The Role of RNA Interference in Stem Cell Biology: Beyond the Mutant Phenotypes

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YJMBI-65322; No. of pages: 12; 4C: 3, 5

Maxime Bodak 1,2,†, Daniel Cirera-Salinas 1,†, Janina Luitz 1,3,† and Constance Ciaudo 1

1 - Swiss Federal Institute of Technology Zurich, Department of Biology, Institute of Molecular Health Sciences, CH-8093 Zurich, Switzerland 2 - Life Science Zurich Graduate School, Molecular Life Science program, University of Zurich, CH-8047 Zurich, Switzerland 3 - Life Science Zurich Graduate School, RNA Biology program, University of Zurich, CH-8057 Zurich, Switzerland

Correspondence to Constance Ciaudo: Swiss Federal Institute of Technology Zurich, Department of Biology, Institute of Molecular Health Sciences, RNAi and Genome Integrity, HPL G32.1, Otto-Stern-Weg 7, CH-8093 Zurich, Switzerland. [email protected]. http://dx.doi.org/10.1016/j.jmb.2017.01.014 Edited by David Laurent

Abstract

Complex gene regulation systems ensure the maintenance of cellular identity during early development in mammals. Eukaryotic small RNAs have emerged as critical players in RNA interference (RNAi) by mediating gene silencing during embryonic stem cell self-renewal. Most of the proteins involved in the biogenesis of small RNAs are essential for proliferation and differentiation into the three germ layers of mouse embryonic stem cells. In the last decade, new functions for some RNAi proteins, independent of their roles in RNAi pathways, have been demonstrated in different biological systems. In parallel, new concepts in stem cell biology have emerged. Here, we review and integrate the current understanding of how RNAi proteins regulate stem cell identity with the new advances in the stem cell field and the recent non-canonical functions of the RNAi proteins. Finally, we propose a reevaluation of all RNAi mutant phenotypes, as non-canonical (small non-coding RNA independent) functions may contribute to the molecular mechanisms governing mouse embryonic stem cells commitment. © 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction these small molecules have considerable deleterious effects including genetic instability, sterility, and loss The first report of RNA interference (RNAi) in 1998 of cellular identity. Recent research indicates that and the discovery of its effector molecules, the small miRNAs have important roles in regulating stem cell interfering RNAs (siRNAs) [1,2], have revolutionized self-renewal and differentiation by repressing the our understanding of gene regulation. Since then, the translation of selected mRNAs in stem cells and biological importance of these silencing small RNAs differentiating daughter cells [4]. More importantly, (sRNAs) and other classes of non-coding sRNAs mouse embryonic stem cells (mESCs) deficient of has been evidenced through a staggering array of Dicer or Dgcr8, two essential genes involved in miRNA publications implicating these molecules in virtually all biogenesis, fail to produce miRNAs and manifest aspects of cell biology. MicroRNAs (miRNAs), in defects in proliferation and differentiation, which particular, influence many biological processes includ- confirms the essential role for the RNAi genes in early ing immune responses, hormonal regulation, phase mouse development [5–7]. Unfortunately, the Dicer transitioning and patterning, and embryonic and and Dgcr8 mutant mESCs reported by different post-embryonic development [3]. Tight and stringent laboratories have a distinct background, making their regulation of the sRNA pathways is a cellular necessity, comparison difficult. Nevertheless, the aforementioned as perturbations in the expression and/or activity of Dicer-andDgcr8-deficient mESCs present differences 0022-2836/© 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). J Mol Biol (2017) xx, xxx–xxx

