Available online at www.sciencedirect.com

ScienceDirect Journal of Genetics and Genomics 42 (2015) 99e105

JGG REVIEW

Large Noncoding RNAs Are Promising Regulators in Embryonic Stem Cells Ya-Pu Li, Yangming Wang* Peking-Tsinghua Center for Life Sciences, Institute of Molecular Medicine, Peking University, Beijing 100871, China Received 8 December 2014; revised 4 February 2015; accepted 5 February 2015 Available online 12 February 2015

ABSTRACT Embryonic stem cells (ESCs) hold great promises for treating and studying numerous devastating diseases. The molecular basis of their potential is not completely understood. Large noncoding RNAs (lncRNAs) are an important class of gene regulators that play essential roles in a variety of physiologic and pathologic processes. Dozens of lncRNAs are now identified to control ESC self-renewal and differentiation. Research on lncRNAs may provide novel insights into manipulating the cell fate or reprogramming somatic cells into induced pluripotent stem cells (iPSCs). In this review, we summarize the recent research efforts in identifying functional lncRNAs and understanding how they act in ESCs, and discuss various future directions of this field. KEYWORDS: Embryonic stem cells; Induced pluripotent stem cells; Large noncoding RNAs; Self-renewal; Differentiation

INTRODUCTION Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass (ICM) of a mammalian blastocyst (Nichols and Smith, 2012). Because of their unlimited self-renewal capacity and pluripotency, defined as their ability to differentiate into most cell types of an organism, these cells hold great promises for regenerative medicine and serve as excellent in vitro model systems to study mammalian early development. The key to harness their potential is to develop methods to control their self-renewal and differentiation into favored cell lineages. This requires understanding molecular mechanisms governing their self-renewal and pluripotency. Previous studies have revealed essential roles of key signaling pathways (Hassani et al., 2014), transcription factors (Ng and Surani, 2011), epigenetic regulators (Tee and Reinberg, 2014) and microRNAs (miRNAs) (Guo et al., 2014b) in the establishment and maintenance of * Corresponding author. Tel: þ86 10 6276 6945; Fax: þ86 10 6276 7143. E-mail address: [email protected] (Y. Wang).

pluripotency. What is more, recent research efforts start to reveal that various post-transcriptional processes such as RNA export (Wang et al., 2013a), alternative splicing (Han et al., 2013) and alternative polyadenylation (Lackford et al., 2014) are also involved in regulating the pluripotency and differentiation of ESCs. All these factors are weaved into a robust regulatory network in ESCs. ESCs also express a large number of large noncoding RNAs (lncRNAs). Their function and how they fit into the established regulatory network are currently emerging and require more intensive investigation. LncRNAs ARE A NOVEL CLASS OF GENE REGULATORS LncRNAs refer to RNA transcripts that are longer than 200 nucleotides and not translated into polypeptides (Wapinski and Chang, 2011; Rinn and Chang, 2012). The 200-nucleotide cutoff is somewhat arbitrarily set to distinguish them from small regulatory RNAs such as miRNAs, piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs) and small interfering RNAs (siRNAs). Ribosomal RNAs and some small nuclear RNAs are noncoding and also longer than 200

http://dx.doi.org/10.1016/j.jgg.2015.02.002 1673-8527/Copyright Ó 2015, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.

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nucleotides, but generally they are not considered as lncRNAs. The polypeptide coding potential is usually determined by bioinformatic calculations rather than experimental approaches (Clamp et al., 2007; Kong et al., 2007; Lin et al., 2011). However, new technologies like ribosome profiling are now used to experimentally evaluate the coding potential of a lncRNA (Guttman et al., 2013; Ingolia et al., 2014). The ENCODE project provides a surprising result that human genome only contains w20000 protein coding genes (ENCODE Project Consortium, 2012). The number of protein coding genes in human is very similar to some simple organisms like Caenorhabditis elegans (Hillier et al., 2005). Interestingly, human and mouse genomes also encode a large number of lncRNAs (possibly >20000) (Okazaki et al., 2002; Carninci et al., 2005; Derrien et al., 2012; Djebali et al., 2012). In contrast to protein coding genes, lncRNAs are usually species-specific and less conserved (Ulitsky et al., 2011; Kutter et al., 2012; Pauli et al., 2012). The number of noncoding RNAs seems to correlate well with the complexity of an organism (Taft et al., 2007), therefore representing a potential cause for the phenotypic differences between organisms. More interestingly, many lncRNAs are restricted to specific cell lineages (Derrien et al., 2012), suggesting a functional role in cell fate determination. CLASSIFICATION OF LncRNAs LncRNAs can be classified into 5 classes according to their relative locations to protein coding genes in the genome: sense, antisense, bidirectional, intronic and intergenic (Fig. 1).

