Figure 8.13: Western blot confirms that hnRNPA2B1 and SOX2 specifically interact with RMST To validate findings from mass spectrometry, a Western blot using the respective antibodies was performed following biotinylated RNA pulldown Figure 8.14: RNA immunoprecipitation (RIP) established in vivo binding of RMST with hnRNPA2 and SOX2 Fold enrichment relative to the isotype IgG RIP is presented, and the respective p-values are indicated (A) hnRNPA2-FLAG was ectopically expressed in ReN-VM cells, and enrichment of RMST was observed in FLAG RIP, indicating physical association (B) Enrichment of RMST in SOX2 RIP also confirmed in vivo association of SOX2 and the lncRNA   138  Since RMST physically interacts with both hnRNPA2B1 and SOX2, the question of the assembly of the “RMST complex” arose It was found that hnRNPA2 and SOX2 assembled in an RNA-independent fashion In three independent coimmunoprecipitation (co-IP) experiments, SOX2 is co-immunoprecipitated with hnRNPA2-FLAG in RNase-treated cell lysate (Figure 8.15) In accordance with this observation, Fang et al (2011) found that SOX2 forms protein-protein interactions with several heterogenous nuclear ribonucleoproteins, including hnRNPA2B1 These evidences suggest that the RMST complex is composed of the RNA-binding protein, hnRNPA2B1 binding to RMST, and SOX2 in turn associates with hnRNPA2B1 through its protein interaction domain (Figure 8.16) Figure 8.15: Co-immunoprecipitation (Co-IP) of hnRNPA2-FLAG and SOX2 in the absence of RNA Cell lysate was treated with RNase, and the co-IP of hnRNPA2 and SOX2 indicated that the two proteins could associate by protein-protein interactions, rather than through a lncRNA scaffold   139  Figure 8.16: Proposed model of the RMST complex hnRNPA2B1 is a RNAbinding protein and probably binds directly to RMST SOX2 and hnRNPA2B1 may interact via protein-protein interactions The transcription factor SOX2 may then bind to target chromatin regions to affect gene expression 8.2.5 RMST and SOX2 co-regulate a common pool of genes During neurogenesis, RMST expression was upregulated Since RMST interacts with hnRNPA2B1 and SOX2, they can be envisaged to engage in a RMST complex, in which these three components together would regulate neurogenesis Previous reports indicate a crucial role for SOX2 in neurogenesis Depletion of SOX2 in neural progenitors resulted in impaired neurogenesis in the central as well as peripheral nervous system (Cavallaro et al., 2008; Cimadamore et al., 2011; Ferri et al., 2004; Puligilla et al., 2010) Therefore, it was expected that the knockdown of SOX2 in the ReN-VM neural stem cells would result in decreased neurogenesis To establish that the protein components of the RMST complex also played a role in neuronal differentiation, siRNAs targeting hnRNPA2B1 and SOX2 were introduced into ReNVM cells, and the effect on neuronal differentiation was assayed days post transfection by immunofluorescence (Figure 8.17)   140  Figure 8.17: Knockdown of components of the RMST complex prevents neurogenesis ReN-VM neural stem cells were transfected with the indicated siRNAs TUJ1 and MAP2, both markers for neurons, were assayed by immunofluorescence days later In the non-target siRNA (si-NT) control, TUJ1+ and MAP2+ neurons were observed When expressions of RMST, hnRNPA2B1 and SOX2 were depleted, there were significantly fewer TUJ1+ cells, indicating a loss of neurogenesis The scale bar indicates 100 µm In the bottom panel, the efficiencies of knockdown are indicated As