Mapping alternative polyadenylation in human cells using direct RNA sequencing technology.

Alternative cleavage and polyadenylation (APA) is a widespread mechanism to generate mRNA isoforms with alternative 3' untranslated regions. Here, we detail a protocol for detecting APA genome wide using direct RNA sequencing technology including computational analysis. We describe steps for RNA sample and library preparation, nanopore sequencing, and data analysis. Experiments and data analysis can be performed over a period of 6-8 days and require molecular biology and bioinformatics skills. For complete details on the use and execution of this protocol, please refer to Polenkowski et al.1.

THOC5 complexes with DDX5, DDX17, and CDK12 to regulate R loop structures and transcription elongation rate.

THOC5, a member of the THO complex, is essential for the 3'processing of some inducible genes, the export of a subset of mRNAs and stem cell survival. Here we show that THOC5 depletion results in altered 3'cleavage of >50% of mRNAs and changes in RNA polymerase II binding across genes. THOC5 is recruited close to high-density polymerase II sites, suggesting that THOC5 is involved in transcriptional elongation. Indeed, measurement of elongation rates in vivo demonstrated decreased rates in THOC5-depleted cells. Furthermore, THOC5 is preferentially recruited to its target genes in slow polymerase II cells compared with fast polymerase II cells. Importantly chromatin-associated THOC5 interacts with CDK12 (a modulator of transcription elongation) and RNA helicases DDX5, DDX17, and THOC6 only in slow polymerase II cells. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate a novel function of THOC5 in transcription elongation.

Genome-Wide Methylation Mapping Using Nanopore Sequencing Technology Identifies Novel Tumor Suppressor Genes in Hepatocellular Carcinoma.

Downregulation of multiple tumor suppressor genes (TSGs) plays an important role in cancer formation. Recent evidence has accumulated that cancer progression involves genome-wide alteration of epigenetic modifications, which may cause downregulation of the tumor suppressor gene. Using hepatocellular carcinoma (HCC) as a system, we mapped 5-methylcytosine signal at a genome-wide scale using nanopore sequencing technology to identify novel TSGs. Integration of methylation data with gene transcription profile of regenerated liver and primary HCCs allowed us to identify 10 potential tumor suppressor gene candidates. Subsequent validation led us to focus on functionally characterizing one candidate-glucokinase (GCK). We show here that overexpression of GCK inhibits the proliferation of HCC cells via induction of intracellular lactate accumulation and subsequently causes energy crisis due to NAD+ depletion. This suggests GCK functions as a tumor suppressor gene and may be involved in HCC development. In conclusion, these data provide valuable clues for further investigations of the process of tumorigenesis in human cancer.

C20orf204, a hepatocellular carcinoma-specific protein interacts with nucleolin and promotes cell proliferation.

In most human cancers, a large number of proteins with driver mutations are involved in tumor development, implying that multiple fine tuners are involved in cancer formation and/or maintenance. A useful strategy for cancer therapy may therefore be to target multiple cancer type-specific fine tuners. Furthermore, genome-wide association studies of tumor samples have identified a large number of long noncoding (lnc)RNA associated with various types of tumor. In this context we have previously found that C20orf204 (a splice variant of Linc00176) RNA contains a 189 amino acid (AA) long open reading frame (C20orf204-189AA) that is expressed predominantly in hepatocellular carcinoma (HCC). We report here that a protein, C20orf204-189AA, was detected in the nucleus of 14 out of 20 primary HCC, but not in control livers. Strikingly, overexpression of C20orf204-189AA enhanced cell proliferation and ribosomal RNA transcription. C20orf204-189AA is co-localized, and interacted with nucleolin via the C-terminal and with ribosomal RNA via the N-terminal domain. Furthermore, the expression of C20orf204-189AA upregulates the protein level of nucleolin. Nucleolin and C20orf204 mRNA levels in HCC are correlated with tumor differentiation grade and patient survival, suggesting that C20orf204-189AA is a cancer type-specific fine tuner in some HCC that presents itself for potential targeting therapy and cancer biomarker. Thus, cancer cells exhibit remarkable transcriptome alterations partly by adopting cancer-specific splicing isoforms of noncoding RNAs and may participate in tumor development.