So far there is no association or enrichment reported for specific biological functions with any particular classes of lncRNAs. LncRNAs are usually transcribed by RNA polymerase II and processed in a similar way as mRNAs. Some lncRNAs do not have a polyA tail and are characterized as polyA minus lncRNAs. The lack of polyA tail poses a challenge for stabilizing these lncRNAs in the cellular environment full of RNases. Some lncRNAs are stabilized by highly structured elements at their ends. For example, Malat1 and Neat1 form a triplex structure at their 30 ends which are recognized and protected by various protein factors (Wilusz et al., 2012; Brown et al., 2014). Yin et al. (2012) identified a cluster of polyA minus lncRNAs called sno-lncRNAs in human ESCs that are derived from introns and protected from RNA degradation by bearing snoRNA sequences at both 50 and 30 ends (Yin et al., 2012). In addition, there exist thousands of circular lncRNAs in cells. Like other lncRNAs, the function of most circular lncRNAs is not known, with some appearing to be miRNA sponges (Hansen et al., 2013; Memczak et al., 2013; Guo et al., 2014a). Currently, there seems to be no unifying biogenesis pathway for lncRNAs as seen for small noncoding RNAs like miRNAs and siRNAs. Perturbing the biogenesis pathway of small noncoding RNAs has proven to be valuable means to identify functions of endogenous siRNAs, piRNAs and miRNAs (Kim et al., 2009). Therefore, it is currently in urgent need to define the biogenesis pathways for lncRNAs. Elucidating the biogenesis pathways may also help classify lncRNAs into functional groups. In ESCs, it is particularly important to investigate how various signaling pathways, transcription factors, epigenetic modifiers and miRNAs regulate the expression of lncRNAs (Fig. 2). This will further our understanding on the mechanism of known ESC regulators. NUMEROUS LncRNAs ARE FUNCTIONAL IN ESCs

Fig. 1. Classification of lncRNAs by genomic localization. Sense and antisense lncRNAs overlap with a protein-coding gene. Bidirectional/divergent lncRNAs and its neighboring protein genes are located at opposite strands and their transcription start sites are less than 1 kb away from each other. Intronic lncRNAs are located in an intron of a protein coding gene. Intergenic lncRNAs (lincRNAs) are at least 5 kb away from any protein coding genes.

Evidence is mounting that lncRNAs play important roles in ESCs. First, there are numerous lncRNAs appearing to be differentially expressed in ESCs and somatic cells. Using microarray technology, the Mattick group identified 174 lncRNAs enriched in ESCs (Dinger et al., 2008). Second, many lncRNAs are regulated by key pluripotency transcription factors such as OCT4, SOX2 and NANOG. In fact, around 10% of OCT4 or NANOG targets are potentially lncRNAs. In another study, Guttman et al. (2011) found that OCT4, NANOG and SOX2 co-occupy w12% of ESC expressed lncRNAs. More interestingly, Zheng et al. (2014) discovered that the expression of hundreds of lncRNAs relies on an oncogenic transcription factor cMyc in ESCs. Finally, the direct evidence for roles of lncRNAs in ESCs is from functional screening using small hairpin RNAs (shRNAs) against candidate lncRNAs. Lander group and Rana group independently identified dozens of lncRNAs that are potential regulators of ESC self-renewal or differentiation (Guttman et al., 2011; Lin et al., 2014). Interestingly, there were no overlapping lncRNAs identified by both groups, perhaps due to different reporter systems and strategies used in two studies.

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Fig. 2. LncRNAs may interact at multiple levels with known regulatory networks of ESCs.

Alternatively, this could suggest that these screenings are far from exhaustion and more functional lncRNAs are expected to be found in ESCs. Current efforts focus on identifying lncRNAs important for the ESC self-renewal. The self-renewal of ESCs includes two aspects: one is to maintain the cell identify, and the other is to proliferate. The maintenance of ESC identity is usually measured by expression of key pluripotency transcription factors and alkaline phosphatase, repression of transcription factors of differentiated lineages, and colony formation. The proliferation of ESCs is dependent on the rate of cell cycle progression, metabolism and apoptosis. ESCs generally have an extremely short G1 phase and lack a functional restriction point at the G1/S transition (Schratt et al., 2001; Tiscornia and

Izpisu´a Belmonte, 2010; Wang and Blelloch, 2011). The unique cell cycle structure is hypothesized to contribute to the self-renewal state of ESCs. In addition, ESCs show enhanced glycolysis for energy supply and growth (Kondoh et al., 2007; Folmes et al., 2011; Zhou et al., 2012; Cao et al., 2015). The enhanced glycolysis is also proposed to have an important role in maintaining the self-renewal state of ESCs and regaining pluripotency during reprogramming. Future studies should focus on elucidating the specific function of lncRNAs in all aspects of self-renewal, including expression of key pluripotency factors, cell cycle progression, metabolism, and repression of differentiation or cell death (Fig. 3). By knocking out 18 lncRNAs genetically, Sauvageau et al. (2013) found that several lncRNAs may be essential for the survival of mice and

Fig. 3. LncRNAs may play important roles in a variety of processes in ESCs or during reprogramming.

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proper tissue differentiation during development. In addition, Klattenhoff et al. (2013) identified a heart associated lincRNA Bvht that is important for the progression of nascent mesoderm into a cardiac fate. These studies foretell that lncRNAs play essential roles for lineage commitment of ESCs. As this is the most fundamental question and pressing issue for regenerative medicine, we expect more efforts should be put forth to identify differentiation related lncRNAs and their working mechanisms. LncRNAs INTERACT WITH OTHER IMPORTANT MOLECULAR REGULATORS TO EXECUTE THEIR FUNCTION IN ESCs Although dozens of lncRNAs have been found to be important for ESC self-renewal or differentiation, their exact mechanisms of action are still largely unknown. In general, lncRNAs regulate gene expression in trans or in cis. Cis-regulatory lncRNAs are lncRNAs that regulate the expression of neighboring genes from the same allele. These lncRNAs often function by tethering to the locus where they are transcribed. The notable example is Xist and HOTTIP (Wang et al., 2011; Engreitz et al., 2013). Trans-regulatory lncRNAs regulate the expression of genes which are not physically neighboring to them. It was largely accepted that lncRNAs mostly affect gene expression in cis. However, gene expression analysis after knocking down hundreds of lincRNAs in ESCs reveals that most lincRNAs do not regulate the expression of their neighboring genes (Guttman et al., 2011), therefore most likely function in trans. With the support from studies on other trans-regulatory lincRNAs such as HOTAIR (Rinn et al., 2007; Tsai et al., 2010a), it is now clear that lncRNAs can function both in cis and in trans. Generally, lncRNAs act as decoys, guides and scaffolds to interfere with or enhance the function of other regulators including protein and miRNAs (Wang and Chang, 2011). Both cis- and trans-acting lncRNAs can function by directly interacting with DNA, RNA or proteins (Table 1). Depending on different partners, lncRNAs regulate chromatin modifications, transcription, mRNA stability, translation and miRNA activity. In ESCs, lncRNAs have been found associated with epigenetic modifiers, RNA binding proteins and miRNAs. Table 1 Examples of lncRNAs interacting with various factors Type of interacting factors

