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• W2032900712 abstract "•Retrotransposition gave rise to an oocyte-specific Dicer isoform (DicerO) in mice•DicerO is N-terminally truncated and has higher activity than somatic Dicer•DicerO controls the endogenous RNAi pathway and is essential for mouse oocytes•Low Dicer activity and low dsRNA abundance constrain endogenous RNAi in mammals In mammals, a single Dicer participates in biogenesis of small RNAs in microRNA (miRNA) and RNAi pathways. In mice, endogenous RNAi is highly active in oocytes, but not in somatic cells, which we ascribe here to an oocyte-specific Dicer isoform (DicerO). DicerO lacks the N-terminal DExD helicase domain and has higher cleavage activity than the full-length Dicer in somatic cells (DicerS). Unlike DicerS, DicerO efficiently produces small RNAs from long double-stranded (dsRNA) substrates. Expression of the DicerO isoform is driven by an intronic MT-C retrotransposon promoter, deletion of which causes loss of DicerO and female sterility. Oocytes from females lacking the MT-C element show meiotic spindle defects and increased levels of endogenous small interfering RNA (endo-siRNA) targets, phenocopying the maternal Dicer null phenotype. The alternative Dicer isoform, whose phylogenetic origin demonstrates evolutionary plasticity of RNA-silencing pathways, is the main determinant of endogenous RNAi activity in the mouse female germline. In mammals, a single Dicer participates in biogenesis of small RNAs in microRNA (miRNA) and RNAi pathways. In mice, endogenous RNAi is highly active in oocytes, but not in somatic cells, which we ascribe here to an oocyte-specific Dicer isoform (DicerO). DicerO lacks the N-terminal DExD helicase domain and has higher cleavage activity than the full-length Dicer in somatic cells (DicerS). Unlike DicerS, DicerO efficiently produces small RNAs from long double-stranded (dsRNA) substrates. Expression of the DicerO isoform is driven by an intronic MT-C retrotransposon promoter, deletion of which causes loss of DicerO and female sterility. Oocytes from females lacking the MT-C element show meiotic spindle defects and increased levels of endogenous small interfering RNA (endo-siRNA) targets, phenocopying the maternal Dicer null phenotype. The alternative Dicer isoform, whose phylogenetic origin demonstrates evolutionary plasticity of RNA-silencing pathways, is the main determinant of endogenous RNAi activity in the mouse female germline. A single mammalian gene encoding ribonuclease (RNase) III Dicer controls biogenesis of two small RNA classes involved in RNA silencing: microRNAs (miRNAs) and small interfering RNAs (siRNAs). First expressed as long primary transcripts, miRNAs are processed into short hairpin precursor miRNAs (pre-miRNAs) by the nuclear Microprocessor complex, which consists of RNase III Drosha and its cofactor DGCR8 (Jinek and Doudna, 2009Jinek M. Doudna J.A. A three-dimensional view of the molecular machinery of RNA interference.Nature. 2009; 457: 405-412Crossref PubMed Scopus (562) Google Scholar, Krol et al., 2010Krol J. Loedige I. Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay.Nat. Rev. Genet. 2010; 11: 597-610Crossref PubMed Scopus (3542) Google Scholar). Pre-miRNAs are cleaved by Dicer to produce mature miRNAs, which act as sequence-specific guides in effector complexes containing one of the four Argonaute (AGO) proteins and additional protein factors (Jinek and Doudna, 2009Jinek M. Doudna J.A. A three-dimensional view of the molecular machinery of RNA interference.Nature. 2009; 457: 405-412Crossref PubMed Scopus (562) Google Scholar, Krol et al., 2010Krol J. Loedige I. Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay.Nat. Rev. Genet. 