Identification and Characterization of Novel Genotoxic Stress-Inducible Nuclear Long Noncoding RNAs in Mammalian Cells

Whole transcriptome analyses have revealed a large number of novel transcripts including long and short noncoding RNAs (ncRNAs). Currently, there is great interest in characterizing the functions of the different classes of ncRNAs and their relevance to cellular processes. In particular, nuclear long ncRNAs may be involved in controlling various aspects of biological regulation, such as stress responses. By a combination of bioinformatic and experimental approaches, we identified 25 novel nuclear long ncRNAs from 6,088,565 full-length human cDNA sequences. Some nuclear long ncRNAs were conserved among vertebrates, whereas others were found only among primates. Expression profiling of the nuclear long ncRNAs in human tissues revealed that most were expressed ubiquitously. A subset of the identified nuclear long ncRNAs was induced by the genotoxic agents mitomycin C or doxorubicin, in HeLa Tet-off cells. There were no commonly altered nuclear long ncRNAs between mitomycin C- and doxorubicin-treated cells. These results suggest that distinct sets of nuclear long ncRNAs play roles in cellular defense mechanisms against specific genotoxic agents, and that particular long ncRNAs have the potential to be surrogate indicators of a specific cell stress

Strategy for the selection of ncRNAs.

<p>(A) A schematic of the selection procedure for ncRNAs. They were selected based on their lack of susceptibility to nonsense-mediated RNA decay (NMD) and nuclear localization. The identities of the transcripts in each category are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034949#pone-0034949-t001" target="_blank">Table 1</a>. (B) Schematic of aberrant mRNAs harboring a premature termination codon (PTC) and surveillance complexes containing UPF factors. EJC indicates an exon junction complex, which is an essential component for NMD in mammalian cells. (C) Details of the sub-cellular localization analysis of transcripts.</p

Expression of the genes neighboring stress-inducible nuclear long ncRNAs under MMC treatment.

<p>HeLa Tet-off (TO) cells were treated with MMC and subjected to qRT-PCR. (A) UT18-neighboring genes were co-regulated, whereas (B) UT6-neighboring genes were not. Error bars show the experimental error of two experiments.</p

Alteration of nuclear long ncRNA expression by anticancer agents in HeLa Tet-off (TO) cells.

<p>(A, B) HeLa Tet-off (TO) cells were treated with 20 µg/ml MMC (A), and 5 or 20 µg/ml MMC (B) and subjected to qRT-PCR. RNA levels were normalized to those of <i>β-actin</i>, and are presented relative to non-treated cells. <i>GADD45a</i> was used as a positive control induced by MMC. (C, D) HeLa Tet-off (TO) cells were treated with 1.0 nM DOX (C) and 0.5 or 1.0 nM DOX (D) and subjected to qRT-PCR. RNA levels were normalized to those of <i>GAPDH</i>, and are presented relative to non-treated cells (con). <i>p21</i> was used as a positive control induced by DOX. Error bars show the experimental error of two experiments.</p

Novel noncoding RNA candidates.

<p>Novel noncoding RNA candidates.</p

Examples of ncRNA candidates.

<p>(A, C) UCSC genome browser analysis of UT20 (A) and UT21 (C). (B, D) Northern blot analysis of UT20 (B) and UT21 (D). Total RNA from HeLa Tet-off cells was analyzed. The positions of the ribosomal RNAs (rRNAs) are shown to the left of the blots. Asterisks indicate bands corresponding to the expected size.</p

Procedure for the identification of ncRNA candidates.

<p>(A) Outline of our gene-prediction method from human full-length cDNAs and ESTs mapped to the human genome. ORF, open reading frame. (B) Classification of 180 ncRNA candidates analyzed manually using the UCSC genome browser.</p