Example of lncRNAs

Example of lncRNAs in ESCs

Chromatin modifiers

Xist, HOTAIR, HOTTIP

w30% ESC-enriched lincRNAs

TFs and RBPs

Xist, Malat1

TUNA

Signaling pathway proteins

Lnc-DC

None

DNA

pRNA

TUNA

RNA

Antisense Uchl1, TINCR, linc-MD1

Linc-RoR

TF: transcription factor; RBP: RNA binding protein.

Interacting with epigenetic modifiers Interaction with different epigenetic modifiers (readers, writers and erasers) using different domains of lncRNAs is proposed to be a general mechanism for lncRNAs that modify the epigenetic landscape. It is widely perceived that lncRNAs bestow non-specific epigenetic regulators with sequencespecificity on genome, since lncRNAs can interact with DNA in a duplex or triplex form by canonical or Hoogsteen base-pairing. HOTAIR regulates the transcription in trans by enhancing the PRC2 activity at Hox D locus. Close investigation has found that the 50 and 30 ends of HOTAIR bind the PRC2 complex (H3K27 methylase) and the LSD1 (H3K4me3 demethylase) complex, respectively (Tsai et al., 2010b). During reprogramming, lincRNA-p21 acts as a barrier to the induction of pluripotency by sustaining H3K9me3 and/or CpG methylation at multiple pluripotency gene promoters. The direct role of lincRNA-p21 in regulating epigenetic modifications is supported by the fact that it is physically associated with the H3K9 methyltransferase SETDB1 and the maintenance DNA methyltransferase DNMT1 (Bao et al., 2015). In ESCs, w30% of lincRNAs were found to be associated with at least one epigenetic regulatory complexes (Guttman et al., 2011), suggesting wide-spread existence of the interaction between a subclass of lncRNAs and epigenetic modulators. However, how these interactions are translated into regulatory signals and even the precise function of these interactions are still waiting for investigation. Interacting with RNA binding proteins LncRNAs can also interact with proteins other than epigenetic regulators. For example, hnRNPU and YY1 tether Xist to the chromosome to facilitate its gene-silencing function (Hasegawa et al., 2010; Jeon and Lee 2011). In ESCs, TUNA (Tcl1 upstream neuron-associated lincRNA) forms a complex with three RNA-binding proteins (PTBP1, hnRNP-K, and NCL) and mediates the recruitment of these proteins at the promoters of Nanog, Sox2, and Fgf4. Knockdown of TUNA or the individual RBPs by shRNAs leads to decrease in the expression level of these genes and inhibits ESC self-renewal (Lin et al., 2014). More interestingly, TUNA knockdown also inhibits the differentiation of mouse ESCs into neural lineages. Chromatin isolation by RNA purification (ChIRP) experiments (Chu et al. 2012) confirmed that TUNA physically interacts with the promoters of Nanog, Sox2, and Fgf4 to activate their transcription by recruiting PTBP1, hnRNP-K, and NCL. Previously, it was shown that a promoter associated RNA (pRNA) interacts with the target site of TTF-I in the rDNA promoter directly through DNA:RNA triplex (Schmitz et al., 2010). It would be interesting to determine whether TUNA binds to the promoter directly or through protein intermediates. To answer this question, it requires novel methods that would discriminate direct or indirect binding between RNA and DNA components. This may be achieved through using different crosslinking reagents as a recent study nicely distinguishing direct and indirect binding between RNA and RNA

