A Novel miRNA Processing Pathway Independent of Dicer Requires Argonaute2 Catalytic Activity

Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA.
Science (Impact Factor: 33.61). 05/2010; 328(5986):1694-8. DOI: 10.1126/science.1190809
Source: PubMed


Dicer is a central enzyme in microRNA (miRNA) processing. We identified a Dicer-independent miRNA biogenesis pathway that
uses Argonaute2 (Ago2) slicer catalytic activity. In contrast to other miRNAs, miR-451 levels were refractory to dicer loss of function but were reduced in MZago2 (maternal-zygotic) mutants. We found that pre-miR-451 processing requires Ago2 catalytic activity in vivo. MZago2 mutants showed delayed erythropoiesis that could be rescued by wild-type Ago2 or miR-451-duplex but not by catalytically
dead Ago2. Changing the secondary structure of Dicer-dependent miRNAs to mimic that of pre-miR-451 restored miRNA function
and rescued developmental defects in MZdicer mutants, indicating that the pre-miRNA secondary structure determines the processing pathway in vivo. We propose that Ago2-mediated
cleavage of pre-miRNAs, followed by uridylation and trimming, generates functional miRNAs independently of Dicer.

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    • "For example, some cellular pre-miRNAs are produced via splicing (mirtrons ) or Pol II transcription initiation/termination (m 7 Gcapped pre-miRNA and other endogenous shRNAs), thereby bypassing the Drosha cleavage step (Okamura et al. 2007; Ruby et al. 2007; Babiarz et al. 2008; Xie et al. 2013). Dicer cleavage is replaced by AGO-2 cleavage in vertebrate miR-451 biogenesis (Cheloufi et al. 2010; Cifuentes et al. 2010). In animal viruses, several miRNA biogenesis pathways skip the Drosha cleavage step by using Pol III to produce pre-miRNAs directly or tRNApre-miRNA chimeric transcripts that are further processed by RNaseZ (Bogerd et al. 2010; Kincaid et al. 2012). "
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    ABSTRACT: Herpesvirus saimiri (HVS) is an oncogenic γ-herpesvirus that produces microRNAs (miRNAs) by cotranscription of precursor miRNA (pre-miRNA) hairpins immediately downstream from viral small nuclear RNAs (snRNA). The host cell Integrator complex, which recognizes the snRNA 3' end processing signal (3' box), generates the 5' ends of HVS pre-miRNA hairpins. Here, we identify a novel 3' box-like sequence (miRNA 3' box) downstream from HVS pre-miRNAs that is essential for miRNA biogenesis. In vivo knockdown and rescue experiments confirmed that the 3' end processing of HVS pre-miRNAs also depends on Integrator activity. Interaction between Integrator and HVS primary miRNA (pri-miRNA) substrates that contain only the miRNA 3' box was confirmed by coimmunoprecipitation and an in situ proximity ligation assay (PLA) that we developed to localize specific transient RNA-protein interactions inside cells. Surprisingly, in contrast to snRNA 3' end processing, HVS pre-miRNA 3' end processing by Integrator can be uncoupled from transcription, enabling new approaches to study Integrator enzymology. © 2015 Xie et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & development 07/2015; 29(14):1552-64. DOI:10.1101/gad.266973.115 · 10.80 Impact Factor
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    • "This results in truncated hairpins called Ago-cleaved pre-miRNAs (ac-pre-miRNAs) (Diederichs & Haber, 2007). There is little evidence that ac-pre-miRNAs generate mature miRNAs with an exception of ac-pre-miR-451 that is shorter than others and trimmed further into mature miRNA (Cheloufi et al, 2010; Cifuentes et al, 2010; Yang et al, 2010; Yoda et al, 2013). Thus, it remains unclear whether acpre-miRNAs have a certain biological role in general or whether they are mostly degradation intermediates. "
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    ABSTRACT: Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single-molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre-miRNAs) in the absence of Lin28. We find that the overhang of a pre-miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre-miRNAs, which have a 1-nt 3′ overhang, TUT7 restores the canonical end structure (2-nt 3′ overhang) through mono-uridylation, thereby promoting miRNA biogenesis. For pre-miRNAs where the 3′ end is further recessed into the stem (as in 3′ trimmed pre-miRNAs), TUT7 generates an oligo-U tail that leads to degradation. In contrast to Lin28-stimulated oligo-uridylation, which is processive, a distributive mode is employed by TUT7 for both mono- and oligo-uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7-RNA interaction, thus explaining how TUT7 differentiates pre-miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre-miRNAs.
    The EMBO Journal 05/2015; 34(13). DOI:10.15252/embj.201590931 · 10.43 Impact Factor
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    • "As for many other complex biological processes, exceptions to the canonical pathway have been discovered for the biogenesis of several cellular miRNAs [reviewed in (Xie and Steitz, 2014)]. Examples include Drosha-independent but splicing-dependent pre-miRNAs called mirtrons (Okamura et al., 2008; Ruby et al., 2007), m 7 Gcapped pre-miRNAs and endogenous short hairpin (sh)RNAs that are transcribed directly as pre-miRNAs (Babiarz et al., 2008; Xie et al., 2013) and AGO2 slicing activity-dependent miR- 451, which bypasses Dicer cleavage (Cheloufi et al., 2010; Cifuentes et al., 2010). Not surprisingly, viruses employ various alternative mechanisms to produce their own miRNAs. "
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    ABSTRACT: Eukaryotic cells produce several classes of long and small noncoding RNA (ncRNA). Many DNA and RNA viruses synthesize their own ncRNAs. Like their host counterparts, viral ncRNAs associate with proteins that are essential for their stability, function, or both. Diverse biological roles-including the regulation of viral replication, viral persistence, host immune evasion, and cellular transformation-have been ascribed to viral ncRNAs. In this review, we focus on the multitude of functions played by ncRNAs produced by animal viruses. We also discuss their biogenesis and mechanisms of action. © 2015 Tycowski et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & development 03/2015; 29(6):567-584. DOI:10.1101/gad.259077.115 · 10.80 Impact Factor
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