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Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells.

ABSTRACT: Long noncoding RNAs (lncRNAs), which have emerged in recent years as a new and crucial layer of gene regulators, regulate various biological processes such as carcinogenesis and metastasis. HOTAIR (Hox transcript antisense intergenic RNA), a lncRNA overexpressed in most human cancers, has been shown to be an oncogenic lncRNA. Here, we explored the role of HOTAIR in HeLa cells and searched for proteins regulated by HOTAIR. To understand the mechanism of action of HOTAIR from a systems perspective, we employed a quantitative proteomic strategy to systematically identify potential targets of HOTAIR. The expression of 170 proteins was significantly dys-regulated after inhibition of HOTAIR, implying that they could be potential targets of HOTAIR. Analysis of this data at the systems level revealed major changes in proteins involved in diverse cellular components, including the cytoskeleton and the respiratory chain. Further functional studies on vimentin (VIM), a key protein involved in the cytoskeleton, revealed that HOTAIR exerts its effects on migration and invasion of HeLa cells, at least in part, through the regulation of VIM expression. Inhibition of HOTAIR leads to mitochondrial dysfunction and ultrastructural alterations, suggesting a novel role of HOTAIR in maintaining mitochondrial function in cancer cells. Our results provide novel insights into the mechanisms underlying the function of HOTAIR in cancer cells. We expect that the methods used in this study will become an integral part of functional studies of lncRNAs.

SUBMITTER: Zheng P

PROVIDER: S-EPMC4458712 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells.

Zheng Peng P Xiong Qian Q Wu Ying Y Chen Ying Y Chen Zhuo Z Fleming Joy J Gao Ding D Bi Lijun L Ge Feng F

Molecular & cellular proteomics : MCP 20150311 6

Long noncoding RNAs (lncRNAs), which have emerged in recent years as a new and crucial layer of gene regulators, regulate various biological processes such as carcinogenesis and metastasis. HOTAIR (Hox transcript antisense intergenic RNA), a lncRNA overexpressed in most human cancers, has been shown to be an oncogenic lncRNA. Here, we explored the role of HOTAIR in HeLa cells and searched for proteins regulated by HOTAIR. To understand the mechanism of action of HOTAIR from a systems perspective ...[more]

PMID: 25762744

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Project description:UnlabelledChronic hepatitis B virus (HBV) infection is a major factor in hepatocellular carcinoma (HCC) pathogenesis by a mechanism not yet understood. Elucidating mechanisms of HBV-mediated hepatocarcinogenesis is needed to gain insights into classification and treatment of HCC. In HBV replicating cells, including virus-associated HCCs, suppressor of zeste 12 homolog (SUZ12), a core subunit of Polycomb repressive complex2 (PRC2), undergoes proteasomal degradation. This process requires the long noncoding RNA, Hox transcript antisense intergenic RNA (HOTAIR). Intriguingly, HOTAIR interacts with PRC2 and also binds RNA-binding E3 ligases, serving as a ubiquitination scaffold. Herein, we identified the RNA helicase, DEAD box protein 5 (DDX5), as a regulator of SUZ12 stability and PRC2-mediated gene repression, acting by regulating RNA-protein complexes formed with HOTAIR. Specifically, knockdown of DDX5 and/or HOTAIR enabled reexpression of PRC2-repressed genes epithelial cell adhesion molecule (EpCAM) and pluripotency genes. Also, knockdown of DDX5 enhanced transcription from the HBV minichromosome. The helicase activity of DDX5 stabilized SUZ12- and PRC2-mediated gene silencing, by displacing the RNA-binding E3 ligase, Mex-3 RNA-binding family member B (Mex3b), from HOTAIR. Conversely, ectopic expression of Mex3b ubiquitinated SUZ12, displaced DDX5 from HOTAIR, and induced SUZ12 down-regulation. In G2 phase of cells expressing the HBV X protein (HBx), SUZ12 preferentially associated with Mex3b, but not DDX5, resulting in de-repression of PRC2 targets, including EpCAM and pluripotency genes. Significantly, liver tumors from HBx/c-myc bitransgenic mice and chronically HBV-infected patients exhibited a strong negative correlation between DDX5 messenger RNA levels, pluripotency gene expression, and liver tumor differentiation. Notably, chronically infected HBV patients with HCC expressing reduced DDX5 exhibited poor prognosis after tumor resection, identifying DDX5 as an important player in poor prognosis HCC.ConclusionThe RNA helicase DDX5, and E3 ligase Mex3b, are important cellular targets for the design of novel, epigenetic therapies to combat HBV infection and poor prognosis HBV-associated liver cancer. (Hepatology 2016;64:1033-1048).

Project description:The regulation of latency is central to herpesvirus biology. Recent transcriptome-wide surveys have uncovered evidence for promiscuous transcription across the entirety of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome and postulated the existence of multiple viral long noncoding RNAs (lncRNAs). Next-generation sequencing studies are highly dependent on the specific experimental approach and particular algorithms of analysis and therefore benefit from independent confirmation of the results. The antisense-to-latency transcript (ALT) lncRNA was discovered by genome-tiling microarray (Chandriani et al., J Virol 86:7934-7942, 2010, https://doi.org/10.1128/JVI.00645-10). To characterize ALT in detail, we physically isolated this lncRNA by a strand-specific hybrid capture assay and then employed transcriptome sequencing and novel reverse transcription-PCR (RT-PCR) assays to distinguish all RNA species in the KSHV latency region. These methods confirm that ALT initiates at positions 120739/121012 and encodes a single splice site, which is shared with the 3'-coterminal K14-vGPCR/ORF74 mRNA, terminating at 130873 (GenBank accession number GQ994935), resulting in an ∼10,000-nucleotide transcript. No shorter ALT isoforms were identified. This study also identified a novel intron within the LANA 5' untranslated region using a splice acceptor at 127888. In summary, ALT joins PAN/nut1/T1.1 as a bona fide lncRNA of KSHV with potentially important roles in viral gene regulation and pathogenesis. Increasing data support the importance of noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and lncRNAs, which have been shown to exert critical regulatory functions without coding for recognizable proteins. Defining the sequences of these ncRNAs is essential for future studies aiming to functionally characterize a specific ncRNA. Most lncRNA studies are highly dependent on high-throughput sequencing and bioinformatic analyses, few studies follow up on the initial predictions, and analyses are at times discordant. The manuscript characterizes one key viral lncRNA, ALT, by physically isolating ALT and by a sequencing-independent assay. It provides for a simple assay to monitor lncRNA expression in experimental and clinical samples. ALT is expressed antisense to the major viral latency transcripts encoding LANA as well as the viral miRNAs and thus has the potential to regulate this key part of the viral life cycle.

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Dataset Information

Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells.

Publications

Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells.

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