Please cite this article as: M. Bodak, et al., The Role of RNA Interference in Stem Cell Biology: Beyond the Mutant Phenotypes, J. Mol. Biol. (2017), http://dx.doi.org/10.1016/j.jmb.2017.01.014 2 Roles of RNAi pathways in Stem Cell Biology in their phenotype (e.g., proliferation and cell cycle tion [22]. In BMP4 plus LIF condition, mESCs from defects, impaired differentiation) [6–8], suggesting that the 129 background were successfully derived and the role of DICER in the siRNA pathway could have cultured in a feeder-free and serum-free environment a specific function in mESCs homeostasis [7] or [22]. Remarkably, LIF has also been shown to promote that DICER and/or DGCR8 proteins might have differentiation by stimulating the mitogen-activated non-canonical functions other than their specific role protein kinase/Extracellular Signal-Regulated Kinase in the miRNA pathway. Moreover, recent break- (ERK pathway) [23]. Indeed, suppression of the ERK throughs in stem cell biology now allow to distinguish pathway improved mESC self-renewal [24].Finally,it between the exit from pluripotency (an event necessary was reported that mESCs produce significant amount for mESC commitment) and the differentiation of of another potent activator of the ERK pathway: the mESCs (their capacity to activate differentiation fibroblast growth factor-4 [24,25]. Therefore, new programs). approaches using ERK pathway repressors started to In this perspective, we present previous and be employed. In conclusion, a new culture condition emerging concepts in RNAi and stem cell biology combining LIF, BMP4, and ERK pathway inhibitors fields. Finally, we propose that these new concepts allowed the reproducible derivation of mESCs from should call for a reinvestigation of the RNAi mutant C57BL/6 and CBA strains in a feeder-free and serum- phenotypes in the light of their putative non-canonical free environment [26]. functions in stem cell biology. In the early 2000s, studies revealed a new antagonist of mESC self-renewal, the glycogen synthase kinase-3 (GSK3), and further investigations showed that its State-Of-The-Art and New Advances in inhibition enhanced mESC propagation [27–30]. GSK3 Stem Cell Biology negatively regulates a range of cellular and intracellular pathways, including the canonical Wnt/ß-catenin path- In 1981, two groups successfully isolated cells way [31,32]. The big breakthrough happened in 2008, directly from mouse blastocyst for the first time [9,10]. when Ying and colleagues defined a new medium Derived from the whole delayed blastocyst [9] or from combining three inhibitors: an FGF receptor inhibitor the inner cell mass (ICM) isolated by immunosurgery (SU5042), an ERK pathway inhibitor (PD184352), and [10], early embryonic cells exhibited the capacity to a GSK3 inhibitor (CHIR99021) [33]. Mouse ESCs generate well-differentiated teratomas and presented cultured in this new medium named 3i (for three a normal karyotype [9–11]. The ICM-isolated cells inhibitors) displayed a stronger self-renewal compared were named mESCs [10]. The first studies implement- to mESCs cultured in serum plus LIF or LIF and BMP4 ed the culture protocols of mESCs using feeder cells conditions and even propagated in the absence of LIF and serum-supplemented medium [9]. The establish- or serum [33]. Afterwards, a single high potency and ment of these culture conditions highlighted either selectively MEK stands for MAP (Mitogen-Activated the presence (in the serum and in the conditioned Protein) Kinase/ERK (Extracellular Signal-Regulated medium) or the production (by the feeder cells) of Kinase) Kinase (MEK) inhibitor (PD0325901) and a factors favoring the survival of mESCs [9,10,12,13]. GSK3 inhibitor were shown to be sufficient to support Despite these mESC isolation breakthroughs the long-term culture of mESCs [34]; therefore, this new [9,10], the original empirically established protocols culture condition was coined 2i. Thus, it is now and culture conditions did not allow isolation of ESCs acknowledged that in 2i medium, mESCs reach a from other mammals. At the beginning, ESCs could ground-state naïve pluripotency, resembling to the be derived only from mouse embryos of inbred 129 epiblast of the mature mouse blastocyst [35]. strain and more rarely from the C57BL/6 strain [14]. In addition, the 2i culture condition overcame the non- Deconvolution of the mixture of the factors present in permissive strain barrier and enabled the derivation the serum and of the interactions between mESCs of ESCs from other mouse backgrounds [33,36–38] and the feeder cells allowed researchers to identify a and from other rodent species [39,40]. More recently, key differentiation-inhibiting protein: the cytokine the 2i media has permitted the derivation of haploid leukemia inhibitory factor (LIF) [15,16]. Addition of mESCs [41]. These cells are a tremendous valuable LIF to the serum allowed derivation and long-term tool because they allow the usage of genetic screens, culture of mESCs in the absence of feeder cells which will extend genome exploration into develop- [15,16]. However, this improvement was still limited mentally and medically relevant pathways [42]. to the 129 mouse strains. Briefly, LIF promotes self-renewal by repressing the differentiation programs via the stimulation of the signal transducer Impact of these New Discoveries on the and activator of transcription 3 [17–20]. Moreover, Pluripotency Network in mESCs LIF alone was not sufficient to sustain mESC culture, as serum withdraw led to the differentiation of the Several transcription factors are critical for the cells [21]. Later, the bone morphogenetic factor-4 maintenance of the naïve pluripotent state of mESCs (BMP4) was found to also support mESC propaga- including OCT4, SOX2, and NANOG (OSN). Their

