MicroRNA precursor programmed silencing complex assembly and regulatory pathways in mammals

The assembly of microRNA Ribonucleoproteins (miRNPs) or RNA-Induced Silencing Complexes (RISC) is essential for the function of miRNAs and initiates from processing of precursor miRNAs (pre-miRNAs) by Dicer or by the ribonucleolytic activity of Ago2. Here, we report the generation of a novel in-vitro miRNP/RISC assembly assay programmed by pre-miRNAs from mammalian cell lysates. Parallel studies in cells lacking Dicer that were reconstituted with wild type or catalytically inactive Dicer, confirm the operation of the reported pathways operate in vivo. We find that the miRNA Loading Complex (miRLC) is the primary machinery linking pre-miRNA processing to miRNP/RISC assembly in mammals. We show that a miRNA Precursor Deposit Complex (miPDC) plays a crucial role for the Ago2-dependent miRNA biogenesis and promotes miRNP assembly of certain Dicer-dependent miRNAs. Furthermore, we find that 5’-uridine, 3’-mid base pairing and 5’-mid mismatches within pre-miRNAs promote their assembly into miPDC. Our studies provide a comprehensive view of miRNP/RISC assembly pathways in mammals and our assay provides a versatile platform for further mechanistic dissection of such pathways in mammals. Post-transcriptional regulation of gene expression by the miRNA requires its binding to the Ago protein to form the miRNP or the RISC, whose assembly requires the association of the pre-miRNA with Ago and the processing of the pre-miRNA within the miRLC or the miPDC. The miRNP/RISC assembly is a tightly regulated process and one emerging regulatory paradigm is through tailing of pre-miRNAs. Here, we report the full-length sequence of authentic pre-miRNAs through high-throughput sequencing of Ago-interacting pre-miRNAs. We observe widespread tailing of pre-miRNAs at their 3’ ends, which may suppress the miRNP/RISC assembly through inhibition of Dicer processing. We also find that a group of Ago2-cleaved pre-miRNAs (ac-pre-miRNAs) are tailed at the Ago2-cleavage site. Our findings suggest that tailing of pre-miRNAs and ac-pre-miRNAs may play an important regulatory role in the miRNP/RISC assembly in mammals