Ncrna Classification Essay

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1. Introduction

The human genome project revealed that approximately 90% of the sequence is actively transcribed, but only 1%–1.5% is translated to protein products [1]. The transcriptional products which are not transcribed are referred to as non-coding RNAs (ncRNA). The most commonly used classification is based on RNA length: the dividing line is set at 200 nucleotides (nt). Longer RNAs are considered to be long ncRNAs (lincRNA), and shorter RNAs are considered short ncRNA (microRNAs, small nucleolar RNAs and piwi-interacting RNA) [2,3]. Promoter-associated RNAs (paRNAs) are generally 200–500 nt long. paRNAs were found in yeast and Arabidopsis and span the size of ≈250–500 nucleotides (nt) [3,4]. Napoli et al. showed the presence of low copy RNA transcripts in the region from −400 to +120 (520 nt) relative to the transcription start site (TSS) of the c-myc gene [5]. Seila et al. showed divergent transcription around active promoters and active TSS, both in abundance and size. The low abundance RNA are around 500 nt [6]. They are transcribed from sequences positioned in the promoter regions of genes. Those lncRNAs were first identified by Han et al. [7] and described as “sense-stranded RNA transcripts corresponding to the known promoter region” that may serve as a target for siRNAs targeting promoter regions and inducing transcriptional gene silencing.

A second and potentially overlapping class of paRNAs are transcription start site-associated RNAs, that are 20–90 nt long and localized within −250 to +50 of TSSs. A third class of paRNA are transcription initiation RNAs which are 18 nt in length and have their highest density just downstream of TSSs [3,4,8]. LincRNAs are known to have dynamic expression patterns in different cell types, tissues and differentiation stages [9]. These transcripts appear in low copy number per cell, are often poorly conserved throughout evolution and are very unstable [1]. Their functional importance is far from being understood, but an increasing number of studies have shown their ability to regulate diverse functions such as X chromosome silencing [10], pluripotency [11] and epigenetic gene regulation [12]. LincRNAs can be classified, based on their genomic position in relation to protein-coding genes, as intronic or intergenic and also in accordance with their orientation (in respect to protein-coding transcripts) as sense or antisense [13].

PaRNAs are lincRNAs with sequence complementarity to parts of gene promoters. A few studies suggested that paRNAs promote silencing of gene transcription from their cognate promoter [7,14,15], whereas only one work suggested that paRNA promotes transcription of the c-myc gene [5]. Recent studies have found that paRNAs serve as scaffolds for antisense transcripts that regulate gene transcription as reviewed in [8,16]. In this current research, we set out to identify and isolate new paRNAs from an in vitro model of melanocyte melanoma and assess the association between paRNA expression and transcription of the cognate gene.

2. Materials and Methods

2.1. Cell Lines and Melanoma Biopsies

Melanoma cell lines were generated directly from metastatic melanoma lesions of patients at the surgical branch of the National Institute of Health (NIH, Bethesda, MD, USA) (mel526, mel624, mel33B1 [14,15]) or at the Ella Institute for Melanoma Research at the Sheba Medical Center (Tel-Hashomer, Israel) (014mel, 15AY) [15]. MNT-1 cell lines were generously given by Dr. Patrizio Giacomini of the immunology lab at the “Regina Elena” National Cancer institute Rome, Italy [16].

Melanoma lines were grown in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum (FBS), 1% l-glutamate, 1% penicillin-streptomycin mixture (P/S mix, full medium) and 2.5% HEPES solution. All materials were purchased from Biological Industries Ltd. (Beit Haemek, Israel).

Three different batches of normal human epidermal melanocytes (NHEM) cell line were purchased from PromoCell (c-12400; PromoCell, Heidelberg, Germany) and grown in Melanocyte Growth Medium containing 0.6% supplement mix (PromoCell, cat No. c-24010 and c-39415, respectively), and 1% P/S mix.

