Roles of long non coding RNAs in human embryonic stem cell pluripotency and neural differentiation 3

Chapter V – Identification of Long Non-coding RNAs Associated with Pluripotency and Neural Differentiation 5.1 Introduction   The mammalian transcriptome comprises a vast number of long non-coding RNAs (lncRNAs), which are defined as transcripts greater than 200 nucleotides with little or no protein-coding potential (Carninci et al., 2005) They participate in numerous biological processes that coordinate gene expression, through epigenetic modification (Gupta et al., 2010; Khalil et al., 2009; Mohammad et al., 2010; Tsai et al., 2010; Wang et al., 2011), mRNA splicing (Tripathi et al., 2010), control of transcription (Orom et al., 2010) or translation (Gong and Maquat, 2011) and genomic imprinting (Mohammad et al., 2010; Pandey et al., 2008; Redrup et al., 2009) Nevertheless, to date only a tiny fraction of lncRNAs has been functionally validated in biological or disease processes LncRNAs are emerging players in embryogenesis and in developmental processes (Amaral and Mattick, 2008; Dinger et al., 2008) Recent studies in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) indicate that lncRNAs are integral members of the ESC self-renewal regulatory circuit (Guttman et al., 2011; Sheik Mohamed et al., 2010) In addition, Loewer et al (2010) showed that a large intergenic non-coding RNA (lincRNA), lincRNA-RoR, enhanced the reprogramming of fibroblasts into iPSCs LncRNAs such as MALAT1, Evf2 and Nkx2.2AS, have also been reported to specify neural cell fate and function (Bernard et   70  al., 2010; Bond et al., 2009; Rapicavoli et al., 2010; Tochitani and Hayashizaki, 2008) LncRNAs are also dynamically expressed during neuronal-glia fate specification, and they appear to regulate the expression of protein-coding genes within the same genomic locus, suggesting lncRNA function (Mercer et al., 2010) Additional evidence suggesting functional roles of lncRNAs in the brain includes a computational analysis of in situ hybridization data from the Allen Brain Atlas, which identified 849 lncRNAs showing specific expression in the mouse brain (Mercer et al., 2008) Furthermore, neural lncRNAs have been shown to be regulated by transcription factors (Johnson et al., 2009) and epigenetic processes (Mercer et al., 2010) So far, most efforts aimed at understanding lncRNA functions in pluripotency and neural differentiation focused on the mouse as a model system (Dinger et al., 2008; Guttman et al., 2011; Mercer et al., 2010; Sheik Mohamed et al., 2010; Tochitani and Hayashizaki, 2008) To date, the roles of lncRNAs in human embryonic and neural developmental gene networks have not been investigated Given the generally poor evolutionary conservation of lncRNAs (Pang et al., 2006), there is a clear need to investigate whether lncRNAs are also important in human embryonic and neuronal developmental networks The enriched and highly homogenous cultures of neural progenitors and neurons derived from hESCs provided an ideal source of cells for expression profiling to identify lncRNAs that are necessary for pluripotency and neural development I hypothesized that if the lncRNAs were important in pluripotency, they should be highly expressed in undifferentiated hESCs and downregulated upon differentiation Likewise, lncRNAs involved in neuronal differentiation would probably be silenced in hESCs and upregulated as the cells acquire a neuronal identity In this chapter, I   71  describe the identification of lncRNAs possibly involved in pluripotency or neurogenesis by means of microarray expression profiling 5.2 Results 5.2.1 Microarray expression profiling identifies differentially expressed lncRNAs The highly enriched cultures of NPCs and neurons described in Chapter IV, together with undifferentiated hESCs, were used for global expression profiling, to examine gene expression changes as hESCs differentiate into NPCs and subsequently into neurons For this purpose, two types of microarrays were utilized A custom-designed microarray was used for detecting lncRNA transcripts, while an Illumina beadchip microarray was used for protein-coding transcripts The lncRNA microarray design included 6671 transcripts identified in a number of published sources, and described in a previous publication (Jia et al., 2010) Importantly, the