Identification of Novel Micropeptides Derived from Hepatocellular Carcinoma-Specific Long Noncoding RNA.

Identification of cancer-specific target molecules and biomarkers may be useful in the development of novel treatment and immunotherapeutic strategies. We have recently demonstrated that the expression of long noncoding (lnc) RNAs can be cancer-type specific due to abnormal chromatin remodeling and alternative splicing. Furthermore, we identified and determined that the functional small protein C20orf204-189AA encoded by long intergenic noncoding RNA Linc00176 that is expressed predominantly in hepatocellular carcinoma (HCC), enhances transcription of ribosomal RNAs and supports growth of HCC. In this study we combined RNA-sequencing and polysome profiling to identify novel micropeptides that originate from HCC-specific lncRNAs. We identified nine lncRNAs that are expressed exclusively in HCC cells but not in the liver or other normal tissues. Here, DNase-sequencing data revealed that the altered chromatin structure plays a key role in the HCC-specific expression of lncRNAs. Three out of nine HCC-specific lncRNAs contain at least one open reading frame (ORF) longer than 50 amino acid (aa) and enriched in the polysome fraction, suggesting that they are translated. We generated a peptide specific antibody to characterize one candidate, NONHSAT013026.2/Linc013026. We show that Linc013026 encodes a 68 amino acid micropeptide that is mainly localized at the perinuclear region. Linc013026-68AA is expressed in a subset of HCC cells and plays a role in cell proliferation, suggesting that Linc013026-68AA may be used as a HCC-specific target molecule. Our finding also sheds light on the role of the previously ignored 'dark proteome', that originates from noncoding regions in the maintenance of cancer.

Myc/Max dependent intronic long antisense noncoding RNA, EVA1A-AS, suppresses the expression of Myc/Max dependent anti-proliferating gene EVA1A in a U2 dependent manner.

The Myc gene has been implicated in the pathogenesis of most types of human cancerous tumors. Myc/Max activates large numbers of pro-tumor genes; however it also induces anti-proliferation genes. When anti-proliferation genes are activated by Myc, cancer cells can only survive if they are downregulated. Hepatocellular carcinoma (HCC) specific intronic long noncoding antisense (lnc-AS) RNA, the EVA1A-AS gene, is located within the second intron (I2) of the EVA1A gene (EVA-1 homolog A) that encodes an anti-proliferation factor. Indeed, EVA1A, but not EVA1A-AS, is expressed in normal liver. Depletion of EVA1A-AS suppressed cell proliferation of HepG2 cells by upregulation of EVA1A. Overexpression of EVA1A caused cell death at the G2/M phase via microtubule catastrophe. Furthermore, suppressed EVA1A expression levels are negatively correlated with differentiation grade in 365 primary HCCs, while EVA1A-AS expression levels are positively correlated with patient survival. Notably, both EVA1A and EVA1A-AS were activated by the Myc/Max complex. Eva1A-AS is transcribed in the opposite direction near the 3'splice site of EVA1A I2. The second intron did not splice out in a U2 dependent manner and EVA1A mRNA is not exported. Thus, the Myc/Max dependent anti-proliferating gene, EVA1A, is controlled by Myc/Max dependent anti-sense noncoding RNA for HCC survival.

Colony-stimulating factor-1 receptor provides a growth advantage in epithelial cancer cell line A431 in the presence of epidermal growth factor receptor inhibitor gefitinib.