MelST, melST-R and melST-M-transformed melanocytes [17] were generously donated by Dr. Robert Weinberg’s lab (Whitehead Institute for Biomedical research Cambridge, MA, USA) and were grown in full medium. In brief, the melST line, immortalized but non-malignant melanocytes, was developed through the transfection of NHEM with the SV40ER and hTERT cDNA. The two additional melST sub-lines were created using the RAS protein (melST-R) or the active form of the c-Met receptor (melST-M) and both demonstrate a fully transformed malignant phenotype [17].

All cell lines were grown at 37 °C, 8% CO2 and 99% humidity.

All of the biopsy samples were a gift from Dr. Luca Quagliata, at the Molecular Pathology Unit, Institute of Pathology, University Hospital Basel, Switzerland.

The research use of the clinical specimens was approved (N.310/10) by the ethical committee of the University Hospital of Basel, Switzerland. RNA samples were sent to us on dried ice and incubated at −80 °C until used.

2.2. RNA Purification and Enhancement

Total RNA was extracted from cells using the Total RNA Purification Kit (cat# 17200, Norgen Biotek Corp, Thorold, ON, Canada). For the deep sequencing project and for paRNA detection, cells were harvested using Trizol® reagent (InvitrogenTM, Thermo-Fisher Scientific Inc., Waltham, MA, USA). All purifications were performed according to the manufacturer’s protocols. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the PrimeScriptTM RT reagent Kit (cat. No. RR037, TaKaRa Bio Inc., Otsu, Japan), using both random hexamer and OligodT. The SYBR Green method was used to detect genes’ mRNA and paRNAs. An amount of 1 µL or 2 µL (gene expression or paRNA expression, respectively) of RT product was amplified in a 96-well plate containing 5 µL Power SYBR® Green Master mix (Applied Biosystems Inc., Foster City, CA, USA), and 5 pmol primers. Plates were incubated on the ABI 7900HT thermocycler (Applied Biosystems Inc., Foster City, CA, USA) for 45 cycles. Primers sequences are detailed in Table 1.

For mRNA-paRNA detection and correlation assay, 600 ng of DNA-free RNA (cleaned with the Turbo DNA-free Kit, (Ambion Thermo-Fisher Scientific Inc., Waltham, MA, USA) were used. Using the same threshold for all runs for each sample, the average cycle threshold (CT) was calculated for at least three different runs, and the correlation between the gene’s mRNA and paRNA was measured. On the mRNA assay alone, different samples were normalized using the RPLPO gene.

2.3. Deep Sequencing (NGS)

All the procedures for the deep sequencing (deep-seq) (also known as Next Generation Sequencing, or NGS) were performed by the Functional Genomics Laboratory, at Tel-Aviv University, Tel-Aviv, Israel.

Total RNA from NHEM cells and 014mel melanoma cell line were used for deep-seq. The first enrichment for potential paRNA was performed by leaving out RNAs corresponding to rRNAs and tRNA. Then, by size separation using gel electrophoresis, only RNAs corresponding to 200–500 nt were extracted for further treatment and analysis. Illumina’s Directional mRNA-Seq Sample Preparation protocol was used with one adjustment: After the RNA gel extraction, neither RNA purification (Poly-A “fishing”) nor RNA fragmentation were used and samples were directly treated with polynucleotide Kinase and Antarctic Phosphatase. Quality control analysis was performed on all libraries using Agilent Technologies 2100 Bioanalyzer (Santa Clara, CA, USA).

2.4. Cloning and Plasmids

PTER+ plasmid was used for all cloning [18], except for paTYR sense, where pcDNA3 plasmid was used. Genomic fragments matching the paRNAs were amplified by PCR, using the PhusionTM master mix (cat# F-531S, Finnzymes OY, 02150 Espoo, Finland) with 10 pmol of the following primers:
  • HSPC152 F-5′-CGACGGTGTTAGGCGC 3′