expected, knockdown of any of the components of the RMST complex resulted in the loss of neurogenesis, indicated by decrease in the number of TUJ1+ and MAP2+ cells The similarity in loss-of-function phenotype, together with RNAprotein interactions, suggested the possibility that RMST and SOX2 could be regulating a common pool of genes To this end, a microarray experiment was carried out ReN-VM cells were transfected with RMST siRNA (si-RMST), SOX2 siRNA (siSOX2) or non-target siRNA (si-NT) RNA was extracted 48 hours after transfection   141  of siRNAs Microarray expression data was normalized to the si-NT dataset, and the extent of overlap between differentially expressed genes in the si-RMST and si-SOX2 datasets was examined To summarize the microarray findings, 1171 genes were differentially detected (FDR < 0.05; absolute fold change > 1.5) in the si-RMST cells, while there were 626 differentially expressed genes in the si-SOX2 cells There was a 100% overlap between the datasets in that the 626 genes differentially expressed in siSOX2 were also differentially expressed in si-RMST Of the 1170 differentially expressed genes in si-RMST cells, 632 were upregulated while 539 were downregulated A similar trend was observed in the si-SOX2 cells, where 372 genes were upregulated, while 254 were downregulated (Table 8.1) The extent of overlap was also large (more than 60%), indicating that RMST and SOX2 possibly regulate a common pool of genes (Figure 8.18) In addition, a gene ontology (GO) analysis of the 152 genes downregulated upon both RMST and SOX2 knockdown indicated an enrichment of GO terms related to neurogenesis and neuronal function (Table 8.2) Taken together, the loss-of-function studies, coupled with whole genome gene expression analyses, indicate that RMST and SOX2 regulate a common set of target genes that are essential for neurogenesis   142  Table 8.1: Table summarizing microarray findings upon knockdown of RMST or SOX2 in neural stem cells si-RMST si-SOX2 overlap differentially expressed 1171 626 626 upregulated 632 372 331 downregulated 539 254 152 Figure 8.18: RMST and SOX2 regulate a common pool of targets The extent of overlap between differentially expressed genes in si-SOX2 and si-RMST datasets was examined by means of a microarray experiment Red circles indicate si-RMST, while blue circles indicate si-SOX2 datasets (A) Regardless of direction of fold change, the overlap between si-SOX2 and si-RMST was 100%, with 626 differentially expressed genes in si-SOX2 also misexpressed in si-RMST (B) Of these, 331 genes were upregulated in both si-RMST and si-SOX2 (C) 152 genes were downregulated in both si-RMST and si-SOX2   143  Table 8.2: Gene Ontology (GO) analysis of the 152 genes in the si-RMST and siSOX2 overlap Gene Ontology Biological Process 10 Neuron migration Regulation of Notch signaling pathway Cellular response to stress Forebrain development Neurogenesis Positive regulation of Notch signaling pathway Telencephalon development Rho protein signal transduction Brain development Oligodendrocyte differentiation Gene Ontology Term GO:0001764 GO:0008593 GO:0033554 GO:0030900 GO:0022008 GO:0045747 GO:0021537 GO:0007266 GO:0007420 GO:0048709 p-value 5.28E-06 6.55E-06 2.97E-05 2.97E-05 1.29E-04 1.46E-04 1.48E-04 1.67E-04 1.81E-04 1.89E-04 The top 10 terms, and their respective p-values are shown Gene clusters categorized into biological processes at levels 3-9 when analyzed with FatiGO 8.2.6 RMST does not regulate SOX2 expression Since RMST and