Fig. 1. MiRNA and siRNA pathways in mammals. Schematic representation of the canonical miRNA, non-canonical mirtron, and m7G-capped pathways. The exogenous and endo-siRNA pathways are also represented. down-regulation is essential to exit from the plur- state and underpins pluripotency by preparing mESCs ipotency state and to differentiate into the three for lineage commitment [50,51]. Importantly, mESCs embryonic germ layers [43]. Indeed, when mESCs, cultured in 2i plus LIF media show substantially uniform derived from the E4.5 epiblast [44], are cultured in expression of key pluripotency factors (e.g., NANOG serum supplemented with LIF and/or on feeder cells, and STELLA) [47]. Furthermore, transcription factors they present a mosaic of some pluripotent factors' typically down-regulated at the onset of mESC expression pattern [45–47]. Particularly, it has been differentiation such as KLF4, ESRRB, and REX1 are shown that NANOG expression is heterogeneous in also homogenously expressed in 2i medium in contrast serum plus LIF cultures [48]. Similarly, the expression to serum plus LIF condition [35]. As a consequence of STELLA and REX1, other important pluripotency of the homogeneity observed in the pluripotency- transcription factors, presents dynamic heterogeneity associated gene expression between the two growth in serum plus LIF culture condition [45,49]. The fact that conditions, cellular morphology also differs [47]. Mouse mESCs, cultured in serum plus LIF, express various ESCs cultured in 2i media are morphologically uniform lineage-specific genes in a heterogeneous manner and present as homogenous clusters of small, tightly indicates that a proportion of cells undergo priming and/ packed cells, whereas serum plus LIF cultured mESCs or differentiation in response to the complex mix of are rather flattened and exhibit heterogeneous mor- serum components [45–47]. It has been proposed that phologies [47]. the simultaneous heterogeneous expression of The culture conditions (serum plus LIF or 2i plus LIF) lineage-specific genes and pluripotency factors across also influence the epigenetic status of the mESCs [52]. the culture reflects an inherent fluidity of the pluripotent The most highly expressed genes under both