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components using a psoralen derivative and formaldehyde as crosslinker, respectively (Engreitz et al., 2014). Recently, a provoking study identified lnc-DC which regulates human dendritic cell differentiation by directly interacting with STAT3 protein to modulate the STAT3 phosphorylation status (Wang et al., 2014). It opens some exciting possibilities that lncRNAs may modulate the activity of various signaling pathways to control cellular behavior. Since signaling pathways play essential roles in ESCs, it would be interesting to uncover lncRNAs that directly regulate the activity of various signaling pathways such as MEK/ERK, LIF-STAT3, BMP and WNT pathways in ESCs. Interacting with miRNAs LncRNA can also function through interacting with other RNA molecules by base-pairing. In fact, small noncoding RNAs such as miRNAs mainly act by this way (Bartel, 2009). Antisense Uchl1 and TINCR have been shown to bind other mRNAs through complementarity to regulate translation or mRNA stability (Carrieri et al., 2012; Kretz et al., 2013). Other than binding to large RNAs, multiple lncRNAs have been shown to act as miRNA sponges to restrict miRNA activity (Tay et al., 2014). Linc-RoR is found to support human ESC self-renewal and promote reprogramming (Loewer et al., 2010; Wang et al., 2013b). Interestingly, it functions as a miRNA sponge to sustain the expression of the core transcription factors Oct4, Nanog, and Sox2 through binding to miR-145, a miRNA that negatively regulates expression of these transcription factors (Xu et al., 2009). Since there are many miRNAs that regulate ESC self-renewal and differentiation (Guo et al., 2014b), future studies may uncover more lncRNAs acting as miRNA sponges to affect cell fate decision. When uncovering lncRNAs functioning as miRNA sponges, their expression levels in cells must be taken into consideration, as recent studies show that the expression level of a functional miRNA sponge has to reach a certain threshold value which is determined by concentrations of corresponding miRNAs and their targets (Bosson et al., 2014; Denzler et al., 2014). Conversely and perhaps more interestingly, miRNAs may also modulate lncRNA activity by tethering them to different RNA binding proteins or altering their secondary and even tertiary structures (Ulitsky et al., 2011). CONCLUDING REMARKS LncRNAs represent a new class of gene regulators that may play essential roles in a variety of biological processes including the self-renewal and differentiation of ESCs. Future studies should clarify the specific function related to cell cycle, the induction and maintenance of pluripotency or lineage commitment and working mechanisms of regulatory lncRNAs in ESCs, particularly how they are integrated into the known regulatory networks involving signaling proteins, miRNAs, transcription factors and epigenetic regulators (Fig. 2). This requires the development of novel concepts as well as novel toolboxes that are tailored for lncRNAs. Understanding the

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function and working mechanisms of lncRNAs could yield novel RNA-based strategies in manipulating ESCs or iPSCs to advance regenerative medicine. ACKNOWLEDGMENTS The research in Wang lab is supported by grants from Chinese Ministry of Science and Technology (Nos. 2011CBA01100 and 2012CB966700) and the National Natural Science Foundation of China (No. 31221002). REFERENCES Bao, X., Wu, H., Zhu, X., Guo, X., Hutchins, A.P., Luo, Z., Song, H., Chen, Y., Lai, K., Yin, M., Xu, L., Zhou, L., Chen, J., Wang, D., Qin, B., Frampton, J., Tse, H.F., Pei, D., Wang, H., Zhang, B., Esteban, M.A., 2015. The p53-induced lincRNA-p21 derails somatic cell reprogramming by sustaining H3K9me3 and CpG methylation at pluripotency gene promoters. Cell Res. 25, 80e92. Bartel, D.P., 2009. MicroRNAs: target recognition and regulatory functions. Cell 136, 215e233. Bosson, A.D., Zamudio, J.R., Sharp, P.A., 2014. Endogenous miRNA and target concentrations determine susceptibility to potential ceRNA competition. Mol. Cell 56, 347e359. Brown, J.A., Bulkley, D., Wang, J., Valenstein, M.L., Yario, T.A., Steitz, T.A., Steitz, J.A., 2014. Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix. Nat. Struct. Mol. Biol. 21, 633e640. Carninci, P., Kasukawa, T., Katayama, S., Gough, J., Frith, M.C., Maeda, N., Oyama, R., Ravasi, T., Lenhard, B., Wells, C., Kodzius, R., Shimokawa, K., Bajic, V.B., Brenner, S.E., Batalov, S., Forrest, A.R., Zavolan, M., Davis, M.J., Wilming, L.G., Aidinis, V., Allen, J.E., AmbesiImpiombato, A., Apweiler, R., Aturaliya, R.N., Bailey, T.L., Bansal, M., Baxter, L., Beisel, K.W., Bersano, T., Bono, H., Chalk, A.M., Chiu, K.P., Choudhary, V., Christoffels, A., Clutterbuck, D.R., Crowe, M.L., Dalla, E., Dalrymple, B.P., de Bono, B., Della Gatta, G., di Bernardo, D., Down, T., Engstrom, P., Fagiolini, M., Faulkner, G., Fletcher, C.F., Fukushima, T., Furuno, M., Futaki, S., Gariboldi, M., Georgii-Hemming, P., Gingeras, T.R., Gojobori, T., Green, R.E., Gustincich, S., Harbers, M., Hayashi, Y., Hensch, T.K., Hirokawa, N., Hill, D., Huminiecki, L., Iacono, M., Ikeo, K., Iwama, A., Ishikawa, T., Jakt, M., Kanapin, A., Katoh, M., Kawasawa, Y., Kelso, J., Kitamura, H., Kitano, H., Kollias, G., Krishnan, S.P., Kruger, A., Kummerfeld, S.K., Kurochkin, I.V., Lareau, L.F., Lazarevic, D., Lipovich, L., Liu, J., Liuni, S., McWilliam, S., Madan Babu, M., Madera, M., Marchionni, L., Matsuda, H., Matsuzawa, S., Miki, H., Mignone, F., Miyake, S., Morris, K., MottaguiTabar, S., Mulder, N., Nakano, N., Nakauchi, H., Ng, P., Nilsson, R., Nishiguchi, S., Nishikawa, S., Nori, F., Ohara, O., Okazaki, Y., Orlando, V., Pang, K.C., Pavan, W.J., Pavesi, G., Pesole, G., Petrovsky, N., Piazza, S., Reed, J., Reid, J.F., Ring, B.Z., Ringwald, M., Rost, B., Ruan, Y., Salzberg, S.L., Sandelin, A., Schneider, C., Scho¨nbach, C., Sekiguchi, K., Semple, C.A., Seno, S., Sessa, L., Sheng, Y., Shibata, Y., Shimada, H., Shimada, K., Silva, D., Sinclair, B., Sperling, S., Stupka, E., Sugiura, K., Sultana, R., Takenaka, Y., Taki, K., Tammoja, K., Tan, S.L., Tang, S., Taylor, M.S., Tegner, J., Teichmann, S.A., Ueda, H.R., van Nimwegen, E., Verardo, R., Wei, C.L., Yagi, K., Yamanishi, H., Zabarovsky, E., Zhu, S., Zimmer, A., Hide, W., Bult, C., Grimmond, S.M., Teasdale, R.D., Liu, E.T., Brusic, V., Quackenbush, J., Wahlestedt, C., Mattick, J.S., Hume, D.A., Kai, C., Sasaki, D., Tomaru, Y., Fukuda, S., Kanamori-Katayama, M., Suzuki, M., Aoki, J., Arakawa, T., Iida, J., Imamura, K., Itoh, M., Kato, T., Kawaji, H., Kawagashira, N., Kawashima, T., Kojima, M., Kondo, S., Konno, H., Nakano, K., Ninomiya, N., Nishio, T., Okada, M., Plessy, C., Shibata, K., Shiraki, T., Suzuki, S., Tagami, M., Waki, K., Watahiki, A., Okamura-Oho, Y., Suzuki, H., Kawai, J., Hayashizaki, Y., FANTOM Consortium; RIKEN