Although epidermal growth factor receptor (EGFR) has been identified as a potent "oncogenic driver" in various tumors of epithelial origin, EGFR-targeted therapies are often of limited success. One of the challenges of improving targeted therapies is to overcome bypassing signaling pathways. Analysis of RNA-seq data of 1006 cell lines from the Cancer Cell Line Encyclopedia (CCLE) revealed that more than 12% of carcinoma cell lines expressed markedly elevated mRNA levels of colony-stimulating factor (CSF)-1 receptor (CSF-1R). Since epithelial cells also express CSF-1, elevated levels of CSF-1R may participate in providing alternative growth and survival signals under targeted therapies. To address this question, we ectopically expressed CSF-1R in A431 cells that express EGFR at high levels, but no biologically relevant level of CSF-1R. In the presence of EGFR inhibitor gefitinib, CSF-1R provided a significant growth advantage in A431 cells. As expected, activation of both receptors, EGFR or CSF-1R, induced phosphorylation of extracellular signal-regulated kinase (Erk)1/2, Akt, protein kinase C (PKC) and signal transducer and activator of transcription (STAT)3. However, EGFR, but not CSF-1R, also induced STAT5 phosphorylation. Inhibitor of phosphatidylinositol 3-kinase (PI3K) (AZD8186), MAPK/ERK kinase (MEK)1/2 (U0126), PKCs (Bisindolylmaleimide I or Gö6976) or STAT3 (Stattic) partially reduced proliferation of CSF-1R expressing A431 cells in the presence of gefitinib. Moreover, multi-kinase inhibitor, cabozantinib, suppressed CSF-1R activation and drastically reduced cell growth when combined with gefitinib. These data suggest that CSF-1R has the potential to reduce sensitivity to gefitinib and may be involved in resistance development.

Treatment with MAPKAP2 (MK2) inhibitor and DNA methylation inhibitor, 5-aza dC, synergistically triggers apoptosis in hepatocellular carcinoma (HCC) via tristetraprolin (TTP).

Over 100 putative driver genes that are associated with multiple recurrently altered pathways were detected in hepatocellular carcinoma (HCC), suggesting that multiple pathways will need to be inhibited for any therapeutic method to be effective. In this context, functional modification of the RNA regulating protein, tristetraprolin (TTP) that regulates approximately 2500 genes represents a promising strategy in HCC therapy. Since overexpression of TTP induces cell death in all cell types, it would be useful to target the regulator of TTP. In this study, we applied an inhibitor to MAPKAP2 (MK2) that suppresses TTP function. Importantly, cBIOportal for HCC genomics shows that expression level of the MK2 gene correlates with clinical outcome of HCC. We show that upon treatment with MK2 inhibitor, all 5 HCC cell lines, namely HepG2, Huh7, Hep3B, HLE and HLF, reduced cell growth, especially HepG2 and Hep3B cells underwent apoptosis. Simultaneously, TTP target genes such as c-Myc, IER3 or AKT-1 were downregulated. Depletion of the TTP gene rescued cells from apoptosis and restored the TTP-target mRNA expression in the presence of MK2 inhibitor. Furthermore, MK2 was activated in primary HCC that express TTP at high level. The TTP gene was induced upon treatment with DNA methylation inhibitor, 5-aza dC or interferon in three other cell lines, Huh7, HLE or HLF. Upon treatment with MK2 inhibitor and 5-aza dC or interferon these cells underwent apoptosis. The depletion of TTP in these cells partially rescued them from apoptosis, suggesting that the MK2/TTP pathway plays a role in proliferation and maintenance of HCCs. Notably, under the same conditions human hepatocyte cells (THLE-2) did not undergo apoptosis. These data also suggest that MK2 inhibitor with 5-aza dC or interferon may be a useful tool for therapy against HCC.

Extracellular-signal regulated kinase (Erk1/2), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and tristetraprolin (TTP) comprehensively regulate injury-induced immediate early gene (IEG) response in in vitro liver organ culture.

Differentiated hepatocytes are long-lived and normally do not undergo cell division, however they have the unique capacity to autonomously decide their replication fate after liver injury. In this context, the key players of liver regeneration immediately after injury have not been adequately studied. Using an in vitro liver culture system, we show that after liver injury, p38 mitogen-activated protein kinase (p38MAPK), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and extracellular-signal regulated kinase (Erk)1/2 were activated within 15 min and continued to be phosphorylated for more than 2h. Both p38MAPK and Erk1/2 were activated at the edge of the cut as well as on the liver surface where the mesothelial cell sheet expresses several cytokines. Notably, in human liver Erk1/2 was also activated under the mesothelial cell sheet shortly after liver resections. Furthermore, in in vitro liver slice culture immediate early genes (IEGs) were upregulated within 1-2 h and the S phase marker proliferation-cell-nuclear-antigen (PCNA) appeared 24 h after injury. Although Erk1/2 was activated after injury, in MK2 depleted liver a set of IEGs, such as Dusp1, Cox2, or c-Myc and proliferation marker gene Ki67 were not induced. In addition, in immortalized hepatocyte cells, THLE-2, the same subset of genes was upregulated upon stimulation with lipopolysaccharide (LPS), but not in the presence of MK2 inhibitor. The protein level of tristetraprolin (TTP), a substrate for MK2 that plays a role in mRNA degradation, was increased in the presence of MK2 inhibitor. In this context, the depletion of TTP gene rescued Dusp1, Cox2, or c-Myc upregulation in the presence of MK2 inhibitor. These data imply that MK2 pathway is positively involved in Erk1/2 induced IEG response after liver injury. These data also suggest that in vitro liver culture may be a useful tool for measuring the proliferation potential of hepatocytes in individual liver.