Please cite this article as: M. Bodak, et al., The Role of RNA Interference in Stem Cell Biology: Beyond the Mutant Phenotypes, J. Mol. Biol. (2017), http://dx.doi.org/10.1016/j.jmb.2017.01.014 4 Roles of RNAi pathways in Stem Cell Biology conditions presented the trimethylation of the lysine 4 Importance of Canonical RNAi Pathways of the histone 3 associated with active promoters and the trimethylation of the lysine 36 of the histone H3 in mESCs associated with coding body of transcribed genes. In mammals, two types of canonical RNAi pathways The levels of the trimethylation of the lysine 27 of coexist: miRNA and siRNA pathways, which them- the histone H3 (H3K27me3) have been shown to selves could be divided into canonical or non-canonical increase considerably in mESCs cultured in serum miRNA pathways and into endogenous and exoge- plus LIF media [53]. Further analysis demonstrated a nous siRNA pathways (Fig. 1). global lack of H3K27me3 at promoter regions in 2i MiRNAs have been identified as important post- – media, while in serum plus LIF condition, 60 65% of transcriptional regulators of gene expression [62,63]. – H3K27me3 was observed at promoters [53 55]. They are small and single-stranded non-coding RNAs, Indeed, in serum plus LIF conditions, many promoters which control the expression of their target genes couldbedistinguishedandarecalledbivalentpro- by primarily acting as sequence-specific inhibitors moters (marked with the trimethylation of the lysine 4 of the corresponding mRNA. This inhibitory effect of the histone 3 and H3K27me3), while only a few can occur by transcript destabilization or translational of them were found in 2i condition, mainly due to inhibition or by both via deadenylation, decapping, the previously described loss of H3K27me3 [54]. and exonucleolysis [64]. As illustrated in Fig. 1, the H3K27me3 mark is deposited on histone H3 by the production of a functional mature miRNA involves histone lysine methyltransferase EZH2, a member multiple processing steps. Most animal miRNAs are of the polycomb II complex [56]. In serum plus LIF transcribed in the nucleus by RNA polymerase II condition, genes targeted by polycomb II complex as stem-loop long primary transcripts. Following tran- exhibited an important variability of their expression scription, they are processed sequentially in the [57]. These targeted genes include many develop- nucleus and cytoplasm by two protein complexes mental regulators and lineage specification activators containing RNAse-III-type endonucleases, DROSHA and are repressed with a unique chromatin signature and DICER. Specifically, DROSHA and its partner in mESCs (highly enriched for the H3K27me3 mark) DGCR8, the so-called “Microprocessor complex”,trim [58]. Taken together, the epigenomic and transcrip- the primary transcript (pri-miRNA) to a 70- to 100-nt tional profiles of mESCs cultured in 2i plus LIF con- stem-loop precursor miRNA (pre-miRNA). Subse- dition resemble the epigenetic and transcriptional quently, they are delivered to the cytoplasm environment of the epiblast in the blastocyst, thereby by EXPORTIN 5, where it is cleaved by DICER, allowing the direct capture and preservation of naïve which forms a complex with TRBP, to produce pluripotency in vitro [47]. a ~22-nt miRNA-3p:miRNA-5p duplex (canonical Besides, when 2i and LIF components are removed miRNA pathway). However, a subset of intronic from the culture, naïve mESCs rapidly exit their miRNAs called “mirtrons” and intergenic short hairpin – pluripotent state and differentiate [47,59 61]. The exit RNA-derived miRNAs can circumvent the Micro- from the pluripotent state requires the dissolution of the processor complex. They are produced by splicing core pluripotency transcription factor circuit, but it still and the lariat debranching of short hairpin introns remains unknown how the differentiation machinery (mirtron pathway) or are directly transcribed into the breaks down this network. Betschinger and colleagues precursor short hairpin RNA by RNA polymerase II took advantage of this rapid exit from the pluripotency of [7-methylguanosine (m7G)-capped pathway] [65,66]. mESCs cultured in permissive media and developed These Microprocessor-independent m7G-capped “ ” an exit from pluripotency assay , in which the ability to precursor miRNAs, whose 5′ ends coincide with tran- self-renew in a 2i culture after a period of permissive scription start sites and 3′ ends are most likely culture is assessed by alkaline phosphatase staining generated by transcription termination, are exported [60]. Only the cells that will not commit to differentiation by EXPORTIN 1 to the cytoplasm. In summary, the and will remain in a pluripotent state will survive biogenesis of miRNAs is achieved by at least three and proliferate when reintroduced into the 2i culture pathways: the canonical miRNA pathway and the condition. This new assay has enabled the study of two non-canonical mirtron and m7G-capped path- new regulators of the commitment of mESCs to ways (Fig. 1). differentiation. Once in the cytoplasm, one of the duplex strands In conclusion, the 2i media rapidly emerged as a (miRNA-3p or miRNA-5p) is preferentially incorporated novel and very standard method to culture mESCs into the RNA-induced silencing complex (RISC) in due to its well-defined composition, easily transpos- association with an Argonaute (AGO) family member. able from one laboratory to another and from one Within the RNA-induced silencing complex-AGO background of cells to another. Furthermore, it entity, the miRNA guides the complex to its RNA allows the exploration of the mechanisms involved target, thereby mediating its repression. In animals, in stem cell commitment using exit from pluripotency most miRNAs control gene expression by binding to assays. the 3′ UTR of their target genes through Watson–Crick