104

Y.-P. Li, Y. Wang / Journal of Genetics and Genomics 42 (2015) 99e105

Genome Exploration Research Group and Genome Science Group (Genome Network Project Core Group), 2005. The transcriptional landscape of the mammalian genome. Science 309, 1559e1563. Cao, Y., Guo, W.T., Tian, S., He, X., Wang, X.W., Liu, X., Gu, K.L., Ma, X., Huang, D., Hu, L., Cai, Y., Zhang, H., Wang, Y., Gao, P., 2015. MiR-290/ 371-Mbd2-Myc circuit regulates glycolytic metabolism to promote pluripotency. EMBO J. pii: e201490441. Carrieri, C., Cimatti, L., Biagioli, M., Beugnet, A., Zucchelli, S., Fedele, S., Pesce, E., Ferrer, I., Collavin, L., Santoro, C., Forrest, A.R., Carninci, P., Biffo, S., Stupka, E., Gustincich, S., 2012. Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat. Nature 491, 454e457. Chu, C., Quinn, J., Chang, H.Y., 2012. Chromatin isolation by RNA purification (ChIRP). J. Vis. Exp. 61, 3912. Clamp, M., Fry, B., Kamal, M., Xie, X., Cuff, J., Lin, M.F., Kellis, M., Lindblad-Toh, K., Lander, E.S., 2007. Distinguishing protein-coding and noncoding genes in the human genome. Proc. Natl. Acad. Sci. USA 104, 19428e19433. Denzler, R., Agarwal, V., Stefano, J., Bartel, D.P., Stoffel, M., 2014. Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance. Mol. Cell 54, 766e776. Derrien, T., Johnson, R., Bussotti, G., Tanzer, A., Djebali, S., 2012. The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res. 22, 1775e1789. Dinger, M.E., Amaral, P.P., Mercer, T.R., Pang, K.C., Bruce, S.J., Tilgner, H., Guernec, G., Martin, D., Merkel, A., Knowles, D.G., Lagarde, J., Veeravalli, L., Ruan, X., Ruan, Y., Lassmann, T., Carninci, P., Brown, J.B., Lipovich, L., Gonzalez, J.M., Thomas, M., Davis, C.A., Shiekhattar, R., Gingeras, T.R., Hubbard, T.J., Notredame, C., Harrow, J., Guigo´, R., 2008. Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation. Genome Res. 18, 1433e1445. Djebali, S., Davis, C.A., Merkel, A., Dobin, A., Lassmann, T., Mortazavi, A., Tanzer, A., Lagarde, J., Lin, W., Schlesinger, F., Xue, C., Marinov, G.K., Khatun, J., Williams, B.A., Zaleski, C., Rozowsky, J., Ro¨der, M., Kokocinski, F., Abdelhamid, R.F., Alioto, T., Antoshechkin, I., Baer, M.T., Bar, N.S., Batut, P., Bell, K., Bell, I., Chakrabortty, S., Chen, X., Chrast, J., Curado, J., Derrien, T., Drenkow, J., Dumais, E., Dumais, J., Duttagupta, R., Falconnet, E., Fastuca, M., Fejes-Toth, K., Ferreira, P., Foissac, S., Fullwood, M.J., Gao, H., Gonzalez, D., Gordon, A., Gunawardena, H., Howald, C., Jha, S., Johnson, R., Kapranov, P., King, B., Kingswood, C., Luo, O.J., Park, E., Persaud, K., Preall, J.B., Ribeca, P., Risk, B., Robyr, D., Sammeth, M., Schaffer, L., See, L.H., Shahab, A., Skancke, J., Suzuki, A.M., Takahashi, H., Tilgner, H., Trout, D., Walters, N., Wang, H., Wrobel, J., Yu, Y., Ruan, X., Hayashizaki, Y., Harrow, J., Gerstein, M., Hubbard, T., Reymond, A., Antonarakis, S.E., Hannon, G., Giddings, M.C., Ruan, Y., Wold, B., Carninci, P., Guigo´, R., Gingeras, T.R., 2012. Landscape of transcription in human cells. Nature 489, 101e108. ENCODE Project Consortium, 2012. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57e74. Engreitz, J.M., Pandya-Jones, A., McDonel, P., Shishkin, A., Sirokman, K., Surka, C., Kadri, S., Xing, J., Goren, A., Lander, E.S., Plath, K., Guttman, M., 2013. The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome. Science 341, 1237973. Engreitz, J.M., Sirokman, K., McDonel, P., Shishkin, A.A., Surka, C., Russell, P., Grossman, S.R., Chow, A.Y., Guttman, M., Lander, E.S., 2014. RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent pre-mRNAs and chromatin sites. Cell 159, 188e199. Folmes, C.D., Nelson, T.J., Martinez-Fernandez, A., Arrell, D.K., Lindor, J.Z., Dzeja, P.P., Ikeda, Y., Perez-Terzic, C., Terzic, A., 2011. Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming. Cell Metab. 14, 264e271. Guo, J.U., Agarwal, V., Guo, H., Bartel, D.P., 2014a. Expanded identification and characterization of mammalian circular RNAs. Genome Biol. 15, 409. Guo, W.T., Wang, X.W., Wang, Y., 2014b. Micro-management of pluripotent stem cells. Protein Cell 5, 36e47. Guttman, M., Donaghey, J., Carey, B.W., Garber, M., Grenier, J.K., Munson, G., Young, G., Lucas, A.B., Ach, R., Bruhn, L., Yang, X.,

