In Vivo Analysis of RNA Proximity Proteomes Using RiboPro
Xianzhi Lin,Kate Lawrenson +1 more
TL;DR: RiboPro, a flexible method that leverages the RNA-binding specificity of inactive Cas13b and proximity labeling activity of peroxidase APEX to permit rapid and unbiased discovery of target RNA binding proteins in vivo is reported.
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Abstract: Functional and mechanistic annotation of uncharacterized long noncoding RNAs is challenging and often requires identification of interacting proteins. Here we report RiboPro, a flexible method that leverages the RNA-binding specificity of inactive Cas13b and proximity labeling activity of peroxidase APEX to permit rapid and unbiased discovery of target RNA binding proteins in vivo. RiboPro of poly(A)+ RNA reveals insights into poly (A)+ RNA nucleocytoplasmic transport, localization, turnover, and higher-order structure.
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Citations
Deciphering molecular interactions by proximity labeling.
TL;DR: Proximity labeling (PL) is a technique for tagging the endogenous interaction partners of specific protein "baits" via genetic fusion to promiscuous enzymes that catalyze the generation of diffusible reactive species in living cells as discussed by the authors.
374
CLIP and complementary methods
Markus Hafner,Maria Katsantoni,Maria Katsantoni,Tino Köster,James Marks,Joyita Mukherjee,Joyita Mukherjee,Dorothee Staiger,Jernej Ule,Jernej Ule,Mihaela Zavolan,Mihaela Zavolan +11 more
- 04 Mar 2021
TL;DR: The prospect of integrating data obtained by CLIP with complementary methods to gain a comprehensive view of RNP assembly and remodelling, unravel the spatial and temporal dynamics of RNPs in specific cell types and subcellular compartments and understand how defects in RNPs can lead to disease are discussed.
RNA–protein interaction mapping via MS2- or Cas13-based APEX targeting
TL;DR: Two complementary methods for tagging endogenous proteins in the vicinity of specific cellular RNAs, for subsequent identification by mass spectrometry and validated the interaction between hTR and the N6-methyladenosine demethylase AL KBH5 and showed that ALKBH5 is able to erase the m6A modification on endogenous hTR.
131
RNA-protein interaction mapping via MS2 or Cas13-based APEX targeting
TL;DR: Two approaches were used to target APEX2 to specific cellular RNAs and validated the unexpected interaction between hTR and the N6-methyladenosine demethylase ALKBH5, which showed that endogenous hTR is modified by m6A, which can be erased by ALKBh5, and that AL KBH5 influences both telomerase complex assembly and activity.
28
RNA-Centric Methods: Toward the Interactome of Specific RNA Transcripts
TL;DR: An overview of methods to identify RNA-protein interactions can be found in this article, with a particular focus on strategies that provide insights into the interactome of specific RNA transcripts, including the potential of CRISPR-RNA targeting systems to investigate endogenous RNA−protein interactions.
22
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