Table 1.

Proximity labeling methods for the detection of RNA–protein interactions

Proximity Labeling for Mapping Subcellular Transcriptomes
Method	Description	Model Organism/ Cell Type	Achievements	Strengths	Weaknesses	Ref.
APEX-RIP, Proximity-CLIP	Targeting of a labeling enzyme (e.g., APEX2) to subcellular compartments in order to biotinylate proximal proteins. After crosslinking proteins and RNA, biotinylated proteins are enriched by streptavidin beads and bound RNAs are identified via RNA-Seq.	HEK293T	Identification of compartment specific RNAs, e.g., in the nucleus, cytoplasm, mitochondrial matrix and at the ER membrane.	Proximity-Clip allows identification of RBP-protected regions of RNA targets. Short labeling time. No need of specific antibody.	Limited detection of RNAs in non-membrane bound cellular regions.	[81,82]
APEX-seq, CAP-seq	Targeting of a labeling enzyme (e.g., APEX2, miniSOG) to subcellular compartments or complexes to directly label RNAs. Biotinylated RNAs are enriched by streptavidin beads and identified using RNA-Seq.	HEK293T	Identification of RNAs localized to various locations, including nucleolus, nuclear lamina, nuclear pore, the outer mitochondrial membrane, the mitochondrial matrix, the ER lumen, the ER cytosolic interface and RNA granules.	No crosslinking required. Can identify proximal RNAs in insoluble and open cellular regions. Short labeling time.	Interactomes of individual RBPs cannot be assessed.	[83,84,85]
Proximity Labeling of RNA–Protein Interactions: Finding the RNA Partners
Method	Description	Model Organism/ Cell Type	Achievements	Strengths	Weaknesses	Ref.
TRIBE	An RBP of interest is fused to the catalytic domain of the RNA editing enzyme ADAR. ADAR edits target RNAs (A-to-I editing) bound by the RBP, which can be identified by RNA-Seq.	Drosophila S2 cells and neurons	Identification of RNAs bound to Drosophila RBPs: Hrp48, dFMR1 and NonA.	No crosslinking required. No specific substrate required for labeling. Can be used to identify the RNA region close to the RBP binding site.	The edited sequence is biased due to the binding and editing preference of ADARcd. ADAR can also edit RNAs in the vicinity but not bound by the RBP. Cannot be used to detect dynamic interactions.	[88,89]
RNA tagging	An RBP of interest is fused to the uridine polymerase PUP-2. PUP-2 attaches an uracil tail to RNAs bound by the RBP which allow their identification by RNA-Seq.	S. cerevisiae	Identification of RNA targets of yeast pumilio proteins as well as RNAs localized to ER and mitochondrial surfaces.	No crosslinking required. Counting of added uracil residues allows differentiation of true and false interactors.	Might miss proteins that interact close to the 5′ of the RNA. Can stress cells. Cannot be used to detect dynamic interactions.	[90,91,92]
Proximity Labeling of RNA–Protein Interactions: Finding the Protein Partners
Method	Description	Model Organism/ Cell Type	Achievements	Strengths	Weaknesses	Ref.
RaPID, RNA-BioID	An RNA sequence of interest is tagged with either BoxB or MS2 aptamers. The aptamers recruit a viral coat protein fused to a labeling enzyme (BirA*, BASU, APEX2) which biotinylates associated proteins.	HEK293T, huh7, mouse embryonic fibroblasts	Identification of proteins binding various RNA motifs, the UTR of the Zika virus RNA genome, human telomerase RNA, or β-actin mRNA.	Allows identification of weak or transient interactions. High specificity and affinity of MCP or λ N-peptide for their corresponding aptamer. No crosslinking required.	Aptamer insertion might affect RNA function or regulation. Technically challenging to genomically integrate the aptamer cassette at correct location.	[62,64,93,94]
CARPID, dCas13d-dsRBD-APEX2, RPL	Catalytically inactive Cas13 fused to a labeling enzyme (BioID2, BASU, APEX, APEX2) is targeted to an RNA of interest using guide RNAs.	HEK293T	Identification of proteins binding to Xist, MALAT1, DANCR, hTR and U1 snRNA.	No need for changes in target RNA. Probing of different endogenous RNAs can easily be achieved by changing the gRNA. Can be used to probe a specific region on the RNA. No crosslinking required.	Background biotinylation from off-target gRNAs or unbound Cas13-labeling complex possible. Thorough optimization of the Cas13-labeling enzyme construct required.	[93,95,96,97]