SOX2 share a large set of target genes, one question that arose was whether RMST regulates SOX2 expression To this end, SOX2 expression following RMST knockdown in ReN-VM cells was measured RNA was isolated 48 hours after transfection of siRNAs Quantification by qPCR showed that upon RMST knockdown, transcript levels of SOX2 and hnRNPA2B1 remained unchanged Similarly, knockdown of SOX2 had no effect on RMST or hnRNPA2B1 expression (Figure 8.19) This demonstrates that RMST did not regulate downstream target expression by altering cellular levels of the transcription factor SOX2   144  Figure 8.19: Transcript levels of SOX2 remained unchanged following RMST knockdown Knockdown of hnRNPA2B1 (bars shown in light blue) did not alter expression levels of RMST and SOX2 significantly Similarly, knockdown of RMST (bars shown in dark blue) did not result in a change in SOX2 and hnRNPA2B1 levels in the cells 8.3 Discussion 8.3.1 RMST forms part of a complex that is required for neurogenesis In this chapter, I established that RMST interacts with hnRNPA2B1, as well as SOX2, and loss of any of these three components of the RMST complex resulted in loss of neurogenesis In accordance with literature, Sox2 induces neuronal formation, and the loss of Sox2 had been reported to cause neurodegeneration and impaired neurogenesis in the mouse brain (Ferri et al., 2004; Puligilla et al., 2010) SOX2 has also been reported to associate with hnRNPA2B1 via protein-protein interactions (Fang et al., 2011), and this interaction was also observed in this study via the co-IP experiment hnRNPA2 and hnRNPB1 (referred to as hnRNPA2B1) are spliced isoforms, and are RNA-binding proteins that regulate alternative splicing (Chen et al., 2010; Tauler et al., 2010) Therefore, apart from serving as the adaptor molecule linking RMST to   145  SOX2, it is also probable that hnRNPA2B1 may regulate the splicing of RMST into its three isoforms However, in this current study, the functions of individual isoforms are not established 8.3.2 RMST may change the binding patterns of SOX2 to chromatin From the microarray experiment described in Figure 8.17, it was evident that RMST and SOX2 share many common targets, supporting the notion that the two are part of the RMST complex that regulates genes crucial for neurogenesis There are at least two possibilities in which RMST could function in the complex First, association of RMST with SOX2 could alter binding of the transcription factor to chromatin Preliminary evidence suggests that depletion of RMST in ReN-VM cells reduced SOX2 occupancy at some target genes (Figure 8.20), indicating that this is a plausible mechanism in which RMST may modulate neurogenesis Figure 8.20: Depletion of RMST resulted in decreased SOX2 occupancy at target genes Cells were transfected with either a non-target siRNA or RMST siRNA, and SOX2 ChIP was performed 48 hours later Fold enrichment at SOX2 targets (pDLX1, pJAG1 and pGLI2) were decreased upon RMST depletion pNECDIN was a negative control (Engelen et al., 2011)   146  The second possibility is that RMST may recruit SOX2 to specific gene loci to regulate transcription Several lncRNAs, including HOTAIR, KCNQ1OT1 and Evf2 (Bond et al., 2009; Chu et al., 2011; Mohammad et al., 2010), have been reported to recruit transcription factors or chromatin modifiers to specific regions in the genome to regulate gene expression 8.3.3 RMST may bind to proteins other than hnRNPA2B1 and SOX2 Depletion of the lncRNA RMST resulted in differential