mRNA export protein THOC5 as a tool for identification of target genes for cancer therapy.

Recent evidence indicates that mRNA export is selective, giving priority to a subset of mRNAs that control diverse biological processes including cell proliferation, differentiation, stress response, and cell survival as well as tumor development. The depletion of a member of the mRNA export complex, the THO complex, impairs the expression of only a subset of genes, but causes dramatic changes in phenotype, such as cell cycle inhibition, abnormal differentiation, and importantly apoptosis of stem cells and cancer cells but not normal epithelial cells, hepatocytes, or fibroblasts. Recent exosome sequence data revealed that over 100 driver gene mutations with a number of signaling pathways are involved in human cancer formation, indicating that multiple signaling pathways will need to be inhibited for cancer therapy. In this review we firstly describe a basic feature and function of the mRNA export complex, THO, secondly, the biological alteration upon depletion of a member of the THO complex in normal and cancer cells, and thirdly, identification of its target genes. Finally we describe our recent data on selection of targeting candidates from THOC5 dependent genes for application in cancer therapy.

Murine precision-cut liver slices (PCLS): a new tool for studying tumor microenvironments and cell signaling ex vivo.

BACKGROUND: One of the most insidious characteristics of cancer is its spread to and ability to compromise distant organs via the complex process of metastasis. Communication between cancer cells and organ-resident cells via cytokines/chemokines and direct cell-cell contacts are key steps for survival, proliferation and invasion of metastasized cancer cells in organs. Precision-cut liver slices (PCLS) are considered to closely reflect the in vivo situation and are potentially useful for studying the interaction of cancer cells with liver-resident cells as well as being a potentially useful tool for screening anti-cancer reagents. Application of the PCLS technique in the field of cancer research however, has not yet been well developed. RESULTS: We established the mouse PCLS system using perfluorodecalin (PFD) as an artificial oxygen carrier. Using this system we show that the adherence of green fluorescent protein (GFP) labeled MDA-MB-231 (highly invasive) cells to liver tissue in the PCLS was 5-fold greater than that of SK-BR-3 (less invasive) cells. In addition, we generated PCLS from THOC5, a member of transcription/export complex (TREX), knockout (KO) mice. The PCLS still expressed Gapdh or Albumin mRNAs at normal levels, while several chemokine/growth factor or metalloprotease genes, such as Cxcl12, Pdgfa, Tgfb, Wnt11, and Mmp1a genes were downregulated more than 2-fold. Interestingly, adhesion of cancer cells to THOC5 KO liver slices was far less (greater than 80% reduction) than to wild-type liver slices. CONCLUSION: Mouse PCLS cultures in the presence of PFD may serve as a useful tool for screening local adherence and invasiveness of individual cancer cells, since single cells can be observed. This method may also prove useful for identification of genes in liver-resident cells that support cancer invasion by using PCLS from transgenic liver.

THOC5 controls 3'end-processing of immediate early genes via interaction with polyadenylation specific factor 100 (CPSF100).

