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Not Dicer but Argonaute Is Required for a Microrna Production

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Not Dicer but Argonaute Is Required for a Microrna Production Cell Research (2010) 20:735-737. npg © 2010 IBCB, SIBS, CAS All rights reserved 1001-0602/10 $ 32.00 RESEARCH HIGHLIGHT www.nature.com/cr A new twist in the microRNA pathway: Not Dicer but Argonaute is required for a microRNA production Gabriel D Bossé1, Martin J Simard1 1Laval University Cancer Research Centre, Hôtel-Dieu de Québec (CHUQ), Quebec City, Québec G1R 2J6, Canada Cell Research (2010) 20:735-737. doi:10.1038/cr.2010.83; published online 15 June 2010 Found in all metazoans, microRNAs A Canonical pathway B Ago2-dependent pathway or miRNAs are small non-coding RNA Nucleus Cytoplasm Nucleus Cytoplasm of ~22 nucleotides in length that com- Exp.5 Exp.5 pletely reshaped our understanding of gene regulation. This new class of gene pre-miR-451 regulator is mostly transcribed by the pre-miRNA RNA polymerase II producing a long stem-loop structure, called primary- or Ago2 pri-miRNA, that will first be processed Ago2 Dicer in the cell nucleus by a multiprotein TRBP complex called microprocessor to gen- erate a shorter RNA structure called Ago2 RISC precursor- or pre-miRNA. The precisely Ago2 RISC processed pre-miRNA will next be ex- ported into the cytoplasm by Exportin 5 and loaded onto another processing machine containing the ribonuclease III enzyme Dicer, an Argonaute protein Ago2 Ago2 and other accessory cellular factors mRNA mRNA (Figure 1A; [1]). Dicer will mediate the Translation inhibition Translation inhibition cleavage of the pre-miRNA to form the mature miRNA that will then be bound Figure 1 (A) Canonical microRNA biogenesis. In mammals, the pre-miRNA is by the Argonaute protein to form, most loaded onto a multiprotein complex consisting minimally of Dicer, Tar RNA Bind- likely with other cellular factors, the ef- ing Protein (TRBP) and Ago2. The RNase III enzyme Dicer processes the pre- fector silencing complex or RISC able cursor by cleaving the stem-loop to produce the mature miRNA. Ago2 bind this miRNA to form the RISC complex to target specific mRNA.(B) Ago2-dependent to modulate protein expression upon microRNA biogenesis. For miR-451, the precursor is loaded directly onto Ago2, binding by sequence complementarity and this Argonaute cleaves the precursor RNA of this miRNA. The mature miR- to the specific region of 3′ untranslated 451 is bound by Ago2 to form the RISC complex that targets erythropoietic- region (UTR) of mRNA to alter protein specific mRNAs. synthesis. In animals, some Argonaute proteins can cleave RNA molecules while others that does not require RNA cleavage [2]. domain confers the endonucleolytic ac- have lost their catalytic activity and par- This family of proteins possesses four tivity of Argonaute proteins and requires ticipate in a gene regulatory mechanism structural domains: the N-terminus, coordination of divalent cations by PAZ, MID and PIWI domains. The PAZ Aspartate-Aspartate-Histidine (DDH) Correspondence: Martin J Simard domain is implicated in the binding of motif for catalysis. Interestingly, all four E-mail: [email protected] single-stranded RNA while the PIWI Argonautes found in mammals can form npg 736 a functional RISC but only Ago2 diplays enzyme cannot recognize and process form the RISC complex. an endonuclease activity [3, 4]. short pre-miRNA efficiently [10]. Sec- The recent work of Cheloufi and The discovery of a new class of ond, they also observed that the mature colleagues represents an important small RNAs in mammal oocytes [5] miR-451 includes the sequence of the breakthrough in the understanding of and embryonic stem cells [6] that have loop region and expands into the com- the catalytically competent Argonaute the potential to cleave complementary plementary strand of the precursor. The Ago2 and thus in the miRNA biogen- RNA sequences has provided part of authors concluded from these findings esis in animals. In addition to clearly the reason why Ago2 has conserved its that the biogenesis of miR-451 must be demonstrating the biological relevance enzymatic activity. However, the dele- unusual, most likely not inferring Dicer of Argonaute cleavage in the matura- tion of Ago2 does not only lead to sterile to process the precursor structure and tion of miRNAs as previously proposed animals but lethality is observed during produce mature miRNA. [11-14], this study represents the first gestation [3, 7, 8] suggesting a role of To further test this hypothesis, the evidence of a miRNA maturation that Ago2 apart of the female germline. authors used embryonic stem cells car- is independent of Dicer. This unex- In order to understand the catalytic rying a conditional Dicer allele. Using pected miRNA biogenesis seems to be function of Ago2 in animals, the Han- that system, they observed that the pro- conserved