expression of more than 1000 putative target genes In contrast, depletion of SOX2 resulted in only 626 misexpressed genes (Figure 8.17A) This suggested that RMST could associate with other nuclear proteins, apart from hnRNPA2B1 and SOX2 However, from the RIP experiments in Chapter VII, it is indicated that RMST are neither bound by SUZ12, a component of the PRC2 repressive complex, nor the 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ZNF462 FLJ20021 PLA2G3 FYN PPTC7 PIK3R2 ADAM19 PCDH17 CCDC34 MTCP1 RAB13 TMTC2 FANCI ARHGEF6 KIF5C MIF4GD ENKUR SLC5A3 SLC44A1 SV2A MAPK10 LOC728475 TYSND1 FAM59A DYRK2 DNAJC30 SLC35F1 LOC389168 XPR1 EHBP1 LOC730202 DLX1 TMEM107 SIK3 BANP RECQL MRE11A FRS3 ASXL2 LOC729065 ASCL1 TOP3A KCNJ10 CLCN2 TUBB3 PRR11 SBK1 COL4A3BP TBX2 CCNY ELOVL2 ST8SIA5 LOC643995 C14orf80 DOCK7 MAP4K4 FOXO3 PCGF6 TMEM5 BEND5 PTPLAD1 QKI ZNF483 CNR1 LOC654126 RALGDS STK39 MMAB C9orf140 B4GALT5 ZNF586 C20orf100 HSPC268 RHOJ HIRIP3 MOCS1 ATP6V0E2 HEY2 KLHL13 169  Appendix II: List of 331 genes upregulated upon RMST and SOX2 knockdown CLDN1 IFI27 IL32 IFI44L VCAM1 CXCL10 MYBPC2 HERC5 EPSTI1 SAMD9 MX1 OASL GBP1 OAS2 OAS1 LOX AFAP1L2 ANGPTL4 LYSMD3 HERC6 TNFAIP2 IL15RA GXYLT2 IFI44 SLC44A3 ZNF429 DKFZP686E215 PPARA MEG3 HIST1H2AC ZNF493 RELB FBN1 ISG15 MYL9 IFIT1 TMEM229B IFIH1 LGALS9 BIRC3 ABTB1 LOC730087 LOC646731 AFTPH RAB11FIP1 MKRN3 IL15 LOC645402   LOC652741 C5orf56 IFITM1 FAM176A MATN3 PRSS35 TDRD7 BST2 ANK1 NFKB2 TAGLN MYC DDX60L N4BP2L2 OPRL1 CUL4B RARRES3 FAM5C TMEM166 OAS3 STX3 EEA1 CYP1B1 SERTAD4 LOC100129681 CLIP4 ECE1 DRAM1 RDH8 CD83 KDELR3 STAT1 DDX58 KPNA5 PLSCR4 PLAUR C19orf66 PARP12 PCTP TNKS1BP1 TTC39C CYorf15B IFIT3 UBA7 METRNL H19 NQO1 VPS4B TNS3 COL5A3 ENPP5 THBS1 DDX60 CROT TRPM4 ZNF37A PARP10 CLCN3 FAM46B ULBP2 IFI6 CARD10 WDR37 TMTC1 APOL2 ANKRD46 ROR1 NNMT LOC100129445 PGM2L1 LAMB2 QPCT CCL2 TTYH2 ZNF404 PPIC LAMB3 LOC100131091 TUFT1 CCDC19 IRAK2 GOLT1B ABCC3 C19orf12 LEPREL1 ITSN1 C17orf91 EXOC5 RSAD2 ZNFX1 EDEM1 OPTN USP18 ATP2B4 DUSP5 PRIC285 DCP2 BTN3A3 IRF9 TNPO1 CCDC126 ABCF3 GCHFR ELOVL4 ST6GALNAC6 SP110 IFI35 CHST15 TPM1 SDC4 LOC652968 C8orf58 IRF7 LYG1 EPHA2 CASP7 MBD1 GOLGA4 ZC3HAV1 ERAP2 CHPF2 IRF1 PLSCR1 C1orf109 CD46 TRIM21 NPHP3 ABCC5 C1orf133 ACSL4 PGCP HERPUD1 ALCAM FBXO32 GCA MBP PIM2 S1PR3 PRPF4B RNF24 SYTL2 CAMTA1 HLA-B XKR8 BLCAP FZD4 FAM167A 170  ZNF83 ST13 UQCC SEZ6L2 PIGZ PLCD1 CHRNA1 LOC643310 SPHAR FLNB ATF1 SEMA5B RAB11FIP2 CYP2R1 SEMA4F ENPP4 TCIRG1 TPD52L1 OGFR GAL PARP9 CNN2 GLRB VLDLR PHLDA2 MAP1LC3B LOC389386 LOC652900 LIN37 BRMS1L STAT2 RNF149 JUP OLFML2B IL18BP SPTLC1 JPH2 LPPR2 NXN CHCHD7 XRN1 LCTL TBL1XR1 PARP14 ISG20 CYR61 HLA-F SLC39A14 TNC LOC341230 SHB BTRC ATP7B   IER3 VWA1 GOLGA3 FDX1 SMAGP GALNTL1 SRPR NHLRC3 EPB41L1 PSMB8 LOC340274 TMF1 MAOB MGC4677 PCK2 LDLR INPP5A ADARB1 PLA2G15 GPR126 FAM111A EIF5A2 ZNF697 TMEM44 C10orf6 STK3 RAB4A YWHAE ZNF721 PDLIM7 RALB MIR221 C19orf28 DNAJB9 CRIM1 PHF1 SLC9A7 FAT1 FAM135A COG6 TRIM56 MPP1 ANKRD49 FER1L3 PDLIM1 GALNT10 LOC399959 ITGAV RASSF7 TNFRSF12A TYW3 PHLPP2 PCGF5 COQ10B ZNF385A PLEKHA1 TRIB1 ATG16L1 PXMP3 DICER1 ASS1 VPS36 AHNAK2 CRY2 RNF19B STK10 NAT13 C9orf3 LBH CDS2 AKAP8 COL6A1 GPR137C C6orf106 IFNGR1 IFIT2 SRXN1 TOM1L1 CGNL1 KIAA1641 LOC648740 KITLG PARD6G 171  ... established in the mouse model system, and very few human lncRNAs have been studied with such extent In this thesis, I focused on examining lncRNAs involved in the pluripotency maintenance of human embryonic. .. 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