non-coding status of these transcripts was independently validated in that study In total, the microarray contained 43800 probes such that each lncRNA was represented by to probes, which achieved high sensitivity and specificity To summarize the microarray findings, comparing the NPC to hESC stages, we found 25% of protein-coding probes detected above background (6153 out of 24526) and 4500 probes (18%) were significantly differentially detected (FDR < 0.01; fold change > 2) Of the lncRNA subset, 16% of probes were detected above background (7017 out of 43800), with 9% (3885 probes) being differentially detected   72  (p < 0.05; fold change > 2) When DA neuron stage was compared to the NPC stage, 24% of protein-coding probes were detected above background (5852 out of 24526), with 13% of these (3076 probes) being differentially detected Similarly, a smaller percentage (11.5%) of lncRNA probes (5058 probes) was expressed above background with 6% being differentially expressed (2622 probes) Altogether, a total of 5051 differentially regulated mRNAs and 934 differentially regulated lncRNAs were identified (Figure 5.1) As a further confirmation that the neural cell types derived from hESCs were expressing neural genes, a gene ontology (GO) analysis of the mRNA genes upregulated in the neurons compared to undifferentiated hESCs was performed This indicated an enrichment of GO terms related to neuronal differentiation and is presented in Table 5.1 Table 5.1 Genes expressed in H1-derived neurons were highly enriched for Gene Ontology terms relating to neuronal differentiation The top 10 terms are shown Gene Ontology Biological Process 10 GO Term neurogenesis generation of neurons neuron differentiation cell morphogenesis neuron development central nervous system development cell morphogenesis involved in differentiation brain development negative regulation of biosynthetic process positive regulation of gene expression GO:0022008 GO:0048699 GO:0030182 GO:0000902 GO:0048666 GO:0007417 GO:0000904 GO:0007420 GO:0009890 GO:0010628 Percentage of Genes 13.35 12.28 11.39 9.43 9.25 9.07 8.54 6.58 6.41 6.23 Gene clusters categorized into biological processes at level 6-9 when analyzed with FatiGO P-value < 0.01   73  Figure 5.1: Microarray expression profiling identified differentially expressed lncRNAs during neural differentiation of hESCs Amplified total RNA from undifferentiated hESCs (ES), hESC-derived neural progenitors (NPC) and hESCderived neurons (N) were hybridized onto the coding (mRNA) microarray and the lncRNA microarray simultaneously Expression profiling identified 5051 differentially expressed mRNAs and 934 differentially expressed lncRNAs LncRNAs that are important for maintenance of stem cell identity should be highly expressed in ES, while lncRNAs important for neuronal differentiation should be upregulated in the neurons 5.2.2 Identification of lncRNAs associated with pluripotency (pluripotent lncRNAs) One hypothesis is that lncRNA transcripts important for hESC pluripotency maintenance would have an expression pattern similar to that of known pluripotency drivers such as OCT4, NANOG, and ZNF206, which are highly expressed in undifferentiated hESCs and downregulated upon differentiation (Figure 5.2A) To identify lncRNAs that control pluripotency, I filtered for lncRNA transcripts that had at least probes showing a greater than 5-fold downregulation (p < 0.05) when differentiated from hESCs to NPCs 36 lncRNAs were identified (Figure 5.2B and Table 5.2), including the telomerase RNA component TERC (Agarwal et al., 2010), indicating that our custom-designed array was able to identify pluripotency-associated lncRNAs   74  Figure 5.2: Identification of lncRNAs important in pluripotency, neural induction, and neuronal differentiation (A) The expression profiles of known pluripotency markers in ES, NPC and N stages (B) To identify pluripotency lncRNAs, we filtered for transcripts that showed at least 5-fold downregulation upon differentiation These lncRNAs show an expression pattern similar to that of known pluripotency genes (C, D) NPC lncRNAs were identified by at least 3-fold enrichment in NPC compared to ES or N This yielded an expression profile similar to known NPC markers such as NOTCH1 and PAX6 (E, F) Neuronal lncRNAs in this study were defined as transcripts enriched by at