  • HSPC152 R-5′-CGGGTACCTGGAGGCG 3′

  • TYR F-5′-CATTTGCAAGGTCAAATCATC 3′

  • TYR R-5′-AGTACAAAACAGCCAGGAGC 3′

PCR fragments were cloned using the TOPO TA Cloning Kit into pCRII-TOPO plasmid (cat# 450640, InvitrogenTM). For sense-oriented paHSPC, EcoRV and HindIII restriction enzymes were used to clone the segment into pTER plasmid (cat# R01955, R01045, respectively). EcoRV and BamHI (cat# R01365) restriction enzymes were used for antisense-oriented paHSPC and paTYR, and sense-oriented paTYR was cut from TOPO paTYR using EcoRI restriction enzyme (cat# R0101S) and cloned into pcDNA3 plasmid. TYR sense/antisense orientation was determined using sequencing with pcDNA3.1-F primer (5′-CTCTGGCTAACTAGAGAAC-3′, Hylab Ltd., Rehovot, Israel). All enzymes were purchased from New England Biolabs® Inc. (Ipswich, MA, USA).

2.5. siRNA Transfection

The siRNA primer was 5′-ACACAUUUUACUCCUACACAGGCdTdT-3′ and was ordered from Sigma-Aldrich Israel Ltd. (Rehovot, Israel). Transfection of siRNA was performed with X-tremeGENE Transfection Reagent (Roche, CH-4070, Basel, Switzerland).

2.6. Plasmid Transfection

All plasmid transfections were performed using Lipofectamin 2000 reagent by InvitrogenTM, according to the manufacturer′s protocols. For 24 h prior to transfection, cells were seeded in 6-well plates and grown to 60% confluence. Cell growth medium was replaced shortly before transfection to cell growth medium without P/S mix. Four hours post-transfection, the medium was replaced with full growth medium. Incubation time was set on 0–72 h, depending on the assay performed.

Melanoma 014mel sub-lines containing sense or antisense paRNA were created using 300 µg/mL of the selective antibiotic Zeocin or 2 mg/mL neomycin.

2.7. Chromatin-Immunoprecipitation (ChIP) Assay

The 014mel melanoma cells, stably expressing sense or antisense paRNA, were subjected to a ChIP assay using a modified protocol as previously described [18]. In short, cells were seeded in 145 × 20 mm plates and grown to 80%–90% confluence. Chromatin cross-linking was performed using 1% formaldehyde followed by quenching with 0.125 M glycine. Cells were scraped from plates, washed and re-suspended in 1% sodium dodecyl sulfate (SDS)-containing lysis buffer. Lysed cells were sonicated with five sets of 10 s each, to produce DNA segments of ≈1000 bp. Subsequently, 20–25 µg of segmented DNA were taken for overnight immune precipitation at 4 °C using antibodies (Millipore, Billerica, MA, USA) for the three most common histone methylations: H3K4me3, H3K9me3 and H3K27me3, which characterize inactive chromatin. As a control, IgG antibodies were used.

Quantitative analysis was performed by real-time PCR with the same primers for the paRNA detection, using 014mel cells stably expressing an empty plasmid as control.

2.8. Cytosine Methylation

The 014mel cells stably expressing either empty plasmid or sense/antisense-oriented paHSPC plasmid were seeded in 6-well plates, in triplicate, and grown to ≈60% confluence. Cells were then harvested from the wells and DNA was purified using the ArchivePure DNA Cell/Tissue Kit (ref #2900267, 5 Prime Inc. Gaithersburg, MD, USA). After DNA purification, the amount of 500 ng was taken from each sample for bisulfite treatment using the EZ-DNA Methylation-Gold Kit (cat. No D5005, the Epigenetics Company, Zymo Research, Irvine, CA, USA) and 4 µL/tube from converted samples were taken for PCR amplification using two sets of primers:
  • BS F3: 5′-TTGTATATGATTTGTATTTTACGAAGAA

  • BS R4: 5′-AATAAAAAACCTATCGAATCACG

And,
  • BS F6: 5′-AGCGGAGGACGATTTTTT

  • BS R6: 5′-CATGCGAGCTCAGCAGA

For gel detection, 2 µL of PCR products were taken while the rest of the sample was cleaned using the GeneJET Gel Extraction and DNA Cleanup Micro Kit (cat. No K0831, Thermo Scientific Inc., Waltham, MA, USA). Clean PCR products were sent for sequencing analysis and paHSPC expressing a cell sequence was compared to control cells using the BioEdit alignment editor software (North Carolina State University, Ranleigh, NC, USA).

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