2010; 11: 597-610Crossref PubMed Scopus (3542) Google Scholar). These effector complexes target cognate mRNAs to repress their translation. miRNA-mediated translational repression plays numerous essential roles in various somatic cell processes, such as differentiation and cell type specification (Bartel, 2009Bartel D.P. MicroRNAs: target recognition and regulatory functions.Cell. 2009; 136: 215-233Abstract Full Text Full Text PDF PubMed Scopus (15858) Google Scholar). However, maternal miRNAs are dispensable and miRNA activity is globally suppressed in mouse oocytes (Ma et al., 2010Ma J. Flemr M. Stein P. Berninger P. Malik R. Zavolan M. Svoboda P. Schultz R.M. MicroRNA activity is suppressed in mouse oocytes.Curr. Biol. 2010; 20: 265-270Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, Suh et al., 2010Suh N. Baehner L. Moltzahn F. Melton C. Shenoy A. Chen J. Blelloch R. MicroRNA function is globally suppressed in mouse oocytes and early embryos.Curr. Biol. 2010; 20: 271-277Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). A different case represents endogenous siRNAs (endo-siRNAs) produced by Dicer from naturally occurring long double-stranded RNA (dsRNA) substrates, which mediate sequence-specific endonucleolytic cleavage in RNAi pathway. Whereas the processing of dsRNA into siRNAs is inefficient and the level of endo-siRNAs is negligible in somatic cells (Calabrese et al., 2007Calabrese J.M. Seila A.C. Yeo G.W. Sharp P.A. RNA sequence analysis defines Dicer’s role in mouse embryonic stem cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 18097-18102Crossref PubMed Scopus (252) Google Scholar, Nejepinska et al., 2012Nejepinska J. Malik R. Filkowski J. Flemr M. Filipowicz W. Svoboda P. dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells.Nucleic Acids Res. 2012; 40: 399-413Crossref PubMed Scopus (44) Google Scholar), high amounts of endo-siRNAs accumulate in mouse oocytes and early preimplantation embryos (Tam et al., 2008Tam O.H. Aravin A.A. Stein P. Girard A. Murchison E.P. Cheloufi S. Hodges E. Anger M. Sachidanandam R. Schultz R.M. Hannon G.J. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes.Nature. 2008; 453: 534-538Crossref PubMed Scopus (861) Google Scholar, Watanabe et al., 2008Watanabe T. Totoki Y. Toyoda A. Kaneda M. Kuramochi-Miyagawa S. Obata Y. Chiba H. Kohara Y. Kono T. Nakano T. et al.Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes.Nature. 2008; 453: 539-543Crossref PubMed Scopus (897) Google Scholar). The highly active endo-siRNA pathway in mouse oocytes appears to play an essential biological role. A significant portion of oocyte endo-siRNAs arises from protein coding gene-pseudogene pairs and from convergent transcripts, suggesting that RNAi directly targets maternal mRNAs (Tam et al., 2008Tam O.H. Aravin A.A. Stein P. Girard A. Murchison E.P. Cheloufi S. Hodges E. Anger M. Sachidanandam R. Schultz R.M. Hannon G.J. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes.Nature. 2008; 453: 534-538Crossref PubMed Scopus (861) Google Scholar, Watanabe et al., 2008Watanabe T. Totoki Y. Toyoda A. Kaneda M. Kuramochi-Miyagawa S. Obata Y. Chiba H. Kohara Y. Kono T. Nakano T. et al.Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes.Nature. 2008; 453: 539-543Crossref PubMed Scopus (897) Google Scholar). Oocyte-specific knockout of Dicer results in a severe phenotype characterized by chromosome misalignment and spindle defects during metaphase of the first meiotic cleavage, leading to meiotic arrest (Murchison et al., 2007Murchison E.P. Stein P. Xuan Z. Pan H. Zhang M.Q. Schultz R.M. Hannon G.J. Critical roles for Dicer in the female germline.Genes Dev. 2007; 21: 682-693Crossref PubMed Scopus (416) Google Scholar, Tang et al., 2007Tang F. Kaneda M. O’Carroll D. Hajkova P. Barton S.C. Sun Y.A. Lee C. Tarakhovsky A. Lao K. Surani M.A. Maternal microRNAs are essential for mouse zygotic development.Genes Dev. 2007; 21: 644-648Crossref PubMed Scopus (465) Google Scholar). This phenotype is observed also in oocytes deficient in AGO2, which mediates endonucleolytic cleavage of mRNA during RNAi (Kaneda et al., 2009Kaneda M. Tang F. O’Carroll D. Lao K. Surani M.A. Essential role for Argonaute2 protein in mouse oogenesis.Epigenetics Chromatin. 2009; 2: 9Crossref PubMed Scopus (90) Google Scholar). In contrast, oocytes lacking Dgcr8 undergo normal meiotic maturation and fertilization and can support development to the term (Kaneda et al., 2009Kaneda M. Tang F. O’Carroll D. Lao K. Surani M.A. Essential role for Argonaute2 protein in mouse oogenesis.Epigenetics Chromatin. 