The Integrator complex regulates microRNA abundance through RISC loading

MicroRNA (miRNA) homeostasis is crucial for the post-transcriptional regulation of their target genes and miRNA dysregulation has been linked to multiple diseases, including cancer. The mechanistic steps of miRNA biogenesis are well understood at molecular level1,2. Subsequent miRNA duplex loading into members of the Argonaute (Ago) protein family, representing the core of the RNA-induced silencing complex (RISC), is pivotal to miRNA-mediated gene silencing3-5. The Integrator complex has been previously identified as important regulator of RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination, processes dependent on Integrator's catalytical activity6-9. Here we report that absence of the Integrator complex leads to a global loss of mature miRNAs. By incorporating 4-Thiouridine (s4U) in nascent transcripts, we traced miRNA fate from biogenesis to stabilization and identified Integrator to be essential for proper miRNA assembly into RISC. Indeed, Integrator enhances miRNA-Ago2 interaction in vitro, which functionally translates to amplified miRNA target cleavage. These findings demonstrate for the first time cytoplasmic Integrator complex functions and stress an indirect role of Integrator in transcription regulation through modulation of miRNA abundance and stability. Overall design: small RNA-seq of HeLa cells depleted for INTS1, INTS3, INTS6, INTS7, or INTS11 and their control using shRNA in duplicates small RNA-seq of HeLa cells depleted for Drosha and Control using siRNA in duplicates small RNA-seq of shINTS6, shINTS11, and shControl HeLa cells, RNA extracted from nucleus and cytoplasm in duplicates SLAM small RNA-seq: 4-Thiouridine kinetic labeling of small RNAs in shINTS6, shINTS11, and shControl HeLa cells at 0, 0.25, 0.5, 1, 3, 6, 12, 24 hours including noS4U and noIAA controls in duplicates Ago2 RNA Immunoprecipitation (RIP) followed by small RNA-seq from shINTS6, shINTS11, and shControl HeLa cells after 0 or 24h of 4-Thiouridine labeling including noS4U controls in duplicates Total RNA-seq of HeLa cells depleted for INTS1, INTS3, INTS6, INTS7, INTS11 using shRNA in duplicates Total RNA-seq of HeLa cells depleted for Drosha and Control using siRNA in duplicates Enhanced UV crosslinking and immunoprecipitation (eCLIP) of INTS11, CPSF73, IgG and their size-matched input controls in duplicates from HeLa cells. The protocol was optimized for smRNA capture by increased RNase concentrations