Amit, I., Meissner, A., Regev, A., Rinn, J.L., Root, D.,E., Lander, E.S., 2011. lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature 477, 295e300. Guttman, M., Russell, P., Ingolia, N.T., Weissman, J.S., Lander, E.S., 2013. Ribosome profiling provides evidence that large noncoding RNAs do not encode proteins. Cell 154, 240e251. Han, H., Irimia, M., Ross, P.J., Sung, H.K., Alipanahi, B., David, L., Golipour, A., Gabut, M., Michael, I.P., Nachman, E.N., Wang, E., Trcka, D., Thompson, T., O’Hanlon, D., Slobodeniuc, V., BarbosaMorais, N.L., Burge, C.B., Moffat, J., Frey, B.J., Nagy, A., Ellis, J., Wrana, J.L., Blencowe, B.J., 2013. MBNL proteins repress ES-cellspecific alternative splicing and reprogramming. Nature 498, 241e245. Hansen, T.B., Jensen, T.I., Clausen, B.H., Bramsen, J.B., Finsen, B., Damgaard, C.K., Kjems, J., 2013. Natural RNA circles function as efficient microRNA sponges. Nature 495, 384e388. Hasegawa, Y., Brockdorff, N., Kawano, S., Tsutui, K., Tsutui, K., Nakagawa, S., 2010. The matrix protein hnRNP U is required for chromosomal localization of Xist RNA. Dev. Cell 19, 469e476. Hassani, S.N., Totonchi, M., Gourabi, H., Scho¨ler, H.R., Baharvand, H., 2014. Signaling roadmap modulating naive and primed pluripotency. Stem Cells Dev. 23, 193e208. Hillier, L.W., Coulson, A., Murray, J.I., Bao, Z., Sulston, J.E., Waterston, R.H., 2005. Genomics in C. elegans: so many genes, such a little worm. Genome Res. 15, 1651e1660. Ingolia, N.T., Brar, G.A., Stern-Ginossar, N., Harris, M.S., Talhouarne, G.J., Jackson, S.E., Wills, M.R., Weissman, J.S., 2014. Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes. Cell Rep. 8, 1365e1379. Jeon, Y., Lee, J.T., 2011. YY1 tethers Xist RNA to the inactive X nucleation center. Cell 146, 119e133. Kim, V.N., Han, J., Siomi, M.C., 2009. Biogenesis of small RNAs in animals. Nat. Rev. Mol. Cell Biol. 10, 126e139. Klattenhoff, C.A., Scheuermann, J.C., Surface, L.E., Bradley, R.K., Fields, P.A., Steinhauser, M.L., Ding, H., Butty, V.L., Torrey, L., Haas, S., Abo, R., Tabebordbar, M., Lee, R.T., Burge, C.B., Boyer, L.A., 2013. Braveheart, a long noncoding RNA required for cardiovascular lineage commitment. Cell 152, 570e583. Kondoh, H., Lleonart, M.E., Nakashima, Y., Yokode, M., Tanaka, M., Bernard, D., Gil, J., Beach, D., 2007. A high glycolytic flux supports the proliferative potential of murine embryonic stem cells. Antioxid. Redox. Signal. 9, 293e299. Kong, L., Zhang, Y., Ye, Z.Q., Liu, X.Q., Zhao, S.Q., Wei, L., Gao, G., 2007. CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Res. 35, W345eW349. Kretz, M., Siprashvili, Z., Chu, C., Webster, D.E., Zehnder, A., Qu, K., Lee, C.S., Flockhart, R.J., Groff, A.F., Chow, J., Johnston, D., Kim, G.E., Spitale, R.C., Flynn, R.A., Zheng, G.X., Aiyer, S., Raj, A., Rinn, J.L., Chang, H.Y., Khavari, P.A., 2013. Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 493, 231e235. Kutter, C., Watt, S., Stefflova, K., Wilson, M.D., Goncalves, A., Ponting, C.P., Odom, D.T., Marques, A.C., 2012. Rapid turnover of long noncoding RNAs and the evolution of gene expression. PLoS Genet. 8, e1002841. Lackford, B., Yao, C., Charles, G.M., Weng, L., Zheng, X., Choi, E.A., Xie, X., Wan, J., Xing, Y., Freudenberg, J.M., Yang, P., Jothi, R., Hu, G., Shi, Y., 2014. Fip1 regulates mRNA alternative polyadenylation to promote stem cell self-renewal. EMBO J. 33, 878e889. Lin, M.F., Jungreis, I., Kellis, M., 2011. PhyloCSF: a comparative genomics method to distinguish protein coding and non-coding regions. Bioinformatics 27, 275e282. Lin, N., Chang, K.,Y., Li, Z., Gates, K., Rana, Z.A., Dang, J., Zhang, D., Han, T., Yang, C.S., Cunningham, T.J., Head, S.R., Dueste, G., Dong, P.D., Rana, T.M., 2014. An evolutionarily conserved long noncoding RNA TUNA controls pluripotency and neural lineage commitment. Mol. Cell 53, 1005e1019. Loewer, S., Cabili, M.N., Guttman, M., Loh, Y.H., Thomas, K., Park, I.H., Garber, M., Curran, M., Onder, T., Agarwal, S., Manos, P.D., Datta, S., Lander, E.S., Schlaeger, T.M., Daley, G.Q., Rinn, J.L., 2010. Large

