Promoter-Associated RNAs Regulate HSPC152 Gene Expression in Malignant Melanoma

Hamutal Bonen,Raya Leibowitz-Amit,Thomas Lorber,Dror Avni,Yechezkel Sidi,Noam Shomron,Nitzan Kol,Luca Quagliata

doi:10.3390/ncrna2030007

Hamutal Bonen, Raya Leibowitz-Amit + Show 6 more

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https://doi.org/10.3390/ncrna2030007

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Abstract

The threshold of 200 nucleotides (nt) conventionally divides non-coding RNAs (ncRNA) into long ncRNA (lincRNA, that have more than 200 nt in length) and the remaining ones which are grouped as “small” RNAs (microRNAs, small nucleolar RNAs and piwiRNAs). Promoter-associated RNAs (paRNAs) are generally 200–500 nt long and are transcribed from sequences positioned in the promoter regions of genes. Growing evidence suggests that paRNAs play a crucial role in controlling gene transcription. Here, we used deep sequencing to identify paRNA sequences that show altered expression in a melanoma cell line compared to normal melanocytes. Thousands of reads were mapped to transcription start site (TSS) regions. We limited our search to paRNAs adjacent to genes with an expression that differed between melanoma and normal melanocytes and a length of 200–500 nt that did not overlap the gene mRNA by more than 300 nt, ultimately leaving us with 11 such transcripts. Using quantitative real-time PCR (qRT-PCR), we found a significant correlation between the expression of the mRNA and its corresponding paRNA for two studied genes: TYR and HSPC152. Ectopic overexpression of the paRNA of HSPC152 (designated paHSPC) enhanced the expression of the HSPC152 mRNA, and an siRNA targeting the paHSPC152 decreased the expression of the HSPC152 mRNA. Overexpression of paHSPC also affected the epigenetic structure of its putative promoter region along with effects on several biologic features of melanoma cells. The ectopic expression of the paRNA to TYR did not have any effect. Overall, our work indicates that paRNAs may serve as an additional layer in the regulation of gene expression in melanoma, thus meriting further investigation.

Highlights

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]
Our first aim was to search for Promoter-associated RNAs (paRNAs) which are differentially expressed in melanoma in comparison to normal melanocytes
Since the expected amount of paRNA is low compared to total cellular RNA and since we did not know whether these paRNAs contain a poly-A tail, we decided to enrich these RNAs by depletion of the rRNA using the Ribo-Zero (Illumina, San Diego, CA, USA) rRNA Removal Core Kit

Summary

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). Longer RNAs are considered to be long ncRNAs (lincRNA), and shorter RNAs are considered short ncRNA Promoter-associated RNAs (paRNAs) are generally 200–500 nt long. Napoli et al showed the presence of low copy RNA transcripts in the region from400 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.

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