Transcription of immediate early genes (IEGs) in response to extrinsic and intrinsic signals is tightly regulated at multiple stages. It is known that untranslated regions of the RNA can play a role in these processes. Here we show that THOC5, a member of the TREX (transcription/export) complex, plays a role in expression of only a subset of constitutively active genes, however transcriptome analysis reveals that more than 90% of IEG were not induced by serum in THOC5 depleted cells. Furthermore, THOC5 depletion does not influence the expression of the most rapidly induced IEGs, e.g. Fos and Jun. One group of THOC5 target genes, including Id1, Id3 and Wnt11 transcripts, were not released from chromatin in THOC5 depleted cells. Genes in another group, including Myc and Smad7 transcripts, were released with shortening of 3'UTR by alternative cleavage, and were spliced but export was impaired in THOC5 depleted cells. By interactome analysis using THOC5 as bait, we show that upon stimulation with serum THOC5 forms a complex with polyadenylation-specific factor 100 (CPSF100). THOC5 is required for recruitment of CPSF100 to 3'UTR of THOC5 target genes. These data suggest the presence of a novel mechanism for the control of IEG response by THOC5 via 3'end-processing.

An ataxia-telangiectasia-mutated (ATM) kinase mediated response to DNA damage down-regulates the mRNA-binding potential of THOC5.

In response to DNA damage, transcription is blocked by inhibition of RNA polymerase II activity. The regulation of a preexisting pool of mRNAs, therefore, plays a key role in DNA repair, cell cycle arrest, or inhibition of differentiation. THOC5 is a member of the THO complex and plays a role in the export of a subset of mRNA, which plays an important role in hematopoiesis and maintaining primitive cells. Since three serine residues in the PEST domain of THOC5 have been shown to be directly phosphorylated by ataxia-telangiectasia-mutated (ATM) kinase, we examined the THOC5-dependent mRNA export under DNA damage. We show here that DNA damage drastically decreased the cytoplasmic pool of a set of THOC5-dependent mRNAs and impaired the THOC5/mRNA complex formation. The mRNP complex formed with nonphosphorylation mutant (S307/312/314A) THOC5, but not with a C-terminal deletion mutant after DNA damage, suggesting that the C-terminal domain of THOC5, but not its phosphorylation in the PEST domain, is necessary for the regulation of the mRNA-binding potency of THOC5. The cytoplasmic THOC5-dependent mRNAs were recovered by treatment with ATM kinase-specific or p53-specific siRNA, as well as by treatment with ATM kinase inhibitor, KU55933, under DNA damage conditions, suggesting that the ATM-kinase-p53 pathway is involved in this response to the DNA damage. Furthermore, the treatment with KU55933 blocked DNA damage-induced THOC5mRNP complex dissociation, indicating that activation of ATM kinase suppresses the ability of THOC5 to bind to its target mRNAs.

Identification of mRNAs that are spliced but not exported to the cytoplasm in the absence of THOC5 in mouse embryo fibroblasts.

The TREX (transcription/export) complex has been conserved throughout evolution from yeast to man and is required for coupled transcription elongation and nuclear export of mRNAs. The TREX complex in mammals and Drosophila is composed of the THO subcomplex (THOC1, THOC2, THOC5, THOC6, and THOC7), THOC3, UAP56, and Aly/THOC4. In human and Drosophila, various studies have shown that THO is required for the export of heat shock mRNAs, but nothing is known about other mRNAs. Our previous study using conditional THOC5 (or FMIP) knockout mice revealed that the presence of THOC5 is critical in hematopoietic cells but not for terminally differentiated cells. In this study, we describe the establishment of a mouse embryo fibroblast cell line (MEF), THOC5 flox/flox. Four days after infection of MEF THOC5 flox/flox with adenovirus carrying Cre-recombinase gene (Ad-GFP-Cre), THOC5 is down-regulated >95% at the protein level, and cell growth is strongly suppressed. Transcriptome analysis using cytoplasmic RNA isolated from cells lacking functional THOC5 reveals that only 2.9% of all genes were down-regulated more than twofold. Although we examined these genes in fibroblasts, one-fifth of all down-regulated genes (including HoxB3 and polycomb CBX2) are known to play a key role in hematopoietic development. We further identified 10 genes that are spliced but not exported to the cytoplasm in the absence of THOC5. These mRNAs were copurified with THOC5. Furthermore, Hsp70 mRNA was exported in the absence of THOC5 at 37°C, but not under heat shock condition (42°C), suggesting that THOC5 may be required for mRNA export under stress and/or upon signaling-induced conditions.