Systematic characterization of the composition and dynamics of processing body-associated mRNAs

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.