least 3-fold in N, relative to ES and NPC   75  Table 5.2: List of the 36 pluripotency lncRNAs identified from the custom lncRNA array These lncRNAs were at least times upregulated in hESCs compared to NPCs or neurons lncRNA ID AF007835 AF086028 AF090096 AF242771 AF308293 AK026295 AK055335 AK056826 AK091113 AK093557 AL117559 AL833138 AY049742 AY927485 AY927486 AY927487 AY927488 AY927489 AY927490 BC020346 chr1:205,258,369-205,258,868 chr12:54,783,174-54,783,548 chr2:70,742,773-70,743,462 chr12:63,376,238-63,376,597 chr21:35,011,807-35,012,373 chr5:79,470,459-79,471,746 chr19:43,012,031-43,014,637 chr6:14,388,338-14,393,355 chr1:207,668,788-207,672,517 chr17:290,001-292,582 chr6:22,328,989-22,330,599 chr5:142,105,349-142,120,746 chr7:148,475,149-148,475,267 chr19:6,631,188-6,671,573 chr19:6,631,193-6,671,573 chr19:6,631,188-6,671,573 chr19:6,631,192-6,671,565 chr19:6,631,188-6,671,573 chr19:6,631,188-6,671,573 chr19:50,528,532-50,545,485 21 22 23 24 BC026300 BC041913 BC042046 BC107034 chr13:53,593,076-53,605,002 chr3:171,595,099-171,597,312 chr5:67,124,797-67,132,641 chr1:200,251,549-200,251,945 25 26 27 28 29 30 31 32 33 34 35 36   genomic location (hg18/ NCBI36) 10 11 12 13 14 15 16 17 18 19 20 EF565083 EF565113 EF565130 L32067 OR5E1P PAR5 TERC X15675 X64986 X97302 Z36841 Z36849 chr1:198,709,840-198,710,182 chrX:121,203,542-121,204,150 chr10:6,869,854-6,870,131 chrX:110,374,991-110,375,260 chr11:7,827,174-7,827,694 chr15:22,781,100-22,784,472 chr3:170,965,092-170,965,542 chr11:67,144,051-67,151,440 chr11:7,916,927-7,917,269 chr17:43,384,620-43,384,873 chr15:99,273,870-99,274,351 chr19:43,474,581-43,474,928 76  5.2.3 Identification of lncRNAs associated with neural progenitors (NPC lncRNAs) Apart from pluripotent lncRNAs, transcripts whose expression peaked in the NPC stage of neural differentiation were also detected Since these lncRNAs showed the highest level of transcription in the neural progenitors, they were referred to as NPC lncRNAs These NPC lncRNAs were defined as transcripts having at least probes showing a greater than 3-fold expression in NPCs compared to hESCs and neurons, and mirrored the expression of genes known to be important in the maintenance of neural stem cell identity, such as PAX6, POU3F2 and SOX1 (Figures 5.2C-D) Interestingly, SOX2OT was also identified as a NPC lncRNA from the transcriptome-wide study SOX2OT, or SOX2-overlapping transcript, is a 2.4 kb long non-coding RNA, and encompasses the entire SOX2 gene Both SOX2 and SOX2OT are transcribed in the same orientation, with the single-exon SOX2 gene embedded within an intron of SOX2OT A previous report by Amaral et al (2009) had also reported a similar observation that expression of Sox2ot increased as mouse ES cells differentiate into neural lineages This again indicated the sensitivity and specificity of the custom-designed lncRNA microarray   77  Table 5.3: List of the 24 NPC lncRNAs identified from the lncRNA microarray These lncRNAs were at least 3-fold upregulated in NPCs compared to hESCs and neurons lncRNA ID AB005217 AF052141 AF070541 AF090902 AF124366 AK000454 AK022857 AK055853 AK055998 AK056630 AK096049 AK097507 AL110177 AL832845 AL833463 BC009777 BC012900 BC020441 BC034326 BC038746 chr2:133,257,623-133,258,873 chr4:87,152,473-87,154,051 chr18:29,583,598-29,585,156 chr4:54,620,521-54,622,131 chr8:11,316,417-11,317,852 chr17:46,059,392-46,064,300 chr15:53,691,042-53,693,912 chr1:15,525,763-15,542,959 chr5:169,223,891-169,340,322 chr7:99,475,109-99,477,247 chr2:85,831,978-85,868,696 chr14:25,359,412-25,361,787 chr3:40,478,871-40,483,651 chr11:56,713,037-56,715,763 chr15:53,618,374-53,622,121 chr7:120,378,069-120,384,270 chr8:29,246,499-29,248,601 chr6:127,878,629-127,881,771 chr12:105,598,667-105,602,608 chr4:87,259,986-87,360,101 21 22 23 24   genomic location (hg18/ NCBI36) 10 11 12 13 14 15 16 17 18 19 20 BC045770 BX640762 SOX2OT X75685 chr4:7,150,697-7,156,004 chr11:31,768,058-31,789,455 chr3:182,810,845-182,941,699 chr21:16,638,278-16,638,503 78  5.2.4 Identification of lncRNAs associated with neuronal differentiation (neuronal lncRNAs) From the microarray, it was evident that there was a group of neuronal lncRNAs that were highly expressed in neurons and weakly expressed in undifferentiated hESCs and NPCs (Figure 5.1) In addition, the expression profiles of these lncRNAs mirror that of known