2019 ; Han et al., 2020 ; Kaewsapsak et al., 2017 ; Lin et al., 2021 ; Markmiller et al., 2018 ; Padrón et al., 2019 ). However, most proximity-labeling methods rely on expression of recombinant enzymes in living cells, making it difficult to apply this technology to poorly transfectable cell types, non-model organisms, or clinical samples.…”

Progress of CRISPR-Cas13 Mediated Live-Cell RNA Imaging and Detection of RNA-Protein Interactions

“… Lin et al (2021) developed a method known as RNA proximity labeling (RPL) based on the fusion of dPspCas13b with the adjacent marker enzyme APEX2, which can lead to biotinylation of proteins within a distance of 25 nm from the targeted RNA by inference ( Figure 2E ). Cas13b was selected for its high specificity and low miss efficiency, and APEX2 was selected for its high kinetic effects ( Lam et al, 2015 ; Yang et al, 2019 ).…”

“…Cas13b was selected for its high specificity and low miss efficiency, and APEX2 was selected for its high kinetic effects ( Lam et al, 2015 ; Yang et al, 2019 ). The RPL system was used to analyze the nuclear RNA (ncRNA) U1 and poly(A) tail proximal proteins and can rapidly identify RBPs and uncover novel RBPs such as KPNB1 ( Lin et al, 2021 ). To reduce the background noise of the system, Lin et al also took some measures, such as selecting the U1 with high abundance ( Stark et al, 2001 ) and designing three kinds of gRNA to be expressed in separate cell lines in combination with different regions.…”

“…Therefore, inducing the low expression of Cas13 is an important part of the whole imaging system. In addition, the binding of gRNA to target RNA may be competitive with the binding of RBPs, while a single gRNA may reduce the RBP detection rate ( Lin et al, 2021 ). Therefore, a RNA array can be set up as described by Yi et al (2020) in CARPID.…”

“…Therefore, a RNA array can be set up as described by Yi et al (2020) in CARPID. Furthermore, groups of different gRNAs can also be set up to reduce background noise ( Lin et al, 2021 ). Meanwhile, the type of biotin also affects the efficiency of imaging ( Li et al, 2021 ).…”