2009; 2: 9Crossref PubMed Scopus (90) Google Scholar, Murchison et al., 2007Murchison E.P. Stein P. Xuan Z. Pan H. Zhang M.Q. Schultz R.M. Hannon G.J. Critical roles for Dicer in the female germline.Genes Dev. 2007; 21: 682-693Crossref PubMed Scopus (416) Google Scholar, Suh et al., 2010Suh N. Baehner L. Moltzahn F. Melton C. Shenoy A. Chen J. Blelloch R. MicroRNA function is globally suppressed in mouse oocytes and early embryos.Curr. Biol. 2010; 20: 271-277Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, Tang et al., 2007Tang F. Kaneda M. O’Carroll D. Hajkova P. Barton S.C. Sun Y.A. Lee C. Tarakhovsky A. Lao K. Surani M.A. Maternal microRNAs are essential for mouse zygotic development.Genes Dev. 2007; 21: 644-648Crossref PubMed Scopus (465) Google Scholar). The highly active RNAi pathway in mouse oocytes has been in contrast with studies showing that mammalian Dicer is more efficient in processing miRNA precursors than perfect duplex RNA (Ma et al., 2008Ma E. MacRae I.J. Kirsch J.F. Doudna J.A. Autoinhibition of human dicer by its internal helicase domain.J. Mol. Biol. 2008; 380: 237-243Crossref PubMed Scopus (171) Google Scholar, Ma et al., 2012Ma E. Zhou K. Kidwell M.A. Doudna J.A. Coordinated activities of human dicer domains in regulatory RNA processing.J. Mol. Biol. 2012; 422: 466-476Crossref PubMed Scopus (51) Google Scholar). Here, we provide evidence that divergence of small RNA production in mouse oocytes and somatic cells arises from an N-terminally truncated isoform of Dicer that efficiently produces endo-siRNAs from long dsRNAs. This oocyte-specific isoform evolved as a consequence of a specific retrotransposon insertion, and it is essential for oocyte function. Our survey of the mouse maternal transcriptome high-throughput sequencing (HTS) (Smallwood et al., 2011Smallwood S.A. Tomizawa S. Krueger F. Ruf N. Carli N. Segonds-Pichon A. Sato S. Hata K. Andrews S.R. Kelsey G. Dynamic CpG island methylation landscape in oocytes and preimplantation embryos.Nat. Genet. 2011; 43: 811-814Crossref PubMed Scopus (499) Google Scholar) revealed an alternative exon (AltE) located within intron 6 of the Dicer gene (Figure 1A and Figure S1 available online). AltE is derived from an MT-C retroelement of the mammalian apparent long terminal repeat retrotransposon family. MT elements are expressed in oocytes and can serve as alternative promoters for adjacent genes (Peaston et al., 2004Peaston A.E. Evsikov A.V. Graber J.H. de Vries W.N. Holbrook A.E. Solter D. Knowles B.B. Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos.Dev. Cell. 2004; 7: 597-606Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar). AltE serves as the first exon spliced at a conserved donor site (Peaston et al., 2004Peaston A.E. Evsikov A.V. Graber J.H. de Vries W.N. Holbrook A.E. Solter D. Knowles B.B. Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos.Dev. Cell. 2004; 7: 597-606Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar) in frame with exon 7, leading to production of a shortened Dicer variant (DicerO) lacking the N-terminal DExD helicase domain (Figure 1B). Comparative genomic analysis suggests that the MT insertion giving rise to DicerO occurred relatively recently in the Muridae family. The MT insertion is found only in mouse and rat and is absent from genomes of other sequenced members of other rodent families (Figure 1C).Figure S1DicerO Genomic Organization, Related to Figure 1Show full caption(A) Position of MT-C-derived alternative exon (AltE) in the Dicer gene. Dicer gene scheme was modified from the UCSC genome browser (http://genome.ucsc.edu/). 5′ end of AltE was determined by 5′RACE. Note that Dicer gene is in the (-) orientation in the mouse genome.(B) Total RNA HTS data (Smallwood et al., 2011Smallwood S.A. Tomizawa S. Krueger F. Ruf N. Carli N. Segonds-Pichon A. Sato S. Hata K. Andrews S.R. Kelsey G. Dynamic CpG island methylation landscape in oocytes and preimplantation embryos.Nat. Genet. 2011; 43: 811-814Crossref PubMed Scopus (499) Google Scholar) indicate position of AltE in the MT-C element. The MT-C element insertion is rodent-specific. Conservation data were downloaded from the UCSC genome browser.