Fig. 2. Dgcr8, Dicer, and Argonaute genes are essential for early mammalian development. Mouse ESCs are derived from the inner cell mass (ICM) of blastocyst at E3.5 dpc and mimic early mammalian development in vitro. Most of RNAi members are essential for mouse early development, as deletion of any of them (Dgcr8, Dicer, and Ago2) causes embryonic lethality before post-implantation stage. Likewise, mESCs lacking Dgcr8, Dicer,orall Agos are unable to differentiate in vitro into the three embryonic germ layers. base pairing between the target and the 5′-end of the In mammals, endogenous siRNAs (endo-siRNAs) miRNAs: the “seed” sequence. However, recent have been only described in germ lines and in mESCs studies suggest that miRNAs might also repress (endo-siRNA pathway) [75–78]. They are mainly de- mRNA targets by binding to other regions including rived from inverted repeat elements, convergent tran- the 5′ UTR or protein-coding exons [64]. scription, and pseudogenes/gene pairs [79] (Fig. 1). MiRNAs have an important role in regulating stem Endo-siRNAs are processed by DICER from double- cell self-renewal and differentiation by repressing stranded RNAs and may function like their artificial the translation of selected mRNAs in stem cells counterparts used in experimental RNAi. They can be and differentiating daughter cells. The mESC miRNA loaded onto different AGO proteins, with the restriction signature is characterized by miRNAs inhibiting differ- that only AGO2 mediates the endonucleolytic cleav- entiation (miR-290-295 cluster) and others promoting age of their target mRNAs [80]. However, examples differentiation (e.g., miR-296, miR-134, miR-21, of this mode of action are limited to mammals [81]. and let-7) [67,68]. Promoters of many miRNA genes Nevertheless, exogenous siRNAs have been identi- are bound in mESCs by the core pluripotency fied and can act as antiviral defense mechanisms factors (OSN) [69]. Interestingly, there is a reciprocal [82,83]. relationship; hence, several miRNAs that are up- In conclusion, the role of the RNAi protein core regulated during mESCs differentiation have been machinery (DGCR8, DROSHA, DICER, and AGO shown to directly target the mRNAs coding for these proteins) in miRNA and siRNA pathways is considered pluripotency factors (OSN and KLF4) [70]. Indeed, as a canonical function of these proteins, whereas an various miRNAs have been employed to success- sRNA biogenesis-independent function is described fully increase the efficiency of reprogramming [71]. as a non-canonical function of these proteins. During the reprogramming of mouse embryonic In vivo studies with RNAi mutants show embryonic fibroblasts (MEFs), a subset of miRNAs belonging to lethality before the implantation stage [5,7,84].As the miR-290-295 cluster was able to significantly investigations during development have been done at enhance the colony number of induced pluripotent 3.5 days post coïtum (dpc) and 6.5 dpc, the exact time stem cells formation [71]. For a detailed description of frame and molecular mechanisms leading to this the individual miRNAs and their function in stem cell lethality have not been dissected so far (Fig. 2). biology and during differentiation we would like to refer Mouse ESCs that are deficient for RNAi genes fail to to recent reviews [72–74]. produce miRNAs and manifest defects in proliferation,

Please cite this article as: M. Bodak, et al., The Role of RNA Interference in Stem Cell Biology: Beyond the Mutant Phenotypes, J. Mol. Biol. (2017), http://dx.doi.org/10.1016/j.jmb.2017.01.014 6 laect hsatcea:M oa,e l,TeRl fRAItreec nSe elBooy eodteMtn hntps J. Phenotypes, Mutant the Beyond Biology: Cell Stem in Interference RNA of Role The al., et (2017), Bodak, Biol. M. as: Mol. article this cite Please http://dx.doi.org/10.1016/j.jmb.2017.01.014

Table 1. Summary of previously generated and characterized RNAi pathway mutant mESCs