drivers of neuronal differentiation (Figures 5.2E-F), and were therefore indicative of important roles of the lncRNAs in neurogenesis A group of 35 lncRNAs were found to be highly expressed in differentiated neurons compared to NPCs and undifferentiated hESCs (Figure 5.2F), and are listed in Table 5.4 These 35 lncRNAs were filtered based on having at least probes showing a greater than 3-fold upregulation (p < 0.05) compared to NPCs and hESCs   79  Figure 7.2: RNA-seq analysis indicating transcription start and end sites of neuronal lncRNAs (A) The three isoforms of RMST are namely AK056164, AF429305 and AF429306 (B) lncRNA_N1 (AK124684) is a single-exon transcript (C) lncRNA_N2 (AK091713) possibly has alternatively spliced isoforms, and hosts the protein-coding gene BLID in its intron In addition, microRNAs in which lncRNA_N2 overlaps with are shown in red (D) lncRNA_N3 (AK055040) is possibly a part of an elongated transcript   110  Functional neuronal lncRNAs should be expressed in the human brain Therefore, expression of the lncRNAs in a panel of somatic tissues was tested by qPCR This demonstrated that RMST, and lncRNAs_N1-3 were all expressed in brain regions (whole brain, fetal brain, substantia nigra and cerebellum) While expression of RMST and lncRNA_N1 were more confined to brain regions, lncRNA_N2 and lncRNA_N3 were also present in other somatic tissues (Figure 7.3) As with the case of pluripotency lncRNAs, neuronal lncRNAs were not abundant (~0.3 to 26% relative to GAPDH mRNA levels), consistent with their proposed regulatory roles (Figure 7.4) Figure 7.3: Tissue specificity of the neuronal lncRNAs RMST, lncRNA_N1, lncRNA_N2 and lncRNA_N3 The expression of the respective neuronal lncRNAs were measured in a panel of somatic tissues While RMST and lncRNA_N1 showed somewhat brain-specific expression, lncRNA_N2 and lncRNA_N3 were expressed in the brain, as well as other adult tissue types   111  Figure 7.4: Relative abundance of neuronal lncRNAs, compared to that of GAPDH mRNA levels RMST is the most abundant of the neuronal lncRNAs, while the others were only between 0.3% to 3.7% that of GAPDH transcript levels GAPDH is a housekeeping mRNA 7.2.2 Neuronal lncRNAs are required for neuronal differentiation To determine if the neuronal lncRNAs were required for neurogenesis, I investigated the effect of their knockdown on neuronal differentiation siRNAs against each of the neuronal lncRNAs were introduced into the human neural stem cell line ReN-VM One day later, neuronal differentiation was induced in the N2B27 medium Seven days later, neuronal differentiation was assayed at the protein level, by immunostaining of TUJ1+ early post-mitotic neurons and/or MAP2+ late mature neurons, as well as at the mRNA level The experimental procedure is summarized in Figure 7.5 Two siRNAs were tested for each of the four lncRNAs, and the more effective siRNA was subsequently used (Figure 7.6)   112  Figure 7.5: Schematic representation of differentiation of ReN-VM neural stem cells following transfection of siRNAs Neural stem cells were plated one day prior to siRNA transfection Neuronal differentiation was induced in N2B27 medium Transfection of siRNAs was performed again at day 3, for prolong RNAi Efficiency of neuronal differentiation was assessed at day 7, by TUJ1 immunofluorescence   113  Figure 7.6: Neuronal lncRNAs can be efficiently targeted by siRNAs Two siRNA duplexes were used for the knockdown of each pluripotency lncRNA Efficiency of knockdown was compared relative to the non-target siRNA (si-NT) control Subsequently, the more effective siRNA was used   114  While the non-target siRNA (si-NT) control yielded TUJ1+ and MAP2+ neurons, very few stained cells were observed where the neuronal lncRNAs were knocked down (Figure 7.7) This was confirmed by FACS analysis of TUJ1+ cells transfected with the respective siRNAs The si-NT control yielded approximately 25% TUJ1+ neurons, while knockdown of the neuronal lncRNAs resulted in less than 5% TUJ1+ neurons (Figure 7.8) Together, these data indicate that the neuronal lncRNAs were required for efficient neuronal differentiation Quantitative PCR at Day further showed that reduced lncRNA levels in neural progenitors resulted in decreased expression of neurogenic markers including NEUROG2, PAX6, DCX, TUJ1, MAP2, SYP, HES5 and SYPL1, and a simultaneous