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Position of MT-C-derived alternative exon (AltE) in the Dicer gene. Dicer gene scheme was modified from the UCSC genome browser (http://genome.ucsc.edu/). 5′ end of AltE was determined by 5′RACE. Note that Dicer gene is in the (-) orientation in the mouse genome. (B) Total RNA HTS data (Smallwood et al., 2011Smallwood S.A. Tomizawa S. Krueger F. Ruf N. Carli N. Segonds-Pichon A. Sato S. Hata K. Andrews S.R. Kelsey G. Dynamic CpG island methylation landscape in oocytes and preimplantation embryos.Nat. Genet. 2011; 43: 811-814Crossref PubMed Scopus (499) Google Scholar) indicate position of AltE in the MT-C element. The MT-C element insertion is rodent-specific. Conservation data were downloaded from the UCSC genome browser. In mice, DicerO transcript was detected only in oocytes (Figure 2A), where transcript levels increased during oocyte growth and rapidly declined after fertilization (Figure 2B). RT-PCR also indicated oocyte-specific expression of rat DicerO (Figure 2C). DicerO is the dominant Dicer protein isoform in mouse oocytes (Figures 2D and S2A). Consistent with HTS and RT-PCR data, the full-length somatic Dicer (DicerS) protein levels are minimal in mouse oocytes (Figures 2D and S2A). Ectopic expression of myc-tagged DicerO showed that the N-terminal truncation does not affect the characteristic cytoplasmic cellular localization of the protein (Figure S2B).Figure S2DicerO Expression and Activity, Related to Figure 2Show full caption(A) Western blot analysis of NIH 3T3 cells (3T3, 20 μg of a protein lysate) and fully-grown GV oocytes (FGO, 600 oocytes = ∼15 μg of total protein). Arrowhead depicts a faint band corresponding to DicerS protein size. A low level of DicerS is consistent with levels of its mRNA (Figure 1A and 2C). The smear above 250 kDa is an artifact of developing the Western blot signal using the SuperSignal West Femto Maximum Sensitivity Substrate.(B) Ectopic expression of myc-tagged DicerO and DicerS in transiently transfected 3T3 cells. Western blot confirms expression of Dicer isoforms. Immunofluorescence images show localization of Dicer proteins stained with anti-myc antibody (9E10, diluted 1:200, green color) and DNA is stained with DAPI (blue color). The smear above 250 kDa is an artifact from the SuperSignal West Femto Maximum Sensitivity Substrate, which accidentally appeared during developing the Western blot signal.(C) Fluorescence-based assay for monitoring Dicer cleavage activity. The fluorescent in vitro Dicer assay is based on cleavage of a 27 nucleotides long perfect RNA duplex containing a two nucleotide 3′ overhang at one terminus and a blunt end at the other one. At the blunt end, one RNA strand carries a fluorescent group (Cy5) and the other strand a quencher (IowaBlackRQ, IBRQ). During cleavage by Dicer, the two nucleotide 3′ overhang is recognized by the PAZ domain and the cleavage releases a short duplex carrying the fluorophore and the quencher. Subsequent separation of the fluorophore from the quencher yields fluorescence.(D) Fluorescent in vitro Dicer assay results using different Dicer and substrate concentrations. Each recombinant Dicer isoform (80, 250 and 500 nM) was incubated for the indicated times with a 27-bp perfect duplex RNA substrate (30 and 200 nM). Cleaved substrate yields fluorescence, which is shown on the y axis as percentage of the maximal fluorescence gain from the substrate. Independent preparations of recombinant Dicer proteins were used for analyzing DicerO activity under single turnover conditions (500 and 250 nM Dicer with 30 nM substrate) and under excess of substrate (200 nM substrate and 80 nM Dicer).