Y.-P. Li, Y. Wang / Journal of Genetics and Genomics 42 (2015) 99e105 intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells. Nat. Genet. 42, 1113e1117. Memczak, S., Jens, M., Elefsinioti, A., Torti, F., Krueger, J., Rybak, A., Maier, L., Mackowiak, S.D., Gregersen, L.H., Munschauer, M., Loewer, A., Ziebold, U., Landthaler, M., Kocks, C., le Noble, F., Rajewsky, N., 2013. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495, 333e338. Ng, H.H., Surani, M.A., 2011. The transcriptional and signalling networks of pluripotency. Nat. Cell Biol. 13, 490e496. Nichols, J., Smith, A., 2012. Pluripotency in the embryo and in culture. Cold Spring Harb Perspect Biol. 4, a008218. Okazaki, Y., Furuno, M., Kasukawa, T., Adachi, J., Bono, H., Kondo, S., Nikaido, I., Osato, N., Saito, R., Suzuki, H., Yamanaka, I., Kiyosawa, H., Yagi, K., Tomaru, Y., Hasegawa, Y., Nogami, A., Scho¨nbach, C., Gojobori, T., Baldarelli, R., Hill, D.P., Bult, C., Hume, D.A., Quackenbush, J., Schriml, L.M., Kanapin, A., Matsuda, H., Batalov, S., Beisel, K.W., Blake, J.A., Bradt, D., Brusic, V., Chothia, C., Corbani, L.E., Cousins, S., Dalla, E., Dragani, T.A., Fletcher, C.F., Forrest, A., Frazer, K.S., Gaasterland, T., Gariboldi, M., Gissi, C., Godzik, A., Gough, J., Grimmond, S., Gustincich, S., Hirokawa, N., Jackson, I.J., Jarvis, E.D., Kanai, A., Kawaji, H., Kawasawa, Y., Kedzierski, R.M., King, B.L., Konagaya, A., Kurochkin, I.V., Lee, Y., Lenhard, B., Lyons, P.A., Maglott, D.R., Maltais, L., Marchionni, L., McKenzie, L., Miki, H., Nagashima, T., Numata, K., Okido, T., Pavan, W.J., Pertea, G., Pesole, G., Petrovsky, N., Pillai, R., Pontius, J.U., Qi, D., Ramachandran, S., Ravasi, T., Reed, J.C., Reed, D.J., Reid, J., Ring, B.Z., Ringwald, M., Sandelin, A., Schneider, C., Semple, C.A., Setou, M., Shimada, K., Sultana, R., Takenaka, Y., Taylor, M.S., Teasdale, R.D., Tomita, M., Verardo, R., Wagner, L., Wahlestedt, C., Wang, Y., Watanabe, Y., Wells, C., Wilming, L.G., Wynshaw-Boris, A., Yanagisawa, M., Yang, I., Yang, L., Yuan, Z., Zavolan, M., Zhu, Y., Zimmer, A., Carninci, P., Hayatsu, N., Hirozane-Kishikawa, T., Konno, H., Nakamura, M., Sakazume, N., Sato, K., Shiraki, T., Waki, K., Kawai, J., Aizawa, K., Arakawa, T., Fukuda, S., Hara, A., Hashizume, W., Imotani, K., Ishii, Y., Itoh, M., Kagawa, I., Miyazaki, A., Sakai, K., Sasaki, D., Shibata, K., Shinagawa, A., Yasunishi, A., Yoshino, M., Waterston, R., Lander, E.S., Rogers, J., Birney, E., Hayashizaki, Y., FANTOM Consortium; RIKEN Genome Exploration Research Group Phase I & II Team, 2002. Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs. Nature 420, 563e573. Pauli, A., Valen, E., Lin, M.F., Garber, M., Vastenhouw, N.L., Levin, J.Z., Fan, L., Sandelin, A., Rinn, J.L., Regev, A., Schier, A.F., 2012. Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis. Genome Res. 22, 577e591. Rinn, J.L., Kertesz, M., Wang, J.K., Squazzo, S.L., Xu, X., Brugmann, S.A., Goodnough, L.H., Helms, J.A., Farnham, P.J., Segal, E., Chang, H.Y., 2007. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129, 1311e1323. Rinn, J.L., Chang, H.Y., 2012. Genome regulation by long noncoding RNAs. Annu. Rev. Biochem. 81, 145e166. Sauvageau, M., Goff, L.A., Lodato, S., Bonev, B., Groff, A.F., Gerhardinger, C., Sanchez-Gomez, D.B., Hacisuleyman, E., Li, E., Spence, M., Liapis, S.C., Mallard, W., Morse, M., Swerdel, M.R., D’Ecclessis, M.F., Moore, J.C., Lai, V., Gong, G., Yancopoulos, G.D., Frendewey, D., Kellis, M., Hart, R.P., Valenzuela, D.M., Arlotta, P., Rinn, J.L., 2013. Multiple knockout mouse models reveal lincRNAs are required for life and brain development. eLife 2, e01749. Schmitz, K.M., Mayer, C., Postepska, A., Grummt, I., 2010. Interaction of noncoding RNA with the rDNA promoter mediates recruitment of DNMT3b and silencing of rRNA genes. Genes Dev. 24, 2264e2269.