increase in glia markers PDGFRα, NG2CSP, CNPase, MBP and LRRN3 (Figure 7.9) This indicates that loss of the neuronal lncRNAs altered cellular differentiation fate from a neurogenic to a gliogenic program, and suggests that the lncRNAs play a key role in neural cell fate specification   115  Figure 7.7: Knockdown of neuronal lncRNAs prevented neurogenesis ReN-VM neural stem cells were transfected with the indicated siRNAs, and TUJ1, a marker for neurons, was assayed by immunofluorescence days later In the non-target siRNA (si-NT) control, TUJ1+ neurons were observed However, when any of the neuronal lncRNAs were knocked down, very few TUJ1+ cells were observed, indicating a loss of neurogenesis The scale bar indicates 100 µm   116  Figure 7.8: Loss of TUJ1+ cells upon knockdown of neuronal lncRNAs The percentage of TUJ1+ cells from Figure 7.6 was quantified by flow cytometry The number of TUJ1+ cells were significantly reduced upon knockdown of any of the four neuronal lncRNAs * and ** indicate p-values of < 0.05 and < 0.01 respectively Figure 7.9: Knockdown of neuronal lncRNAs resulted in cells adopting a glia fate Following knockdown and differentiation described in Figure 7.4, a panel of neuronal and glia markers were analyzed by qPCR, and fold changes, relative to the si-NT control, were represented in a heatmap Green indicates downregulation and red indicates upregulation   117  7.2.3 Neuronal lncRNAs support neurogenesis by associating with nuclear proteins To investigate the molecular functions of the neuronal lncRNAs, the subcellular localization of the neuronal lncRNAs was first determined by means of RNA fractionation followed by qPCR With the exception of lncRNA_N2, the neuronal lncRNAs were preferentially nuclear-retained (Figure 7.10) Thus RMST, lncRNA_N1 and lncRNA_N3 might be interacting with nuclear factors and/or chromatin to support neurogenesis, while lncRNA_N2 possibly has a role in the cytoplasm Next, I sought to identify physical interactions of nuclear lncRNAs with nuclear proteins in ReN-VM neural stem cells, specifically with SUZ12 and REST, as the former has been reported to be associated with many lncRNAs (Khalil et al., 2009), and the latter is an important transcription factor that represses neurogenesis and is part of the complex bound by the lncRNA HOTAIR (Naruse et al., 1999; Su et al., 2004; Tsai et al., 2010) Three individual RNA immunoprecipitation (RIP) experiments confirmed that lncRNA_N1 and lncRNA_N3 were significantly enriched in both SUZ12 IP and REST IP over isotype IgG IP control (Figure 7.11B and Figure 7.11C, respectively), suggesting these two lncRNAs complex with the repressive PRC2 complex and REST/coREST complex to regulate gene expression and specify neural cell fate specification   118  Figure 7.10: Apart from lncRNA_N2, the other neuronal lncRNAs were nuclearlocalized Cellular RNA was separated into the nuclear and cytoplasmic fractions, and qPCR was performed to compute nuclear/cytoplasmic ratio HOTAIR and KCNQ1OT1 are well-characterized, nuclear-localized RNAs, while GAPDH and ACTB are protein-coding mRNAs expected to be cytoplasmically localized   119  Figure 7.11: RNA immunoprecipitation indicated that lncRNA_N1 and lncRNA_N3 associated with SUZ12 and REST (A) RNA immunoprecipitation or RIP of HOTAIR using anti-SUZ12, a previously reported interaction (Gupta et al., 2010) RIP enrichment was measured by qPCR, and values were normalized to background immunoprecipitation measured by isotype IgG (B-C) RIP of lncRNAs using anti-SUZ12, anti-REST respectively lncRNA_N1 and lncRNA_N3 were enriched in the SUZ12 RIP and REST RIP, indicating interaction * and ** indicate pvalues of < 0.05 and < 0.01 respectively   120  7.2.4 Cytoplasmic lncRNA_N2 affects microRNA expression Of the four neuronal lncRNAs assayed, lncRNA_N2 was the only cytoplasmic transcript and contains the microRNAs (miRNAs) MIR-125B and LET7 within its introns (Figure 7.2C) These miRNAs are known to be important for neurogenesis (Le et al., 2009; Rybak et al., 2008) This suggests the possibility that lncRNA_N2 represents the processing product of the miRNA host transcript, and that knockdown of this transcript could repress neuronal lineage commitment To this end, I performed a knockdown of lncRNA_N2 in neural stem cells, isolated total RNA 48 hours later, and compared