DGCR8 DICER ARGONAUTES References Wang et al. [7] Cirera-Salinas et al. Murchison et al. [8] Kanellopoulou et al. [6] Bodak et al. [85] Su et al. [80] [86] ESC strain of origin Male v6.5 E14TG2a 4E4 C57BL/6-derived E14TG2a Bim-deficient ES cell Bruce-4 ES line, NM5 Genetic background C57BL/6 x 129S4/Sv 129/Ola 129S7/SvEvBrd-Hprt C57BL/6 129/Ola 129S7/SvEvBrd-Hprt Technology Cre-loxP-mediated Paired Cre-loxP-mediated recombination Paired Cre-loxP-mediated recombination CRISPR/Cas9 CRISPR/Cas9 recombination Deletion Exon 3 Exons 22 and 23 Exons 18 to 20 Δ23 = Exon 23 Ago1, Ago3, and Ago4 Δ13 = Exons 17 to 23 locus, and exons 8 to 11 of Ago2 Culture conditions MEF-feeder cells Gelatin-coated plates STO-feeder layer or MEF-feeder cells Gelatin-coated plates MEF-feeder cells or (Serum + LIF) (Serum + LIF/2i + LIF) gelatin coated plates (Serum + LIF) (Serum + LIF/2i + LIF) gelatin coated plates (Serum + LIF) (Serum + LIF) Morphology Similar to WT Flattened in Serum + LIF Not assessed Similar to WT Flattened in Serum + LIF Not assessed SimilartoWTin2i+LIF Similar to WT in 2i + LIF Proliferation Slower compared to WT Cell cycle Impaired – accumulation in G1-phase Not assessed Impaired – Similar to WT accumulation in G1-phase Apoptosis Similar to WT Not assessed Not assessed Not assessed Increased compared to WT Biology Cell Stem in pathways RNAi of Roles Stemness Similar Oct4 transcript Reinforcement of the Similar Oct4 transcript levels Reinforcement of the Similar OCT4, levels compared to WT pluripotency network compared to WT pluripotency network NANOG, SOX2 protein (FACS OCT4/NANOG (FACS OCT4/NANOG levels compared to WT in Serum + LIF in Serum + LIF and and 2i + LIF) 2i + LIF) Differentiation Failed monolayer Failed monolayer Not assessed Failed chimeric mice Failed EB Not assessed differentiation differentiation generation differentiation Failed teratoma Failed NPC Failed teratoma generation differentiation generation Failed EB differentiation Failed EB differentiation Failed EB differentiation Exit from pluripotency Not assessed Impaired Not assessed Not assessed Impaired Not assessed EB: Embryoid body. WT: Wild type. NPC: Neuronal precursor cell. Roles of RNAi pathways in Stem Cell Biology 7 cell cycle, and differentiation (Table 1) [5–7,85,86]. independently of its miRNA function. DGCR8, alone, Nevertheless, the essential role of the RNAi genes in was also shown to bind cassette exons, suggesting a stem cell biology was not studied in light of the new role in the regulation of alternative splicing [93]. Along advances in the field until recently. We have lately these same lines, in the absence of DGCR8, more than demonstrated that Dicer- and Dgcr8-deficient mESCs 300 alternatively spliced events were misregulated are not only incapable of differentiating but also [93]. A study also described a physiological DROSHA- present a strong deficit in their potential to exit from and miRNA-independent function of DGCR8 in the the pluripotency state [85,86]. Moreover, recent control of fly neuronal morphogenesis; however, evidences in the RNAi field suggest that the functions whether this is mechanistically linked to small nucleolar of DROSHA, DGCR8, DICER, and AGO proteins go RNAs biogenesis is still unclear [94]. In addition, well beyond small non-coding RNA biogenesis and DGCR8 has been recently reported to directly interact might contribute to the observed phenotypes in early with the exosome subunit RRP6 in a DROSHA- embryonic development [87–89]. The question, which independent manner [95]. DGCR8 controls the stability arises from these studies, is whether the observed of mature small nucleolar RNAs and human telome- differentiation and exit from pluripotency defects are rase RNAs. This new non-canonical function identified caused by sRNAs or by other non-canonical functions DGCR8 as an adaptor to recruit the exosome complex of the RNAi proteins. to structured RNAs and induce their degradation [95]. In order to assess the canonical and non-canonical