among species as recently non’s group generated a mouse in which duction of mature miR-451 was not af- reported in Zebrafish by Cifuentes and they replaced the endogenous allele by fected by the absence of Dicer whereas colleagues [15]. The implication of a catalytically inactive Ago2 carrying the loss-of-function of Dicer caused a Ago2 in the processing of pre-miRNA mutation in the DDH motif (Ago2ADH) strong decrease of other miRNA levels. provides interesting evidence of the [9]. They observed that the animal un- To confirm this observation, the authors importance of the endonuclease activ- derwent a normal embryogenesis but performed an in vitro assay to measure ity of this Argonaute. It will be next died within a few hours after birth and the efficiency of a recombinant Dicer interesting to know how many animal displayed severe sign of anemia. These protein to processed pre-miRNA into miRNAs require Ago2 to be produced. embryos have an important reduction mature miRNA. While a pre-let-7 RNA Since the cleavage of pre-miR-451 by in red blood cell caused by a defect in molecule was effectively process into Ago2 should generate a 30 nucleotides the maturation of erythroid cells. These a mature miRNA form, recombinant long RNA specie, it will be of great results represent the first evidence that Dicer was unable to generate the ma- interest to understand how this miRNA the catalytic domain of Ago2 is essential ture miR-451 from its precursor. These precursor can be trimmed precisely for the survival of mammals. results clearly indicate that in contrast to generate a 21-23 nucleotides long To next determine the molecular phe- to other miRNAs the biogenesis of functional miRNA. To conclude, this notypes of Ago2ADH mice, Cheloufi and miRNA miR-451 is Dicer independent exciting work is yet another example colleagues performed deep sequencing and thus, the Argonaute protein Ago2 that clearly illustrates that we should analysis of short RNAs in the liver, one could be responsible for the biogenesis be extremely cautious of making “gen- of the organ contributing to erythroid of this miRNA. eral” rule in biology; especially in a fast cells maturation in fetus [9]. Among all To confirm this hypothesis, the au- pacing field like the miRNA-mediated the miRNAs expressed in these tissues, thors performed a thorough analysis of silencing. they surprisingly observed that only one the small RNA species sequenced from miRNA in normal fetal liver, miR-451, the Ago2ADH animal fetal liver [9]. They References is strongly decreased in mice carrying observed a significant accumulation catalytically inefficient Ago2. Since of the pre-miR-451 and confirmed the 1 Winter J, Jung S, Keller S, Gregory RI, ADH Diederichs S. Many roads to maturity: the expression of the precursor miRNA absence of mature miR-451 in Ago2 microRNA biogenesis pathways and form of miR-451 was not affected, the samples. The immunoprecipitation of their regulation. Nat Cell Biol 2009; authors concluded that the mutation of the Ago2 complex from a fetal liver 11:228-234. Ago2 most likely impact the maturation homogenate of the transgenic animal 2 Hutvagner G, Simard MJ. Argonaute of miR-451. A careful analysis of the revealed that pre-miR-451 can bind proteins: key players in RNA silencing. miR-451 precursor and mature RNA to the mutated Argonaute. Finally, in Nat Rev Mol Cell Biol 2008; 9:22-32. structure reveals significant differences vitro assays using both wild-type and 3 Liu J, Carmell MA, Rivas FV, et al. Argonaute2 is the catalytic engine between miR-451 and others miRNAs. catalytically inactive affinity purified of mammalian RNAi. Science 2004; At first, they noted that the pre-miR-451 Ago2 proteins confirm that Ago2 can 305:1437-1441. is shorter by 17 nucleotides than most successfully process the pre-miR-451 4 Meister G, Landthaler M, Patkaniows- of pre-miRNAs, making this molecule to generate miR-451 and this miRNA ka A, et al. Human Argonaute2 mediates unlikely a Dicer substrate since this is also able to be loaded onto Ago2 to RNA cleavage targeted by miRNAs Cell Research | Vol 20 No 7 | July 2010 npg 737 and siRNAs. Mol Cell 2004; 15:185- 8 Alisch RS, Jin P, Epstein M, Caspary 12 Leuschner PJ, Ameres SL, Kueng S, 197. T, Warren ST. Argonaute2 is essential Martinez J. Cleavage of the siRNA pas- 5 Tam OH, Aravin AA, Stein P, et al. for mammalian gastrulation and prop- senger strand during RISC assembly in Pseudogene-derived small interfer- er mesoderm formation. PLoS Genet human cells. EMBO Rep 2006; 7:314- ing RNAs regulate gene expression in 2007; 3:e227. 320. mouse oocytes. Nature 2008; 453:534- 9 Cheloufi S, Dos Santos CO, Chong 13 Matranga C, Tomari Y, Shin C, Bar- 538. MM, Hannon GJ. A dicer-independent tel DP, Zamore PD. Passenger-strand 6 Babiarz JE, Ruby JG, Wang Y, Bartel miRNA biogenesis pathway that re- cleavage facilitates assembly of siRNA DP, Blelloch R.
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