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Western blot analysis of NIH 3T3 cells (3T3, 20 μg of a protein lysate) and fully-grown GV oocytes (FGO, 600 oocytes = ∼15 μg of total protein). Arrowhead depicts a faint band corresponding to DicerS protein size. A low level of DicerS is consistent with levels of its mRNA (Figure 1A and 2C). The smear above 250 kDa is an artifact of developing the Western blot signal using the SuperSignal West Femto Maximum Sensitivity Substrate. (B) Ectopic expression of myc-tagged DicerO and DicerS in transiently transfected 3T3 cells. Western blot confirms expression of Dicer isoforms. Immunofluorescence images show localization of Dicer proteins stained with anti-myc antibody (9E10, diluted 1:200, green color) and DNA is stained with DAPI (blue color). The smear above 250 kDa is an artifact from the SuperSignal West Femto Maximum Sensitivity Substrate, which accidentally appeared during developing the Western blot signal. (C) Fluorescence-based assay for monitoring Dicer cleavage activity. The fluorescent in vitro Dicer assay is based on cleavage of a 27 nucleotides long perfect RNA duplex containing a two nucleotide 3′ overhang at one terminus and a blunt end at the other one. At the blunt end, one RNA strand carries a fluorescent group (Cy5) and the other strand a quencher (IowaBlackRQ, IBRQ). During cleavage by Dicer, the two nucleotide 3′ overhang is recognized by the PAZ domain and the cleavage releases a short duplex carrying the fluorophore and the quencher. Subsequent separation of the fluorophore from the quencher yields fluorescence. (D) Fluorescent in vitro Dicer assay results using different Dicer and substrate concentrations. Each recombinant Dicer isoform (80, 250 and 500 nM) was incubated for the indicated times with a 27-bp perfect duplex RNA substrate (30 and 200 nM). Cleaved substrate yields fluorescence, which is shown on the y axis as percentage of the maximal fluorescence gain from the substrate. Independent preparations of recombinant Dicer proteins were used for analyzing DicerO activity under single turnover conditions (500 and 250 nM Dicer with 30 nM substrate) and under excess of substrate (200 nM substrate and 80 nM Dicer). The architecture of DicerO resembles experimentally truncated human Dicer, which was shown to cleave a 37 bp dsRNA substrate more efficiently than full-length Dicer, suggesting an autoinhibitory function of the helicase domain in long dsRNA processing (Ma et al., 2008Ma E. MacRae I.J. Kirsch J.F. Doudna J.A. Autoinhibition of human dicer by its internal helicase domain.J. Mol. Biol. 2008; 380: 237-243Crossref PubMed Scopus (171) Google Scholar). To examine DicerO cleavage activity, we used a fluorescent in vitro dicing assay (Podolska et al., 2013Podolska K. Sedlak D. Bartunek P. Svoboda P. Fluorescence-Based High-Throughput Screening of Dicer Cleavage Activity.J. Biomol. Screen. 2013; (Published online August 14, 2013)https://doi.org/10.1177/1087057113497400Crossref PubMed Scopus (8) Google Scholar), in which a perfect 27 bp RNA duplex substrate is cleaved to yield fluorescent product (Figure S2C). Consistent with previous results, DicerO was more active than DicerS (Figure 2E and S2D). To assess the biological significance of MT-driven DicerO expression in mouse oocytes, we used TAL effector nuclease (TALEN) technology (Miller et al., 2011Miller J.C. Tan S. Qiao G. Barlow K.A. Wang J. Xia D.F. Meng X. Paschon D.E. Leung E. Hinkley S.J. et al.A TALE nuclease architecture for efficient genome editing.Nat. Biotechnol. 2011; 29: 143-148Crossref PubMed Scopus (1566) Google Scholar) to produce mutant mice lacking the MT element in Dicer intron 6 (Figure 3A). Two TALEN pairs flanking the MT element were used to excise it from the genome (Figure S3), abolishing DicerO expression without affecting DicerS expression (Figure 3B). Mice homozygous for the deletion (ΔMT/ΔMT) were viable and males were fertile demonstrating that the deletion did not affect normal Dicer function in somatic cells or male germline. Females, however, phenocopied mice carrying oocyte-specific conditional deletion of Dicer (Murchison et al., 2007Murchison E.P. Stein P. Xuan Z. Pan H. Zhang M.Q. Schultz R.M. Hannon G.J. Critical roles for Dicer in the female germline.Genes Dev. 2007; 21: 682-693Crossref PubMed Scopus (416) Google Scholar, Tang et al., 2007Tang F. Kaneda M. O’Carroll D. Hajkova P. Barton S.C. Sun Y.A. Lee C. Tarakhovsky A. Lao K. Surani M.A. Maternal microRNAs are essential for mouse zygotic development.Genes Dev. 2007; 21: 644-648Crossref PubMed Scopus (465) Google Scholar): they were sterile and their oocytes showed meiotic spindle defects and increased levels of endo-siRNA targets (Figures 3C–3E). Oocytes lacking DicerO also contained increased levels of MT retrotransposon transcripts (Figure 3E), similar to Dicer−/− oocytes (Murchison et al., 2007Murchison E.P. Stein P. Xuan Z. Pan H. Zhang M.Q. Schultz R.M. Hannon G.J. Critical roles for Dicer in the female germline.Genes Dev. 2007; 21: 682-693Crossref PubMed Scopus (416) Google Scholar, Watanabe et al., 2008Watanabe T. Totoki Y. Toyoda A. Kaneda M. Kuramochi-Miyagawa S. Obata Y. Chiba H. Kohara Y. Kono T. Nakano T. et al.Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes.Nature. 2008; 453: 539-543Crossref PubMed Scopus (897) Google Scholar). Thus, ΔMT/ΔMT mice provide genetic evidence that the MT-C insertion into Dicer intron 6 and the resulting MT-driven expression of DicerO are essential for the female germline. Remarkably, DicerO does not seem to be required for miRNA biogenesis in mouse oocytes, because levels of two abundant maternal miRNAs (miR-30 and Let-7) show a mild increase in ΔMT/ΔMT oocytes (Figure 3F).Figure S3TALEN-Mediated Deletion of the MT-C Element in Dicer Intron 6, Related to Figure 3Show full caption(A) Positions of double-stranded breaks introduced by TALENs. Individual TALEN recognition sites are shown in red letters.(B) Nine founder mice carrying ten deletion alleles were obtained. All alleles were sequenced to determine deleted sequences, which are shown in the last column on the right. Alleles colored in red were used in experiments described in the article and they all yielded the null phenotype.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Positions of double-stranded breaks introduced by TALENs. Individual TALEN recognition sites are shown in red letters. (B) Nine founder mice carrying ten deletion alleles were obtained. All alleles were sequenced to determine deleted sequences, which are shown in the last column on the right. Alleles colored in red were used in experiments described in the article and they all yielded the null phenotype. Given the limitations of the oocyte model system, we characterized DicerO in vivo in Dicer-expressing cell lines derived from embryonic stem cells (ESCs) lacking endogenous Dicer (Murchison et al., 2005Murchison E.P. Partridge J.F. Tam O.H. Cheloufi S. Hannon G.J. Characterization of Dicer-deficient murine embryonic stem cells.Proc. Natl. Acad. Sci. USA. 2005; 102: 12135-12140Crossref PubMed Scopus (673) Google Scholar). This approach allowed us to compare the ability of DicerO and DicerS to produce different classes of small RNAs from endogenous and ectopically expressed substrates. Three stable DicerO rescue lines expressing different levels of DicerO were selected for further study (Figure 4A). The DicerO-2 line highly overexpressed the protein, the DicerO-3 line expressed it at medium levels, and the DicerO-4 line expressed it at low levels. We also generated two lines expressing medium levels of DicerS (Figure 4A). As controls, we used a Dicer null ESC line (Dcr−/−), the parental ESC line heterozygous for Dicer (Dcr+/−; Murchison et al., 2005Murchison E.P. Partridge J.F. Tam O.H. Cheloufi S. Hannon G.J. Characterization of Dicer-deficient murine embryonic stem cells.Proc. Natl. Acad. Sci. USA. 2005; 102: 12135-12140Crossref PubMed Scopus (673) Google Scholar), and an ESC line containing Dicer but lacking canonical miRNAs (Dgcr8−/−; Wang et al., 2007Wang Y. Medvid R. Melton C. Jaenisch R. Blelloch R. DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal.Nat. Genet. 2007; 39: 380-385Crossref PubMed Scopus (800) Google Scholar). DicerO in all lines rescued miRNA expression (Figure 4B) and miRNA activity, as evidenced by suppression of five miRNA targets, including cell cycle regulators p21 and Rbl2 (Shenoy and Blelloch, 2009Shenoy A. Blelloch R. Genomic analysis suggests that mRNA destabilization by the microprocessor is specialized for the auto-regulation of Dgcr8.PLoS ONE. 2009; 4: e6971Crossref PubMed Scopus (39) Google Scholar, Sinkkonen et al., 2008Sinkkonen L. Hugenschmidt T. Berninger P. Gaidatzis D. Mohn F. Artus-Revel C.G. Zavolan M. Svoboda P. Filipowicz W. MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells.Nat. Struct. Mol. Biol. 2008; 15: 259-267Crossref PubMed Scopus (397) Google Scholar; Figure 4C). DicerO also rescued the proliferation defect of Dcr−/− ESCs (Kanellopoulou et al., 2005Kanellopoulou C. Muljo S.A. Kung A.L. Ganesan S. Drapkin R. Jenuwein T. Livingston D.M. Rajewsky K. Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing.Genes Dev. 2005; 19: 489-501Crossref PubMed Scopus (1058) Google Scholar; Figure S4). In addition, miRNAs in DicerO lines efficiently repressed luciferase reporters that carried binding sites either fully or partially complementary to the abundant, ESC-specific miR-294 (Houbaviy et al., 2003Houbaviy H.B. Murray M.F. Sharp P.A. Embryonic stem cell-specific MicroRNAs.Dev. Cell. 2003; 5: 351-358Abstract Full Text Full Text PDF PubMed Scopus (948) Google Scholar; Figure 4D). These results indicate that DicerO expression completely restores suppression mediated by partially complementary binding sites, which are typical for natural miRNA targets.Figure S4DicerO Rescues Growth Defects of Dcr−/− ESCs, Related to Figure 4Show full captionGrowth curves of selected ESC lines. Growing ESCs were analyzed at indicated time points as described in supplemental experimental procedures. Values are normalized to the day 0 (the day after plating cells). Values represent average of two experiments performed in duplicates, error bars = SEM. At the day 6, Dcr+/− and DicerS-4 lines showed slightly lower growth rate compared to the remaining three lines. Numerous factors could account for difference among individual cell lines, including their individual adaptations to cell culture, Dicer expression vector insertion site effects, or changing culturing conditions toward the end of the experiment, which could result in slower growth of Dcr+/− and DicerS-4 cells after day 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Growth curves of selected ESC lines. Growing ESCs were analyzed at indicated time points as described in supplemental experimental procedures. Values are normalized to the day 0 (the day after plating cells). Values represent average of two experiments performed in duplicates, error bars = SEM. At the day 6, Dcr+/− and DicerS-4 lines showed slightly lower growth rate compared to the remaining three lines. Numerous factors could account for difference among individual cell lines, including their individual adaptations to cell culture, Dicer expression vector insertion site effects, or changing culturing conditions toward the end of the experiment, which could result in slower growth of Dcr+/− and DicerS-4 cells after day 4. To analyze endo-siRNA production by DicerO, we used HTS to identify small RNAs in ESC lines expressing DicerO or DicerS. Small RNA libraries from eight cell lines were sequenced to depths of 3–5 × 107 reads using sequencing by oligonucleotide ligation and detection (SOLiD) technology (GSE41207; Table S1). The size distribution of small RNAs in DicerO-expressing cells showed a peak at 21–23 nt reads, similar to results in DicerS and Dcr+/− ESC lines (Figure 5A). The majority of 21–23 nt reads in DicerO lines mapped to miRNAs, as did the majority in DicerS lines, although the proportion of 21–23 nt reads from non-miRNA loci was greater in DicerO lines than in DicerS lines (Figure 5B). The composition of the miRNA population in DicerO lines was generally similar to that in DicerS lines (Figure 5C); however, the abundance of several miRNAs was consistently different across the two types of cell lines (Figure S5). Accordingly, unsupervised clustering of miRNA transcriptomes from all samples led to separate clusters for DicerS and DicerO ESC lines, suggesting that each Dicer isoform" @default.
• W2032900712 created "2016-06-24" @default.
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• W2032900712 date "2013-11-01" @default.
• W2032900712 modified "2023-10-18" @default.
• W2032900712 title "A Retrotransposon-Driven Dicer Isoform Directs Endogenous Small Interfering RNA Production in Mouse Oocytes" @default.
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