functions of the DGCR8 protein in stemness, exit from Non-Canonical Functions of RNAi pluripotency, and differentiation of mESCs, our group Proteins in Mammals complemented Dgcr8-deficient mESCs with wild-type and phosphomutant forms of the DGCR8 protein During the last 2 decades, all the RNAi members [86,96]. As previously described, all complemented were thought to only function in the miRNA and mESCs were able to restore miRNA production [96]. siRNA pathways. Indeed, differences have been Nevertheless, the phosphomutant-complemented observed between Dgcr8−/− and Dicer−/− mutant DGCR8 form was not capable to exit from the pluri- mESCs (Table 1), which were explained by the potency state and differentiate despite the presence potential role of DICER in the endo-siRNAs pathway of miRNAs. The integration of omics data and RNA [7]. However, recent evidence indicates the exis- immunoprecipitation experiments established DGCR8 tence of additional non-canonical functions (small as a direct interactor of the Tcf7l1 mRNA, a core non-coding RNA independent) for the RNAi effector component of the pluripotency network. Furthermore, proteins in various biological systems. The earliest we demonstrated that DGCR8 facilitates the splicing indications of miRNA-independent functions in mam- of Tcf7l1, an event necessary for the differentiation mals came from observations of differing phenotypes of mESCs. Our data revealed a new non-canonical between Drosha- and Dicer-deficient cells [88,90,91]. function of DGCR8 in the modulation of the alternative Before these studies, it was assumed that both splicing of Tcf7l1 mRNA in addition to its established DROSHA and DICER RNAse-III proteins were only function in miRNA biogenesis [86]. essential for miRNA biogenesis. Consequently, the Finally, a nuclear localization of AGO proteins in deletion of both genes should result in similar human and mouse cell lines was also reported phenotypes or weak differences due to the role of (reviewed in Refs. [81,97]). AGO2 is re-localized to DICER in siRNA pathways. Nevertheless, DICER the nucleus via Importins [98–100] and TNRC6A deficiency in the eye, but not DROSHA, leads to [101,102], indicating that these RNAi proteins could macular degeneration [88,91]. On the contrary, Drosha function in the nucleus as reported for plants, fungi, and deletion in neuronal stem cells, but not Dicer,resultsin invertebrates [103]. Moreover, several observations of loss of stem cell identity and precocious differentiation robust transcriptional gene silencing, involving synthet- [90].Furthermore,DROSHAwasfoundtobeessential ic sRNA complementary to the promoter region, have for T-cell development in a miRNA-independent been described in mammals [104]. Striking evidences manner [87]. Finally, a recent review nicely recapitu- indicated the involvement of RNAi in the control of lates all the non-canonical functions for DROSHA and splicing in mammals by affecting the epigenetic DICER in different biological systems [92]. modifications of the chromatin [105,106]. Notably, all Interestingly, Macias et al. identified also new of the aforementioned studies were performed using functions for the Microprocessor. A high-throughput artificial sRNAs. Nevertheless, they clearly suggest a sequencing of RNA isolated by cross-linking immuno- conserved function for the RNAi pathways in the control precipitation experiment designed to identify novel of transcription and splicing in mammals. However, substrates of the Microprocessor revealed that this until recently, there were no evidences for the existence complex binds and regulates a large variety of cellular of endogenous sRNAs involved in an RNAi-mediated RNAs [93]. The Microprocessor-mediated cleavage of control of these processes in mammals, although several classes of RNAs not only regulates transcript multiple reports suggest a role for AGO proteins in the levels but also modulates alternative splicing events nucleus. In addition to its nuclear localization, AGO2-

