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Argonaute-bound microRNAs silence mRNA expression in a dynamic and regulated manner to control organismal development, physiology, and disease. We employed metabolic small RNA sequencing for a comprehensive view on intracellular microRNA kinetics in Drosophila. Based on absolute rate of biogenesis and decay, microRNAs rank among the fastest produced and longest-lived cellular transcripts, disposing up to 10 5 copies per cell at steady-state. Mature microRNAs are produced within minutes, revealing tight intracellular coupling of biogenesis that is selectively disrupted by pre-miRNA-uridylation. Control over Argonaute protein homeostasis generates a kinetic bottleneck that cooperates with non-coding RNA surveillance to ensure faithful microRNA loading. Finally, regulated small RNA decay enables the selective rapid turnover of Ago1-bound microRNAs, but not of Ago2-bound small interfering RNAs (siRNAs), reflecting key differences in the robustness of small RNA silencing pathways. Time-resolved small RNA sequencing opens new experimental avenues to deconvolute the timescales, molecular features, and regulation of small RNA silencing pathways in living cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Argonaute proteins act at the core of nucleic acid-guided interference pathways that regulate gene expression and defend organisms against foreign genetic elements in all domains of life. Here, we review recent biophysical studies on how Argonaute proteins instruct oligonucleotides in the process of target finding, binding, cleavage, and release, as measured at high spatiotemporal resolution by single-molecule approaches. In the context of previous structural, biochemical, and computational studies, a model emerges for how Argonaute proteins manipulate the thermodynamic rules for nucleic acid hybridization to convey efficiency and specificity to RNA- and DNA-guided regulatory processes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: MicroRNAs (miRNAs) are ~22 nucleotide (nt) small RNAs that control development, physiology, and pathology in animals and plants. Production of miRNAs involves the sequential processing of primary hairpin-containing RNA polymerase II transcripts by the RNase III enzymes Drosha in the nucleus and Dicer in the cytoplasm. miRNA duplexes then assemble into Argonaute proteins to form the RNA-induced silencing complex (RISC). In mature RISC, a single-stranded miRNA directs the Argonaute protein to bind partially complementary sequences, typically in the 3' untranslated regions of messenger RNAs, repressing their expression., Results: Here, we show that after loading into Argonaute1 (Ago1), more than a quarter of all Drosophila miRNAs undergo 3' end trimming by the 3'-to-5' exoribonuclease Nibbler (CG9247). Depletion of Nibbler by RNA interference (RNAi) reveals that miRNAs are frequently produced by Dicer-1 as intermediates that are longer than ~22 nt. Trimming of miRNA 3' ends occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. In vivo, depletion of Nibbler by RNAi causes developmental defects., Conclusions: We provide a molecular explanation for the previously reported heterogeneity of miRNA 3' ends and propose a model in which Nibbler converts miRNAs into isoforms that are compatible with the preferred length of Ago1-bound small RNAs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Many microRNAs (miRNAs) are expressed with high spatiotemporal specificity during organismal development, with some being limited to rare cell types, often embedded in complex tissues. Yet, most miRNA profiling efforts remain at the tissue and organ levels. To overcome challenges in accessing the microRNomes from tissue-embedded cells, we had previously developed mime-seq (miRNome by methylation-dependent sequencing), a technique in which cell-specific miRNA methylation in C. elegans and Drosophila enabled chemo-selective sequencing without the need for cell sorting or biochemical purification. Here, we present mime-seq 2.0 for profiling miRNAs from specific mouse cell types. We engineered a chimeric RNA methyltransferase that is tethered to Argonaute protein and efficiently methylates miRNAs at their 3′-terminal 2′-OH in mouse and human cell lines. We also generated a transgenic mouse for conditional expression of this methyltransferase, which can be used to direct methylation of miRNAs in a cell type of choice. We validated the use of this mouse model by profiling miRNAs from B cells and bone marrow plasma cells. Synopsis: Mime-seq 2.0 allows profiling of miRNAs from complex tissues and rare cell populations in the mouse. Cell-specific methylation of miRNAs by the chimeric RNA methyltransferase HENMT1ΔC-T6B enables chemo-selective enrichment by high-throughout sequencing. At-HEN1 efficiently methylates C. elegans and D. melanogaste r miRNAs in vivo, but not in mammalian cells. An engineered HENMT1ΔC-T6B chimera methylates miRNAs in vitro in mammalian cell culture and in vivo in mice. Mime-seq 2.0 combines a simple workflow with high sensitivity to enrich miRNAs even from rare cell populations. A new method based on the expression of a chimeric methyltransferase enables the sequencing of small RNAs from specific cell types in mice. [ABSTRACT FROM AUTHOR]