Summary The torpedo model of transcription termination asserts that the exonuclease Xrn2 attacks the 5′PO4-end exposed by nascent RNA cleavage and chases down the RNA polymerase. We tested this mechanism using a dominant-negative human Xrn2 mutant and found that it delayed termination genome-wide. Xrn2 nuclease inactivation caused strong termination defects downstream of most poly(A) sites and modest delays at some histone and U snRNA genes suggesting that the torpedo mechanism is not limited to poly(A) site-dependent termination. A central untested feature of the torpedo model is that there is kinetic competition between the exonuclease and the pol II elongation complex. Using pol II rate mutants, we found that slow transcription robustly shifts termination upstream, and fast elongation extends the zone of termination further downstream. These results suggest that kinetic competition between elongating pol II and the Xrn2 exonuclease is integral to termination of transcription on most human genes.

Highlights d PNUTS-PP1 phosphatase is a global decelerator of Pol II transcription d PNUTS-PP1 promotes Spt5 dephosphorylation and Pol II braking at poly(A) sites d Termination requires poly(A)-dependent Spt5 dephosphorylation and Pol II braking d Allosteric switch converts Pol II to a ''sitting duck'' terminated by a Xrn2 torpedo

SUMMARY RNA metabolism is controlled by an expanding yet incomplete catalog of RNA binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull-down of twelve nuclear and cytoplasmic RBPs followed by quantitative mass-spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1923 human, 489 fly and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins.

Highlights• STAMP identifies binding sites of full-length RBPs by C-to-U RNA editing.• STAMP coupled to long read sequencing reveals isoform specific RBP targets.• STAMP allows cell-type specific and multiplexed-RBP target identification in single cells.• STAMP with ribosome subunits allows detection of ribosome association at single-cell level.

RNA binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet, the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on UV-crosslinking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP- and cell type-specific RNA-protein interactions for multiple RBPs and cell types in single-pooled experiments. Pairing STAMP with long-read sequencing also yields RBP target sites for full-length isoforms. Finally, conducting STAMP using small ribosomal subunits (Ribo-STAMP) allows analysis of transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.