Zhiyuan Sun, Xiaozhen Wen, Yanping Li, Xiaoxin Xie, Peng Dong, Yi Shu, Shuye Tian, Jiao Yang, Yi Lin, Mengran Wang, Feifei Jiang, Qionghua Zhu, Huanhuan Cui, Jixian Zhai, Yuhui Hu, Liang Fang, Wei Chen
Processing bodies (PBs) are dynamic, membraneless organelles consisting of RNAs and proteins. While PB proteins have been extensively characterized, the methods for systematically profiling PB-associated RNAs are limited. To address this, we developed PB-TRIBE-STAMP, a tool based on two orthogonal RNA editing enzymes. Simultaneously applying APOBEC1-DDX6 and LSM14A-ADAR2dd, PB-TRIBE-STAMP identified 1,639 and 2,577 PB-associated mRNAs in HCT116 and HEK293T cells, respectively. Further biochemical isolation of PBs followed by RNA-seq validated that edited transcripts of these mRNAs were indeed enriched in PBs. Integration of PB-TRIBE-STAMP with long-read sequencing revealed that the PB-associated transcripts possessed shorter poly(A)-tails. Many mRNA 3’ UTR isoforms exhibited isoform-specific PB association patterns. Moreover, we established a TRIBE-ID-based tool to characterize the mRNA-LSM14A/PB association at high temporal resolution and unveiled a higher splicing efficiency of LSM14A-associated XBP1 transcripts during unfolded protein response (UPR). Finally, based on single-cell LSM14A-TRIBE-ID (sc-LSM14A-TRIBE-ID), we demonstrated the dynamic pattern of mRNA-LSM14A/PB association during cell cycle progression. The methods for systematically profiling RNA composition of processing bodies (PBs) are limited. Here, the authors present PB-TRIBE-STAMP and LSM14A-TRIBE-ID, two RNA editing-based methods enabling systematic characterization of dynamic PB-RNAs association.