Identification of Intermediate-Size Non-Coding RNAs Involved in the UV-Induced DNA Damage Response in <em>C. elegans</em>

<div><h3>Background</h3><p>A network of DNA damage response (DDR) mechanisms functions coordinately to maintain genome integrity and prevent disease. The Nucleotide Excision Repair (NER) pathway is known to function in the response to UV-induced DNA damage. Although numbers of coding genes and miRNAs have been identified and reported to participate in UV-induced DNA damage response (UV-DDR), the precise role of non-coding RNAs (ncRNAs) in UV-DDR remains largely unknown.</p> <h3>Methodology/Principal Findings</h3><p>We used high-throughput RNA-sequencing (RNA-Seq) to discover intermediate-size (70–500 nt) ncRNAs (is-ncRNAs) in C. elegans, using the strains of L4 larvae of wild-type (N2), UV-irradiated (N2/UV100) and NER-deficient mutant (<em>xpa-1</em>), and 450 novel non-coding transcripts were initially identified. A customized microarray assay was then applied to examine the expression profiles of both novel transcripts and known is-ncRNAs, and 57 UV-DDR-related is-ncRNA candidates showed expression variations at different levels between UV irradiated strains and non- irradiated strains. The top ranked is-ncRNA candidates with expression differences were further validated by qRT-PCR analysis, of them, 8 novel is-ncRNAs were significantly up-regulated after UV irradiation. Knockdown of two novel is-ncRNAs, ncRNA317 and ncRNA415, by RNA interference, resulted in higher UV sensitivity and significantly decreased expression of NER-related genes in <em>C. elegans</em>.</p> <h3>Conclusions/Significance</h3><p>The discovery of above two novel is-ncRNAs in this study indicated the functional roles of is-ncRNAs in the regulation of UV-DDR network, and aided our understanding of the significance of ncRNA involvement in the UV-induced DNA damage response.</p> </div

qRT-PCR-validated differential expression levels of UV-DDR-related is-ncRNA candidates.

<p>The expression levels of UV-DDR-related is-ncRNA candidates were examined by qRT-PCR in N2 and <i>xpa-1</i> deletion mutant before and after UV irradiation (100 J/m²). Results were normalized to the expression level of U6 and compared with the level of the wild type without UV irradiation. Data presented are means ± SEM of at least three independent experiments. *: p<0.05, **: p<0.01, ***: p<0.001.</p

Summary of mRNA genes analysed by qRT-PCR.

<p>Summary of mRNA genes analysed by qRT-PCR.</p

Functional Characterization of Long Noncoding RNA Lnc_bc060912 in Human Lung Carcinoma Cells

Long noncoding RNAs (lncRNAs) are pervasively transcribed in the human genome. Recent studies suggest that the involvement of lncRNAs in human diseases could be far more prevalent than previously appreciated. Here we have identified a lncRNA termed Lnc_bc060912 whose expression is increased in human lung and other tumors. Lnc_bc060912 is 1.2 kb in length and is composed of two exons. The expression of Lnc_bc060912 was repressed by p53. Lnc_bc060912 suppressed cell apoptosis. Using a recently developed method for RNA-pulldown with formaldehyde cross-linking, we found that Lnc_bc060912 interacted with the two DNA damage repair proteins PARP1 and NPM1. Together, these results suggest that Lnc_bc060912, via PARP1 and NPM1, affects cell apoptosis and may play important roles in tumorigenesis and cancer progression

Three is-ncRNAs contributing to UV resistance.

<p>Candidate ncRNAs were knocked down by RNAi via feeding in wild-type worms. A, L4 larval survival following UV irradiation. Worms of L4 stage larvae were exposed to UV irradiation (0, 50, 100, 150, or 200 J/m²). Worm survival was determined about 24 h after UV treatment. Each point represents the mean of three independent experiments, each performed in duplicate (typically, n>60). Error bars denote the SEM. B, Effects of target is-ncRNAs knockdown on UV-DDR-related genes. Quantitative RT-PCR was used to detect the relative expression level of UV-DDR-related genes in RNAi-treated adult worms. Error bars show the mean ± SEM of at least three independent experiments. *: p<0.05, **: p<0.01, ***: p<0.001.</p

Characteristics of UV-DDR-related is-ncRNAs.

<p>A, Coding potential. Coding potentials of known is-ncRNAs, UV-DDR-related is-ncRNAs and 570 randomly selected mRNAs were analysed using CPC. B, Conservation. Conservation of the genomic transcript sequence for repeat sequences, exons, introns, known is-ncRNAs and 57 UV-DDR-related ncRNAs (UV-ncRNA). C, Conservation and location of the three is-ncRNAs. RefSeq genes are in blue and ncRNAs are in gray. Arrows indicate the direction of transcription. The extent of conservation varies with genomic location and is based on PhastCons scores in UCSC. ncRNA317 is located antisense to the intron of protein-coding gene <i>Y60C6A.1.</i> ncRNA415 is an intergenic transcript. Based on our sequencing and 3′ RACE results, the 5′- and 3′-end of ncRNA415 was extended 22 nt and 74 nt, respectively, as shown. ncRNA341 comprises 3 exons, which are located at positions equivalent to the deeply conserved regions of RefSeq gene <i>F35E12.2</i>.</p