105

Schratt, G., Weinhold, B., Lundberg, A.S., Schuck, S., Berger, J., Schwarz, H., Weinberg, R.A., Ru¨ther, U., Nordheim, A., 2001. Serum response factor is required for immediate-early gene activation yet is dispensable for proliferation of embryonic stem cells. Mol. Cell Biol. 21, 2933e2943. Taft, R.J., Pheasant, M., Mattick, J.S., 2007. The relationship between nonprotein-coding DNA and eukaryotic complexity. Bioessays 29, 288e299. Tay, Y., Rinn, J., Pandolfi, P.P., 2014. The multilayered complexity of ceRNA crosstalk and competition. Nature 505, 344e352. Tee, W.W., Reinberg, D., 2014. Chromatin features and the epigenetic regulation of pluripotency states in ESCs. Development 141, 2376e2390. Tiscornia, G., Izpisu´a Belmonte, J.C., 2010. MicroRNAs in embryonic stem cell function and fate. Genes Dev. 24, 2732e2741. Tsai, M.C., Manor, O., Wan, Y., Mosammaparast, N., Wang, J.K., Lan, F., Shi, Y., Segal, E., Chang, H.Y., 2010. Long noncoding RNA as modular scaffold of histone modification complexes. Science 329, 689e693. Ulitsky, I., Shkumatava, A., Jan, C.H., Sive, H., Bartel, D.P., 2011. Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution. Cell 147, 1537e1550. Wang, K.C., Yang, Y.W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B.R., Protacio, A., Flynn, R.A., Gupta, R.A., Wysocka, J., Lei, M., Dekker, J., Helms, J.A., Chang, H.Y., 2011. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472, 120e124. Wang, K.C., Chang, H.Y., 2011. Molecular mechanisms of long noncoding RNAs. Mol. Cell 43, 904e914. Wang, L., Miao, Y.L., Zheng, X., Lackford, B., Zhou, B., Han, L., Yao, C., Ward, J.M., Burkholder, A., Lipchina, I., Fargo, D.C., Hochedlinger, K., Shi, Y., Williams, C.J., Hu, G., 2013a. The THO complex regulates pluripotency gene mRNA export and controls embryonic stem cell self-renewal and somatic cell reprogramming. Cell Stem Cell 13, 676e690. Wang, P., Xue, Y., Han, Y., Lin, L., Wu, C., Jiang, Z., Xu, J., Liu, Q., Cao, X., 2014. The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation. Science 344, 310e313. Wang, Y., Xu, Z., Jiang, J., Xu, C., Kang, J., Xiao, L., Wu, M., Xiong, J., Guo, X., Liu, H., 2013b. Endogenous miRNA sponge lincRNA-RoR regulates Oct4, Nanog, and Sox2 in human embryonic stem cell self-renewal. Dev. Cell 25, 69e80. Wang, Y., Blelloch, R., 2011. Cell cycle regulation by microRNAs in stem cells. Results Probl. Cell Differ. 53, 459e472. Wapinski, O., Chang, H.Y., 2011. Long noncoding RNAs and human disease. Trends Cell Biol. 21, 354e361. Wilusz, J.E., JnBaptiste, C.K., Lu, L.Y., Kuhn, C.D., Joshua-Tor, L., Sharp, P.A., 2012. A triple helix stabilizes the 3’ ends of long noncoding RNAs that lack poly(A) tails. Genes Dev. 26, 2392e2407. Xu, N., Papagiannakopoulos, T., Pan, G., Thomson, J.A., Kosik, K.S., 2009. MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell 137, 647e658. Yin, Q.F., Yang, L., Zhang, Y., Xiang, J.F., Wu, Y.W., Carmichael, G.G., Chen, L.L., 2012. Long noncoding RNAs with snoRNA ends. Mol. Cell 48, 219e230. Zheng, G.X., Do, B.T., Webster, D.E., Khavari, P.A., Chang, H.Y., 2014. Dicer-microRNA-Myc circuit promotes transcription of hundreds of long noncoding RNAs. Nat. Struct. Mol. Biol. 21, 585e590. Zhou, W., Choi, M., Margineantu, D., Margaretha, L., Hesson, J., Cavanaugh, C., Blau, C.A., Horwitz, M.S., Hockenbery, D., Ware, C., Ruohola-Baker, H., 2012. HIF1a induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition. EMBO J. 31, 2103e2116.