Please cite this article as: M. Bodak, et al., The Role of RNA Interference in Stem Cell Biology: Beyond the Mutant Phenotypes, J. Mol. Biol. (2017), http://dx.doi.org/10.1016/j.jmb.2017.01.014 8 Roles of RNAi pathways in Stem Cell Biology cross-linking immunoprecipitation experiments show deeply acknowledge B. Plaum for language editing and that 12% of the targets are mapped on the introns proofreading. This work was supported by a core grant of mRNA [107]. Moreover, divergent transcription from ETH-Z (supported by Roche). M.B. is supported from active promoters has been observed in mESCs, by a PhD fellowship from the ETH-Z foundation generating both small 20- to 90-nt species and long (ETH-21 13-1), D.C.-S. by the Peter und Traudl noncoding RNA (lncRNAs) [108]. As endo-siRNAs Foundation, and J.L. by a Swiss National Science have been thus far characterized in this cell type, they Foundation grant (31003A_153220). are consequently the most adapted model to study Author contributions: M.B., D.C.-S., J.L., and C.C. RNAi-mediated transcriptional gene silencing and contributed equally to the writing of the manuscript. All alternative splicing. authors read and approved the final manuscript. Conflict of interest: The authors declare that they have no conflict of interest. Conclusions and Future Perspectives Received 13 October 2016; The aforementioned recent developments in stem Received in revised form 13 January 2017; cell biology (2i culture conditions and exit from Accepted 16 January 2017 pluripotency assay) clearly demonstrate that two Available online xxxx particular events are crucial for the commitment of stem cells to differentiation: repression of the pluripo- Keywords: tent network (exit from pluripotency) and activation of mouse embryonic stem cells; the differentiation programs (toward the three germ exit from pluripotency; layers) [20]. Nowadays, the establishment of chemi- RNA interference pathways; cally defined culture conditions for mESCs allowed non-canonical functions of RNAi proteins thecaptureofthe“ground state” or naïve state of pluripotency [35]. †M.B., D.C.-S., and J.L. contributed equally to this work. Most of RNAi mutant mice are lethal at the early implantation stage, which is a very difficult time-point to experimentally address in vivo (Fig. 2). Moreover, these proteins are essential for proliferation and Abbreviations used: differentiation into the three embryonic germ layers of RNAi, RNA interference; siRNA, small interfering RNA; mESCs. Previously, it was assumed that this early sRNA, small RNA; miRNA, microRNA; mESCs, mouse embryonic lethality and the stem cells phenotypes embryonic stem cells; ICM, inner cell mass; LIF, leukemia were caused by an impairment of the miRNA inhibitory factor; BMP4, bone morphogenetic factor-4; biogenesis. However, recently, the question arose as GSK3, glycogen synthase kinase-3; OSN, OCT4, SOX2, to whether the function of RNAi proteins in the miRNA and NANOG; H3K27me3, trimethylation of the lysine 27 of or siRNA pathways or other biological processes is the histone H3; m7G, 7-methylguanosine; AGO, Argonaute; responsible for the developmental/differentiation de- MEFs, mouse embryonic fibroblasts; endo-siRNAs, fects observed. Furthermore, is this differentiation endogenous siRNAs; dpc, days post coïtum; EB, defect the result of an impaired activation of the embryoid body; MEK, MAP (Mitogen-Activated Protein) cell-lineage-specific programs or rather a problem in Kinase/ERK (Extracellular Signal-Regulated Kinase) Ki- the exit from the pluripotent state? In the light of the nase; ERK, Extracellular Signal-Regulated Kinase. novel non-canonical functions recently discovered, we think it is necessary to reassess all RNAi mutant mESCs in the same genetic background and identify References the underlying mechanism leading to the impaired exit from pluripotency and differentiation phenotypes, similar to our recent study in Dgcr8-deficient mESCs [1] A. Fire, S. Xu, M. Montgomery, S. Kostas, S. Driver, C. Mello, [86]. Taken together, these experiments will help Potent and specific genetic interference by double-stranded advance and broaden our view on the functions of RNA in Caenorhabditis elegans, Nature 391 (1998) – the RNAi proteins in stem cell biology and in early 806 811. mammalian development. [2] A.J. Hamilton, D.C. Baulcombe, A species of small antisense RNA in posttranscriptional gene silencing in plants, Science 286 (1999) 950–952. [3] M.S. Ebert, P. a Sharp, Roles for microRNAs in conferring robustness to biological processes, Cell 149 (2012) 515–524, http://dx.doi.org/10.1016/j.cell.2012.04.005. Acknowledgments [4] A. Shenoy, R.H. Blelloch, Regulation of microRNA function in somatic stem cell proliferation and differentiation, Nat. We thank J. Yu and D. Spies for the critical reading Rev. Mol. Cell Biol. 15 (2014) 565–576, http://dx.doi.org/10. of the manuscript and for fruitful discussions. We 1038/nrm3854.

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Please cite this article as: M. Bodak, et al., The Role of RNA Interference in Stem Cell Biology: Beyond the Mutant Phenotypes, J. Mol. Biol. (2017), http://dx.doi.org/10.1016/j.jmb.2017.01.014