MIR125B and LET7A levels to that of the non-target siRNA control Quantitative PCR revealed that lncRNA_N2 was knocked down by about 75%, and MIR-125B and LET7A levels were reduced significantly by about 50% (Figure 7.12) This indicated that the lncRNA_N2 is likely to promote neurogenesis by maintaining MIR125B and LET7A levels in neural progenitors Figure 7.12: Quantification of changes in hosted miRNAs in response to lncRNA_N2 knockdown MiRNAs were quantified using Taqman miRNA qPCR The decrease in MIR-125B and LET7A following lncRNA_N2 RNAi indicates that lncRNA_N2 is responsible for maintaining expression of the neurogenic miRNAs   121  7.3 Discussion 7.3.1 Neuronal lncRNAs act via diverse mechanisms While it has been observed that some lncRNAs act in cis (Ponjavic et al., 2009), a recent report indicated that a unique class of lncRNAs termed large intergenic noncoding RNAs or lincRNAs primarily affect gene expression in trans (Guttman et al., 2011) With the exception of cytoplasmic lncRNA_N2, which may represent the miRNA host transcript that gives rise to miRNAs in that region, there is no significant change of gene expression within a 600 kb window (300 kb upstream and 300 kb downstream) following knockdown of the other three lncRNAs (Figure 7.13) Therefore, while some lncRNAs may work in cis, it appears that the neuronal lncRNAs in this study are trans-acting 7.3.2 Neuronal lncRNAs form part of a repressive complex to silence glia genes Association studies demonstrated that neuronal lncRNAs could interact with repressors such as SUZ12 and REST SUZ12 is part of the PRC2 polycomb repressive complex that induces heterochromatin formation, while REST, and its corepressor coREST function primarily in directing neural differentiation REST and coREST have been implicated not only in neuronal, but also glia lineage specification (Abrajano et al., 2009) It is plausible for nuclear-retained neuronal lncRNAs to act as a tether on which chromatin modifiers and repressors such as the PRC2 complex or the REST complex bind to, and possibly direct the RNA/protein complex to silence glia lineage genes   122  Figure 7.13: Neuronal lncRNAs possibly act in trans Upon knockdown of the lncRNAs (shown in blue), gene expression changes within 300 kb upstream and downstream of the lncRNAs were analyzed, to determine if effects of the lncRNAs were felt locally While it is likely that lncRNAs regulate biological processes through epigenetic modifications, elucidation of molecular mechanisms require more studies, including a genome-wide assessment of histone marks in native and perturbed lncRNA conditions In addition, analysis of H3K27 trimethylation marks at promoters of REST target genes in neural progenitors and neurons would shed light on how the lncRNA/protein complex regulates neurogenesis   123  7.4 Conclusion It has been proposed that lncRNAs may represent a key undiscovered genetic component in the evolution of the human brain (Mattick and Mehler, 2008), but little evidence has been presented for functional roles of lncRNAs in the human nervous system First, through RNAi studies, I established that lncRNAs were required for neuronal differentiation Subsequently, RNA fractionation elucidated the subcellular localization of neuronal lncRNAs RNA immunoprecipitation identified SUZ12 and REST to be protein partners of some of the nuclear lncRNAs, while knockdown and expression analysis suggested that cytoplasmic lncRNA_N2 could be hosting miRNAs in its introns The data presented in this chapter represent the first direct demonstration that lncRNAs are necessary components of neural developmental gene networks in human   124  ... chr 13: 36 ,31 5,670 -36 ,31 7,9 83 chr4:1,179,572-1,181,6 03 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 AK124684 AK125262 AK12 539 2 AL 832 189 AY927 532 BC008027 BC 030 122 BC 031 955... location (NCBI36/hg18) AF242771 AK05 533 5 AK056826 (lncRNA_ES1) AK0911 13 AL117559 AL 833 138 BC02 630 0 (lncRNA_ES3) chr12: 63, 376, 238 - 63, 376,597 chr19: 43, 012, 031 - 43, 014, 637 chr3:171,595,099-171,597 ,31 2 chr5:67,124,797-67, 132 ,641... AK12 539 2 AL 832 189 BC008027 BC 030 122 BC 031 955 BC 033 371 BC 035 156 BC 036 480 BC 038 536 BC041400 BC041859 BC042044 BC0 432 37 BC0 432 54 BC0 433 80 BX649145 L31955 chr8:110,725,520-110,729,489 chr 13: 36 ,31 5,670 -36 ,31 7,983